Improved ethanol tolerance and ethanol production from glycerol in a streptomycin-resistant Klebsiella variicola mutant obtained by ribosome engineering.

PubMed

Suzuki, Toshihiro; Seta, Kohei; Nishikawa, Chiaki; Hara, Eri; Shigeno, Toshiya; Nakajima-Kambe, Toshiaki

2015-01-01

To improve the ethanol tolerance of the Klebsiella variicola strain TB-83, we obtained the streptomycin-resistant, ethanol-tolerant mutant strain TB-83D by a ribosome engineering approach. Strain TB-83D was able to grow in the presence of 7% (v/v) ethanol and it showed higher ethanol production than strain TB-83. Examination of various culture conditions revealed that yeast extract was essential for ethanol production and bacterial growth. In addition, ethanol production was elevated to 32g/L by the addition of yeast extract; however, ethanol production was inhibited by formate accumulation. With regard to cost reduction, the use of corn steep liquor (CSL) markedly decreased the formate concentration, and 34g/L ethanol was produced by combining yeast extract with CSL. Our study is the first to improve ethanol tolerance and productivity by a ribosome engineering approach, and we found that strain TB-83D is effective for ethanol production from glycerol. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genetic engineering of Ganoderma lucidum for the efficient production of ganoderic acids.

PubMed

Xu, Jun-Wei; Zhong, Jian-Jiang

2015-01-01

Ganoderma lucidum is a well-known traditional medicinal mushroom that produces ganoderic acids with numerous interesting bioactivities. Genetic engineering is an efficient approach to improve ganoderic acid biosynthesis. However, reliable genetic transformation methods and appropriate genetic manipulation strategies remain underdeveloped and thus should be enhanced. We previously established a homologous genetic transformation method for G. lucidum; we also applied the established method to perform the deregulated overexpression of a homologous 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene in G. lucidum. Engineered strains accumulated more ganoderic acids than wild-type strains. In this report, the genetic transformation systems of G. lucidum are described; current trends are also presented to improve ganoderic acid production through the genetic manipulation of G. lucidum.

Synthesis and accumulation of aromatic aldehydes in an engineered strain of Escherichia coli.

PubMed

Kunjapur, Aditya M; Tarasova, Yekaterina; Prather, Kristala L J

2014-08-20

Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

Leveraging algal omics to reveal potential targets for augmenting TAG accumulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arora, Neha; Pienkos, Philip T.; Pruthi, Vikas

Ongoing global efforts to commercialize microalgal biofuels have expedited the use of multi-omics techniques to gain insights into lipid biosynthetic pathways. Functional genomics analyses have recently been employed to complement existing sequence-level omics studies, shedding light on the dynamics of lipid synthesis and its interplay with other cellular metabolic pathways, thus revealing possible targets for metabolic engineering. Here, we review the current status of algal omics studies to reveal potential targets to augment TAG accumulation in various microalgae. Here, this review specifically aims to examine and catalog systems level data related to stress-induced TAG accumulation in oleaginous microalgae and informmore » future metabolic engineering strategies to develop strains with enhanced bioproductivity, which could pave a path for sustainable green energy.« less

Leveraging Algal Omics to Reveal Potential Targets for Augmenting TAG Accumulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guarnieri, Michael T; Pienkos, Philip T; Arora, Neha

2018-04-18

Ongoing global efforts to commercialize microalgal biofuels have expedited the use of multi-omics techniques to gain insights into lipid biosynthetic pathways. Functional genomics analyses have recently been employed to complement existing sequence-level omics studies, shedding light on the dynamics of lipid synthesis and its interplay with other cellular metabolic pathways, thus revealing possible targets for metabolic engineering. Here, we review the current status of algal omics studies to reveal potential targets to augment TAG accumulation in various microalgae. This review specifically aims to examine and catalog systems level data related to stress-induced TAG accumulation in oleaginous microalgae and inform futuremore » metabolic engineering strategies to develop strains with enhanced bioproductivity, which could pave a path for sustainable green energy.« less

Leveraging algal omics to reveal potential targets for augmenting TAG accumulation

DOE PAGES

Arora, Neha; Pienkos, Philip T.; Pruthi, Vikas; ...

2018-04-18

Ongoing global efforts to commercialize microalgal biofuels have expedited the use of multi-omics techniques to gain insights into lipid biosynthetic pathways. Functional genomics analyses have recently been employed to complement existing sequence-level omics studies, shedding light on the dynamics of lipid synthesis and its interplay with other cellular metabolic pathways, thus revealing possible targets for metabolic engineering. Here, we review the current status of algal omics studies to reveal potential targets to augment TAG accumulation in various microalgae. Here, this review specifically aims to examine and catalog systems level data related to stress-induced TAG accumulation in oleaginous microalgae and informmore » future metabolic engineering strategies to develop strains with enhanced bioproductivity, which could pave a path for sustainable green energy.« less

Leveraging algal omics to reveal potential targets for augmenting TAG accumulation.

PubMed

Arora, Neha; Pienkos, Philip T; Pruthi, Vikas; Poluri, Krishna Mohan; Guarnieri, Michael T

2018-04-18

Ongoing global efforts to commercialize microalgal biofuels have expedited the use of multi-omics techniques to gain insights into lipid biosynthetic pathways. Functional genomics analyses have recently been employed to complement existing sequence-level omics studies, shedding light on the dynamics of lipid synthesis and its interplay with other cellular metabolic pathways, thus revealing possible targets for metabolic engineering. Here, we review the current status of algal omics studies to reveal potential targets to augment TAG accumulation in various microalgae. This review specifically aims to examine and catalog systems level data related to stress-induced TAG accumulation in oleaginous microalgae and inform future metabolic engineering strategies to develop strains with enhanced bioproductivity, which could pave a path for sustainable green energy. Copyright © 2018. Published by Elsevier Inc.

High-Frequency Testing of Composite Fan Vanes With Erosion-Resistant Coating Conducted

NASA Technical Reports Server (NTRS)

Bowman, Cheryl L.; Sutter, James K.; Naik, Subhash; Otten, Kim D.; Perusek, Gail P.

2003-01-01

The mechanical integrity of hard, erosion-resistant coatings were tested using the Structural Dynamics Laboratory at the NASA Glenn Research Center. Under the guidance of Structural Mechanics and Dynamics Branch personnel, fixturing and test procedures were developed at Glenn to simulate engine vibratory conditions on coated polymer-matrix- composite bypass vanes using a slip table in the Structural Dynamics Laboratory. Results from the high-frequency mechanical bench testing, along with concurrent erosion testing of coupons and vanes, provided sufficient confidence to engine-endurance test similarly coated vane segments. The knowledge gained from this program will be applied to the development of oxidation- and erosion-resistant coatings for polymer matrix composite blades and vanes in future advanced turbine engines. Fan bypass vanes from the AE3007 (Rolls Royce America, Indianapolis, IN) gas turbine engine were coated by Engelhard (Windsor, CT) with compliant bond coatings and hard ceramic coatings. The coatings were developed collaboratively by Glenn and Allison Advanced Development Corporation (AADC)/Rolls Royce America through research sponsored by the High-Temperature Engine Materials Technology Project (HITEMP) and the Higher Operating Temperature Propulsion Components (HOTPC) project. High-cycle fatigue was performed through high-frequency vibratory testing on a shaker table. Vane resonant frequency modes were surveyed from 50 to 3000 Hz at input loads from 1g to 55g on both uncoated production vanes and vanes with the erosion-resistant coating. Vanes were instrumented with both lightweight accelerometers and strain gauges to establish resonance, mode shape, and strain amplitudes. Two high-frequency dwell conditions were chosen to excite two strain levels: one approaching the vane's maximum allowable design strain and another near the expected maximum strain during engine operation. Six specimens were tested per dwell condition. Pretest and posttest inspections were performed optically at up to 60 magnification and using a fluorescent-dye penetrant. Accumulation of 10 million cycles at a strain amplitude of two to three times that expected in the engine (approximately 670 Hz and 20g) led to the development of multiple cracks in the coating that were only detectable using fluorescent-dye penetrant inspection. Cracks were prevalent on the trailing edge and on the convex side of the midsection. No cracking or spalling was evident using standard optical inspection at up to 60 magnification. Further inspection may reveal whether these fine cracks penetrated the coating or were strictly on the surface. The dwell condition that simulated actual engine conditions produced no obvious surface flaws even after up to 80 million cycles had been accumulated at strain amplitudes produced at approximately 1500 Hz and 45g.

Metabolic engineering of a laboratory-evolved Thermobifida fusca muC strain for malic acid production on cellulose and minimal treated lignocellulosic biomass.

PubMed

Deng, Yu; Mao, Yin; Zhang, Xiaojuan

2016-01-01

Malic acid is mainly used as an acidulant and taste enhancer in the beverage and food industry. Previously, a mutant strain Thermobifida fusca muC, obtained by adaptive evolution was found to accumulate malic acid on cellulose with low yield. In this study, the malic acid synthesis pathway in T. fusca muC was confirmed to be from phosphoenolpyruvate to oxaloacetate, followed by reduction of oxaloacetate to malate. To increase the yield of malic acid by the muC strain significantly, the carbon flux from pyruvate was redirected to oxaloacetate by expressing an exogenous pyruvate carboxylase (PCx) gene from Corynebacterium glutamicum ATCC 13032 in the chromosome of T. fusca muC-16. The yield of malic acid in the engineered strain muC-16 was increased by 47.9% compared to the parent strain muC. The muC-16 strain was then grown on ∼100 g/L cellulose and the highest titer of malic acid was 62.76 g/L by batch fermentation. T. fusca muC-16 strain converted milled corn stover to malic acid with the highest titer of 21.47 g/L with minimal treatment. © 2016 American Institute of Chemical Engineers.

EFFECTS OF 2,4-DICHLOROPHENOL, A METABOLITE OF A GENETICALLY ENGINEERED BACTERIUM, AND 2,4-DICHLOROPHENOXYACETATE ON SOME MICROORGANISM-MEDIATED ECOLOGICAL PROCESSES IN SOIL

EPA Science Inventory

A genetically engineered microorganism, Pseudomonas putida PPO301 (pRO103), and the plasmidless parent strain, PPO301, were added at approximately 10 7 CFU/g of soil amended with 500 ppm of 2,4-dichlorophenoxyacete (2,4-D)(500 ug/g). he degradation of 2,4-D and the accumulation o...

Genetic engineering of Ganoderma lucidum for the efficient production of ganoderic acids

PubMed Central

Xu, Jun-Wei; Zhong, Jian-Jiang

2015-01-01

Ganoderma lucidum is a well-known traditional medicinal mushroom that produces ganoderic acids with numerous interesting bioactivities. Genetic engineering is an efficient approach to improve ganoderic acid biosynthesis. However, reliable genetic transformation methods and appropriate genetic manipulation strategies remain underdeveloped and thus should be enhanced. We previously established a homologous genetic transformation method for G. lucidum; we also applied the established method to perform the deregulated overexpression of a homologous 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene in G. lucidum. Engineered strains accumulated more ganoderic acids than wild-type strains. In this report, the genetic transformation systems of G. lucidum are described; current trends are also presented to improve ganoderic acid production through the genetic manipulation of G. lucidum. PMID:26588475

Screening of a thiamine-auxotrophic yeast for alpha-ketoglutaric acid overproduction.

PubMed

Zhou, Jingwen; Zhou, Haiyan; Du, Guocheng; Liu, Liming; Chen, Jian

2010-09-01

To obtain a thiamine-auxotrophic yeast strain that overproduces alpha-ketoglutaric acid (alpha-KG) from glycerol and to investigate nutrient effects on alpha-KG production. Yeast strain WSH-Z06, a thiamine auxotroph that gave high yields of alpha-KG from glycerol, was obtained by screening for ampicillin/kanamycin resistance and thiamine auxotrophy. The strain was identified as Yarrowia lipolytica based on physiological, chemical, and phylogenetic analysis. The ability of the strain to convert glycerol to alpha-KG was analysed by investigating the effects of nutritional factors, including thiamine, riboflavin, nitrogen sources, and calcium ion. Thiamine and calcium ion concentration had the greatest effect on alpha-KG accumulation. Under optimal conditions, a yield of 39.2 g l(-1)alpha-KG was obtained from 100 g l(-1) glycerol, with 16.84 g l(-1) pyruvate as a by-product. The current work provides a method for screening for an alpha-KG overproducer. Nutrients have a significant impact on alpha-KG production in the yeast strain presented here. The alpha-KG-overproducing yeast strain Y. lipolytica WSH-Z06 is a promising parent strain for further metabolic engineering to lower by-product accumulation and accelerate glycerol utilization.

Metabolic Engineering of Synechocystis sp. Strain PCC 6803 for Isobutanol Production

PubMed Central

Varman, Arul M.; Xiao, Yi; Pakrasi, Himadri B.

2013-01-01

Global warming and decreasing fossil fuel reserves have prompted great interest in the synthesis of advanced biofuels from renewable resources. In an effort to address these concerns, we performed metabolic engineering of the cyanobacterium Synechocystis sp. strain PCC 6803 to develop a strain that can synthesize isobutanol under both autotrophic and mixotrophic conditions. With the expression of two heterologous genes from the Ehrlich pathway, the engineered strain can accumulate 90 mg/liter of isobutanol from 50 mM bicarbonate in a gas-tight shaking flask. The strain does not require any inducer (i.e., isopropyl β-d-1-thiogalactopyranoside [IPTG]) or antibiotics to maintain its isobutanol production. In the presence of glucose, isobutanol synthesis is only moderately promoted (titer = 114 mg/liter). Based on isotopomer analysis, we found that, compared to the wild-type strain, the mutant significantly reduced its glucose utilization and mainly employed autotrophic metabolism for biomass growth and isobutanol production. Since isobutanol is toxic to the cells and may also be degraded photochemically by hydroxyl radicals during the cultivation process, we employed in situ removal of the isobutanol using oleyl alcohol as a solvent trap. This resulted in a final net concentration of 298 mg/liter of isobutanol under mixotrophic culture conditions. PMID:23183979

Metabolic Engineering of the Regulators in Nitrogen Catabolite Repression To Reduce the Production of Ethyl Carbamate in a Model Rice Wine System

PubMed Central

Zhao, Xinrui; Zou, Huijun; Fu, Jianwei; Chen, Jian

2014-01-01

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production. PMID:24185848

Metabolic engineering of the regulators in nitrogen catabolite repression to reduce the production of ethyl carbamate in a model rice wine system.

PubMed

Zhao, Xinrui; Zou, Huijun; Fu, Jianwei; Zhou, Jingwen; Du, Guocheng; Chen, Jian

2014-01-01

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production.

A balanced ATP driving force module for enhancing photosynthetic biosynthesis of 3-hydroxybutyrate from CO2.

PubMed

Ku, Jason T; Lan, Ethan I

2018-03-01

Using engineered photoautotrophic microorganisms for the direct chemical synthesis from CO 2 is an attractive direction for both sustainability and CO 2 mitigation. However, the behaviors of non-native metabolic pathways may be difficult to control due to the different intracellular contexts between natural and heterologous hosts. While most metabolic engineering efforts focus on strengthening driving forces in pathway design to favor biochemical production in these organisms, excessive driving force may be detrimental to product biosynthesis due to imbalanced cellular intermediate distribution. In this study, an ATP-hydrolysis based driving force module was engineered into cyanobacterium Synechococcus elongatus PCC 7942 to produce 3-hydroxybutyrate (3HB), a valuable chemical feedstock for the synthesis of biodegradable plastics and antibiotics. However, while the ATP driving force module is effective for increasing product formation, uncontrolled accumulation of intermediate metabolites likely led to metabolic imbalance and thus to cell growth inhibition. Therefore, the ATP driving force module was reengineered by providing a reversible outlet for excessive carbon flux. Upon expression of this balanced ATP driving force module with 3HB biosynthesis, engineered strain produced 3HB with a cumulative titer of 1.2 g/L, a significant increase over the initial strain. This result highlighted the importance of pathway reversibility as an effective design strategy for balancing driving force and intermediate accumulation, thereby achieving a self-regulated control for increased net flux towards product biosynthesis. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Combinatorial metabolic engineering of industrial Gluconobacter oxydans DSM2343 for boosting 5-keto-D-gluconic acid accumulation.

PubMed

Yuan, Jianfeng; Wu, Mianbin; Lin, Jianping; Yang, Lirong

2016-05-17

L-(+)-tartaric acid (L-TA) is an important organic acid, which is produced from the cream of tartar or stereospecific hydrolysis of the cis-epoxysuccinate. The former method is limited by the availability of raw material and the latter is dependent on the petrochemical material. Thus, new processes for the economical preparation of L-TA from carbohydrate or renewable resource would be much more attractive. Production of 5-keto-D-gluconate (5-KGA) from glucose by Gluconobacter oxydans is the first step to produce L-TA. The aim of this work is to enhance 5-KGA accumulation using combinatorial metabolic engineering strategies in G. oxydans. The sldAB gene, encoding sorbitol dehydrogenase, was overexpressed in an industrial strain G. oxydans ZJU2 under a carefully selected promoter, P0169. To enhance the efficiency of the oxidation by sldAB, the coenzyme pyrroloquinoline quinone (PQQ) and respiratory chain were engineered. Besides, the role in sldAB overexpression, coenzyme and respiratory chain engineering and their subsequent effects on 5-KGA production were investigated. An efficient, stable recombinant strain was constructed, whereas the 5-KGA production could be enhanced. By self-overexpressing the sldAB gene in G. oxydans ZJU2 under the constitutive promoter P0169, the resulting strain, G. oxydans ZJU3, produced 122.48 ± 0.41 g/L of 5-KGA. Furthermore, through the coenzyme and respiratory chain engineering, the titer and productivity of 5-KGA reached 144.52 ± 2.94 g/L and 2.26 g/(L · h), respectively, in a 15 L fermenter. It could be further improved the 5-KGA titer by 12.10 % through the fed-batch fermentation under the pH shift and dissolved oxygen tension (DOT) control condition, obtained 162 ± 2.12 g/L with the productivity of 2.53 g/(L · h) within 64 h. The 5-KGA production could be significantly enhanced with the combinatorial metabolic engineering strategy in Gluconobacter strain, including sldAB overexpression, coenzyme and respiratory chain engineering. Fed-batch fermentation could further enlarge the positive effect and increase the 5-KGA production. All of these demonstrated that the robust recombinant strain can efficiently produce 5-KGA in larger scale to fulfill the industrial production of L-TA from 5-KGA.

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.

PubMed

Runguphan, Weerawat; Keasling, Jay D

2014-01-01

As the serious effects of global climate change become apparent and access to fossil fuels becomes more limited, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. In recent years, microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fatty acid-derived biofuels and chemicals from simple sugars. Specifically, we overexpressed all three fatty acid biosynthesis genes, namely acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1) and fatty acid synthase 2 (FAS2), in S. cerevisiae. When coupled to triacylglycerol (TAG) production, the engineered strain accumulated lipid to more than 17% of its dry cell weight, a four-fold improvement over the control strain. Understanding that TAG cannot be used directly as fuels, we also engineered S. cerevisiae to produce drop-in fuels and chemicals. Altering the terminal "converting enzyme" in the engineered strain led to the production of free fatty acids at a titer of approximately 400 mg/L, fatty alcohols at approximately 100mg/L and fatty acid ethyl esters (biodiesel) at approximately 5 mg/L directly from simple sugars. We envision that our approach will provide a scalable, controllable and economic route to this important class of chemicals. Copyright © 2013 International Metabolic Engineering Society. All rights reserved.

A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica.

PubMed

Wagner, James M; Liu, Leqian; Yuan, Shuo-Fu; Venkataraman, Maya V; Abate, Adam R; Alper, Hal S

2018-04-23

Evolutionary approaches to strain engineering inherently require the identification of suitable selection techniques for the product and phenotype of interest. In this work, we undertake a comparative analysis of two related but functionally distinct methods of high-throughput screening: traditional single cell fluorescence activated cell sorting (single cell FACS) and microdroplet-enabled FACS (droplet FACS) using water/oil/water (w/o/w) emulsions. To do so, we first engineer and evolve the non-conventional yeast Yarrowia lipolytica for high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. Following mutagenesis and adaptive evolution, a direct parity-matched comparison of these two selection strategies was conducted. Both single cell FACS and droplet FACS led to significant increases in total riboflavin titer (32 and 54 fold relative to the parental PO1f strain, respectively). However, single cell FACS favored intracellular riboflavin accumulation (with only 70% of total riboflavin secreted) compared with droplet FACS that favored extracellular product accumulation (with 90% of total riboflavin secreted). We find that for the test case of riboflavin, the extent of secretion and total production were highly correlated. The resulting differences in production modes and levels clearly demonstrate the significant impact that selection approaches can exert on final evolutionary outcomes in strain engineering. Moreover, we note that these results provide a cautionary tale when intracellular read-outs of product concentration (including signals from biosensors) are used as surrogates for total production of potentially secreted products. In this regard, these results demonstrate that extracellular production is best assayed through an encapsulation technique when performing high throughput screening. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Regulation of EPS production in Lactobacillus casei LC2W through metabolic engineering.

PubMed

Li, N; Huang, Y; Liu, Z; You, C; Guo, B

2015-12-01

Lactobacillus casei LC2W is an exopolysaccharide(EPS)-producing strain with probiotic effects. The low efficiency and unclear regulation mechanism of EPS biosynthesis have become main constraints for its application in food industry. To investigate the major rate-limiting factors of EPS biosynthesis and to improve its yield, metabolic engineering was applied to this strain. Eight relevant genes related to central metabolism, sugar-nucleotides supply, glycosyltransferase and cofactor engineering were cloned and overexpressed. The results suggested that nox, pfk, rfbB and galT genes were the largest contributors to EPS biosynthesis in this study, which elevated EPS yield by 46·0, 20, 17·4 and 19·6% respectively. Notably, under aerobic condition which was not a suitable condition for lactobacilli to grow in, recombinant strain LC-nox achieved the highest EPS yield of 263·7 mg l(-1) , which was increased by 75% compared to that of the starting strain. The oxygen stress was excluded since the phenomenon was not observed in the control strain under the same condition. Therefore, it was probably that higher NADH oxidase activity led to a decreased NADH availability and reduced lactate concentration, which resulted in the elevation of EPS yield. This study contributed to the understanding of EPS biosynthesis in Lact. casei through metabolic engineering and provided a starting point for introducing cofactor engineering into this strain. Overexpression of NADH oxidase was found to have a most significant effect on the EPS production. It is the first report that EPS could be accumulated to such a high level under aerobic condition in lactobacilli. Our results provided a novel strategy for the improvement of EPS production in lactic acid bacteria. © 2015 The Society for Applied Microbiology.

Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid.

PubMed

Xue, Yong; Zhang, Yan; Cheng, Dan; Daddy, Soumana; He, Qingfang

2014-07-01

p-Coumaric acid is the precursor of phenylpropanoids, which are plant secondary metabolites that are beneficial to human health. Tyrosine ammonia lyase catalyzes the production of p-coumaric acid from tyrosine. Because of their photosynthetic ability and biosynthetic versatility, cyanobacteria are promising candidates for the production of certain plant metabolites, including phenylpropanoids. Here, we produced p-coumaric acid in a strain of transgenic cyanobacterium Synechocystis sp. Pasteur Culture Collection 6803 (hereafter Synechocystis 6803). Whereas a strain of Synechocystis 6803 genetically engineered to express sam8, a tyrosine ammonia lyase gene from the actinomycete Saccharothrix espanaensis, accumulated little or no p-coumaric acid, a strain that both expressed sam8 and lacked slr1573, a native hypothetical gene shown here to encode a laccase that oxidizes polyphenols, produced ∼82.6 mg/L p-coumaric acid, which was readily purified from the growth medium.

Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid

PubMed Central

Xue, Yong; Zhang, Yan; Cheng, Dan; Daddy, Soumana; He, Qingfang

2014-01-01

p-Coumaric acid is the precursor of phenylpropanoids, which are plant secondary metabolites that are beneficial to human health. Tyrosine ammonia lyase catalyzes the production of p-coumaric acid from tyrosine. Because of their photosynthetic ability and biosynthetic versatility, cyanobacteria are promising candidates for the production of certain plant metabolites, including phenylpropanoids. Here, we produced p-coumaric acid in a strain of transgenic cyanobacterium Synechocystis sp. Pasteur Culture Collection 6803 (hereafter Synechocystis 6803). Whereas a strain of Synechocystis 6803 genetically engineered to express sam8, a tyrosine ammonia lyase gene from the actinomycete Saccharothrix espanaensis, accumulated little or no p-coumaric acid, a strain that both expressed sam8 and lacked slr1573, a native hypothetical gene shown here to encode a laccase that oxidizes polyphenols, produced ∼82.6 mg/L p-coumaric acid, which was readily purified from the growth medium. PMID:24927550

Different response to acetic acid stress in Saccharomyces cerevisiae wild-type and l-ascorbic acid-producing strains.

PubMed

Martani, Francesca; Fossati, Tiziana; Posteri, Riccardo; Signori, Lorenzo; Porro, Danilo; Branduardi, Paola

2013-09-01

Biotechnological processes are of increasing significance for industrial production of fine and bulk chemicals, including biofuels. Unfortunately, under operative conditions microorganisms meet multiple stresses, such as non-optimal pH, temperature, oxygenation and osmotic stress. Moreover, they have to face inhibitory compounds released during the pretreatment of lignocellulosic biomasses, which constitute the preferential substrate for second-generation processes. Inhibitors include furan derivatives, phenolic compounds and weak organic acids, among which acetic acid is one of the most abundant and detrimental for cells. They impair cellular metabolism and growth, reducing the productivity of the process: therefore, the development of robust cell factories with improved production rates and resistance is of crucial importance. Here we show that a yeast strain engineered to endogenously produce vitamin C exhibits an increased tolerance compared to the parental strain when exposed to acetic acid at moderately toxic concentrations, measured as viability on plates. Starting from this evidence, we investigated more deeply: (a) the nature and levels of reactive oxygen species (ROS); (b) the activation of enzymes that act directly as detoxifiers of reactive oxygen species, such as superoxide dismutase (SOD) and catalase, in parental and engineered strains during acetic acid stress. The data indicate that the engineered strain can better recover from stress by limiting ROS accumulation, independently from SOD activation. The engineered yeast can be proposed as a model for further investigating direct and indirect mechanism(s) by which an antioxidant can rescue cells from organic acid damage; moreover, these studies will possibly provide additional targets for further strain improvements. Copyright © 2013 John Wiley & Sons, Ltd.

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

PubMed

Bottoms, Scott; Dickinson, Quinn; McGee, Mick; Hinchman, Li; Higbee, Alan; Hebert, Alex; Serate, Jose; Xie, Dan; Zhang, Yaoping; Coon, Joshua J; Myers, Chad L; Landick, Robert; Piotrowski, Jeff S

2018-01-12

Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.

Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation.

PubMed

Ruta, Lavinia Liliana; Lin, Ya-Fen; Kissen, Ralph; Nicolau, Ioana; Neagoe, Aurora Daniela; Ghenea, Simona; Bones, Atle M; Farcasanu, Ileana Cornelia

2017-01-01

In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

Proline accumulation protects Saccharomyces cerevisiae cells in stationary phase from ethanol stress by reducing reactive oxygen species levels.

PubMed

Takagi, Hiroshi; Taguchi, Junpei; Kaino, Tomohiro

2016-08-01

During fermentation processes, Saccharomyces cerevisiae cells are exposed to multiple stresses, including a high concentration of ethanol that represents toxicity through intracellular reactive oxygen species (ROS) generation. We previously reported that proline protected yeast cells from damage caused by various stresses, such as freezing and ethanol. As an anti-oxidant, proline is suggested to scavenge intracellular ROS. In this study, we examined the role of intracellular proline during ethanol treatment in S. cerevisiae strains that accumulate different concentrations of proline. When cultured in YPD medium, there was a significant accumulation of proline in the put1 mutant strain, which is deficient in proline oxidase, in the stationary phase. Expression of the mutant PRO1 gene, which encodes the γ-glutamyl kinase variant (Asp154Asn or Ile150Thr) with desensitization to feedback inhibition by proline in the put1 mutant strain, showed a prominent increase in proline content as compared with that of the wild-type strain. The oxidation level was clearly increased in wild-type cells after exposure to ethanol, indicating that the generation of ROS occurred. Interestingly, proline accumulation significantly reduces the ROS level and increases the survival rate of yeast cells in the stationary phase under ethanol stress conditions. However, there was not a clear correlation between proline content and survival rate in yeast cells. An appropriate level of intracellular proline in yeast might be important for its stress-protective effect. Hence, the engineering of proline metabolism could be promising for breeding stress-tolerant industrial yeast strains. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Response of Saccharomyces cerevisiae to D-limonene-induced oxidative stress.

PubMed

Liu, Jidong; Zhu, Yibo; Du, Guocheng; Zhou, Jingwen; Chen, Jian

2013-07-01

In the present study, we investigated the mode of cell response induced by D-limonene in Saccharomyces cerevisiae. D-limonene treatment was found to be accompanied by intracellular accumulation of reactive oxygen species (ROS). Since ROS impair cell membranes, an engineered strain with enhanced membrane biosynthesis exhibited a higher tolerance to D-limonene. Subsequent addition of an ROS scavenger significantly reduced the ROS level and alleviated cell growth inhibition. Thus, D-limonene-induced ROS accumulation plays an important role in cell death in S. cerevisiae. In D-limonene-treated S. cerevisiae strains, higher levels of antioxidants, antioxidant enzymes, and nicotinamide adenine dinucleotide phosphate (NADPH) were synthesized. Quantitative real-time PCR results also verified that D-limonene treatment triggered upregulation of genes involved in the antioxidant system and the regeneration of NADPH at the transcription level in S. cerevisiae. These data indicate that D-limonene treatment results in intracellular ROS accumulation, an important factor in cell death, and several antioxidant mechanisms in S. cerevisiae were enhanced in response to D-limonene treatment.

Laser speckle technique for burner liner strain measurements

NASA Technical Reports Server (NTRS)

Stetson, K. A.

1982-01-01

Thermal and mechanical strains were measured on samples of a common material used in jet engine burner liners, which were heated from room temperature to 870 C and cooled back to 220 C, in a laboratory furnance. The physical geometry of the sample surface was recorded at selected temperatures by a set of 12 single exposure speckle-grams. Sequential pairs of specklegrams were compared in a heterodyne interferometer which give high precision measurement of differential displacements. Good speckle correlation between the first and last specklegrams is noted which allows a check on accumulate errors.

Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity

DOE PAGES

Johnson, Christopher W.; Salvachua, Davinia; Khanna, Payal; ...

2016-04-22

The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCAmore » decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92+/-0.48 g/L muconate. Furthermore, this study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.« less

Metabolic engineering for high glycerol production by the anaerobic cultures of Saccharomyces cerevisiae.

PubMed

Semkiv, Marta V; Dmytruk, Kostyantyn V; Abbas, Charles A; Sibirny, Andriy A

2017-06-01

Glycerol is used by the cosmetic, paint, automotive, food, and pharmaceutical industries and for production of explosives. Currently, glycerol is available in commercial quantities as a by-product from biodiesel production, but the purity and the cost of its purification are prohibitive. The industrial production of glycerol by glucose aerobic fermentation using osmotolerant strains of the yeasts Candida sp. and Saccharomyces cerevisiae has been described. A major drawback of the aerobic process is the high cost of production. For this reason, the development of yeast strains that effectively convert glucose to glycerol anaerobically is of great importance. Due to its ability to grow under anaerobic conditions, the yeast S. cerevisiae is an ideal system for the development of this new biotechnological platform. To increase glycerol production and accumulation from glucose, we lowered the expression of TPI1 gene coding for triose phosphate isomerase; overexpressed the fused gene consisting the GPD1 and GPP2 parts coding for glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, respectively; overexpressed the engineered FPS1 gene that codes for aquaglyceroporin; and overexpressed the truncated gene ILV2 that codes for acetolactate synthase. The best constructed strain produced more than 20 g of glycerol/L from glucose under micro-aerobic conditions and 16 g of glycerol/L under anaerobic conditions. The increase in glycerol production led to a drop in ethanol and biomass accumulation.

Engineering Ashbya gossypii strains for de novo lipid production using industrial by-products.

PubMed

Lozano-Martínez, Patricia; Buey, Rubén M; Ledesma-Amaro, Rodrigo; Jiménez, Alberto; Revuelta, José Luis

2017-03-01

Ashbya gossypii is a filamentous fungus that naturally overproduces riboflavin, and it is currently exploited for the industrial production of this vitamin. The utilization of A. gossypii for biotechnological applications presents important advantages such as the utilization of low-cost culture media, inexpensive downstream processing and a wide range of molecular tools for genetic manipulation, thus making A. gossypii a valuable biotechnological chassis for metabolic engineering. A. gossypii has been shown to accumulate high levels of lipids in oil-based culture media; however, the lipid biosynthesis capacity is rather limited when grown in sugar-based culture media. In this study, by altering the fatty acyl-CoA pool and manipulating the regulation of the main ∆9 desaturase gene, we have obtained A. gossypii strains with significantly increased (up to fourfold) de novo lipid biosynthesis using glucose as the only carbon source in the fermentation broth. Moreover, these strains were efficient biocatalysts for the conversion of carbohydrates from sugarcane molasses to biolipids, able to accumulate lipids up to 25% of its cell dry weight. Our results represent a proof of principle showing the promising potential of A. gossypii as a competitive microorganism for industrial biolipid production using cost-effective feed stocks. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Single cell assessment of yeast metabolic engineering for enhanced lipid production using Raman and AFM-IR imaging.

PubMed

Kochan, Kamila; Peng, Huadong; Wood, Bayden R; Haritos, Victoria S

2018-01-01

Biodiesel is a valuable renewable fuel made from derivatized fatty acids produced in plants, animals, and oleaginous microbes. Of the latter, yeasts are of special interest due to their wide use in biotechnology, ability to synthesize fatty acids and store large amounts of triacylglycerols while utilizing non-food carbon sources. While yeast efficiently produce lipids, genetic modification and indeed, lipid pathway metabolic engineering, is usually required for cost-effective production. Traditionally, gas chromatography (GC) is used to measure fatty acid production and to track the success of a metabolic engineering strategy in a microbial culture; here we have employed vibrational spectroscopy approaches at population and single cell level of engineered yeast while simultaneously investigating metabolite levels in subcellular structures. Firstly, a strong correlation ( r 2  > 0.99) was established between Fourier transform infrared (FTIR) lipid in intact cells and GC analysis of fatty acid methyl esters in the differently engineered strains. Confocal Raman spectroscopy of individual cells carrying genetic modifications to enhance fatty acid synthesis and lipid accumulation revealed changes to the lipid body (LB), the storage organelle for lipids in yeast, with their number increasing markedly (up to tenfold higher); LB size was almost double in the strain that also expressed a LB stabilizing gene but considerable variation was also noted between cells. Raman spectroscopy revealed a clear trend toward reduced unsaturated fatty acid content in lipids of cells carrying more complex metabolic engineering. Atomic force microscopy-infrared spectroscopy (AFM-IR) analysis of individual cells indicated large differences in subcellular constituents between strains: cells of the most highly engineered strain had elevated lipid and much reduced carbohydrate in their cytoplasm compared with unmodified cells. Vibrational spectroscopy analysis allowed the simultaneous measurement of strain variability in metabolite production and impact on cellular structures as a result of different gene introductions or knockouts, within a lipid metabolic engineering strategy and these inform the next steps in comprehensive lipid engineering. Additionally, single cell spectroscopic analysis measures heterogeneity in metabolite production across microbial cultures under genetic modification, an emerging issue for efficient biotechnological production.

Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

PubMed

Zhang, Guo-Chang; Turner, Timothy L; Jin, Yong-Su

2017-03-01

Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD + -linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

Enhanced pinocembrin production in Escherichia coli by regulating cinnamic acid metabolism

PubMed Central

Cao, Weijia; Ma, Weichao; Wang, Xin; Zhang, Bowen; Cao, Xun; Chen, Kequan; Li, Yan; Ouyang, Pingkai

2016-01-01

Microbial biosynthesis of pinocembrin is of great interest in the area of drug research and human healthcare. Here we found that the accumulation of the pathway intermediate cinnamic acid adversely affected pinocembrin production. Hence, a stepwise metabolic engineering strategy was carried out aimed at eliminating this pathway bottleneck and increasing pinocembrin production. The screening of gene source and the optimization of gene expression was first employed to regulate the synthetic pathway of cinnamic acid, which showed a 3.53-fold increase in pinocembrin production (7.76 mg/L) occurred with the alleviation of cinnamic acid accumulation in the engineered E. coli. Then, the downstream pathway that consuming cinnamic acid was optimized by the site-directed mutagenesis of chalcone synthase and cofactor engineering. S165M mutant of chalcone synthase could efficiently improve the pinocembrin production, and allowed the product titer of pinocembrin increased to 40.05 mg/L coupled with the malonyl-CoA engineering. With a two-phase pH fermentation strategy, the cultivation of the optimized strain resulted in a final pinocembrin titer of 67.81 mg/L. The results and engineering strategies demonstrated here would hold promise for the titer improvement of other flavonoids. PMID:27586788

Enhanced pinocembrin production in Escherichia coli by regulating cinnamic acid metabolism.

PubMed

Cao, Weijia; Ma, Weichao; Wang, Xin; Zhang, Bowen; Cao, Xun; Chen, Kequan; Li, Yan; Ouyang, Pingkai

2016-09-02

Microbial biosynthesis of pinocembrin is of great interest in the area of drug research and human healthcare. Here we found that the accumulation of the pathway intermediate cinnamic acid adversely affected pinocembrin production. Hence, a stepwise metabolic engineering strategy was carried out aimed at eliminating this pathway bottleneck and increasing pinocembrin production. The screening of gene source and the optimization of gene expression was first employed to regulate the synthetic pathway of cinnamic acid, which showed a 3.53-fold increase in pinocembrin production (7.76 mg/L) occurred with the alleviation of cinnamic acid accumulation in the engineered E. coli. Then, the downstream pathway that consuming cinnamic acid was optimized by the site-directed mutagenesis of chalcone synthase and cofactor engineering. S165M mutant of chalcone synthase could efficiently improve the pinocembrin production, and allowed the product titer of pinocembrin increased to 40.05 mg/L coupled with the malonyl-CoA engineering. With a two-phase pH fermentation strategy, the cultivation of the optimized strain resulted in a final pinocembrin titer of 67.81 mg/L. The results and engineering strategies demonstrated here would hold promise for the titer improvement of other flavonoids.

Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi.

PubMed

Zhang, De-Huai; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

2017-02-01

Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enhanced isoprene biosynthesis in Saccharomyces cerevisiae by engineering of the native acetyl-CoA and mevalonic acid pathways with a push-pull-restrain strategy.

PubMed

Lv, Xiaomei; Xie, Wenping; Lu, Wenqiang; Guo, Fei; Gu, Jiali; Yu, Hongwei; Ye, Lidan

2014-09-30

To explore the capacity of isoprene production in Saccharomyces cerevisiae, a rational push-pull-restrain strategy was proposed to engineer the mevalonic acid (MVA) and acetyl-CoA pathways. The strategy can be decomposed into the up-regulation of precursor supply in the acetyl-CoA module and the MVA pathway (push-strategy), increase of the isoprene branch flux (pull-strategy), and down-regulation of the competing pathway (restrain-strategy). Furthermore, to reduce the production cost arising from galactose addition and meanwhile maintain the high expression of Gal promoters, the galactose regulatory network was modulated by Gal80p deletion. Finally, the engineered strain YXM10-ispS-ispS could accumulate up to 37 mg/L isoprene (about 782-fold increase compared to the parental strain) under aerobic conditions with glycerol-sucrose as carbon source. In this way, a new potential platform for isoprene production was established via metabolic engineering of the yeast native pathways. Copyright © 2014 Elsevier B.V. All rights reserved.

Investigation of the fuel feed line failures on the Space Shuttle main engine

NASA Technical Reports Server (NTRS)

Larson, E. W.

1980-01-01

The Space Shuttle Main Engine (SSME) development program experienced two similar appearing fuel feed line failures during the shutdown portion of two engine tests. Failure investigations into each incident showed that a few cycles of high-amplitude transient strain occurring during the start and cutoff portions of each test could have either accumulated damage and led to a fatigue failure after 46 tests, or caused rupture in a low-strength weld joint. The cause of the high strain was traced to a period of unsteady flow separation during the start and cutoff of each test coincident with the oblique shock approaching the nozzle exit. Since elimination of the flow separation was impractical, the steps taken to allow engine development and flight preparations to continue were: (1) establish the safe operating life of the nozzle, (2) reinforce all low-strength welds, and (3) eliminate the use of thin-wall fuel feed lines. In parallel, the feed line was redesigned and fabrication was initiated on units to be incorporated into the development program.

Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering.

PubMed

Xie, Wenping; Lv, Xiaomei; Ye, Lidan; Zhou, Pingping; Yu, Hongwei

2015-07-01

Improved supply of farnesyl diphosphate (FPP) is often considered as a typical strategy for engineering Saccharomyces cerevisiae towards efficient terpenoid production. However, in the engineered strains with enhanced precursor supply, the production of the target metabolite is often impeded by insufficient capacity of the heterologous terpenoid pathways, which limits further conversion of FPP. Here, we tried to assemble an unimpeded biosynthesis pathway by combining directed evolution and metabolic engineering in S. cerevisiae for lycopene-overproduction. First, the catalytic ability of phytoene syntheses from different sources was investigated based on lycopene accumulation. Particularly, the lycopene cyclase function of the bifunctional enzyme CrtYB from Xanthophyllomyces dendrorhous was inactivated by deletion of functional domain and directed evolution to obtain mutants with solely phytoene synthase function. Coexpression of the resulting CrtYB11M mutant along with the CrtE and CrtI genes from X. dendrorhous, and the tHMG1 gene from S. cerevisiae led to production of 4.47 mg/g DCW (Dry cell weight) of lycopene and 25.66 mg/g DCW of the by-product squalene. To further increase the FPP competitiveness of the lycopene synthesis pathway, we tried to enhance the catalytic performance of CrtE by directed evolution and created a series of pathway variants by varying the copy number of Crt genes. Finally, fed-batch fermentation was conducted for the diploid strain YXWPD-14 resulting in accumulation of 1.61 g/L (24.41 mg/g DCW) of lycopene, meanwhile, the by-production of squalene was reduced to below 1 mg/g DCW. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions.

PubMed

Heux, Stéphanie; Sablayrolles, Jean-Marie; Cachon, Rémy; Dequin, Sylvie

2006-09-01

We recently showed that expressing an H(2)O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD(+) reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.

Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae.

PubMed

Ambrosio, Rafael; Ortiz-Marquez, Juan Cesar Federico; Curatti, Leonardo

2017-03-01

The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N 2 and CO 2 from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N 2 from the air into crops. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

DOE PAGES

Tran, Tam N. T.; Breuer, Rebecca J.; Avanasi Narasimhan, Ragothaman; ...

2017-03-20

Background: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storagemore » lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts. Results: In order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Additionally, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media. Conclusion: Our results demonstrate that S. cerevisiae can be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain of S. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids.« less

L-Cysteine Production in Escherichia coli Based on Rational Metabolic Engineering and Modular Strategy.

PubMed

Liu, Han; Fang, Guochen; Wu, Hui; Li, Zhimin; Ye, Qin

2018-05-01

L-cysteine is an amino acid with important physiological functions and has a wide range of applications in medicine, food, animal feed, and cosmetics industry. In this study, the L-cysteine synthesis in Escherichia coliEscherichia coli is divided into four modules: the transport module, sulfur module, precursor module, and degradation module. The engineered strain LH03 (overexpression of the feedback-insensitive cysE and the exporter ydeD in JM109) accumulated 45.8 mg L -1 of L-cysteine in 48 hr with yield of 0.4% g/g glucose. Further modifications of strains and culture conditions which based on the rational metabolic engineering and modular strategy improved the L-cysteine biosynthesis significantly. The engineered strain LH06 (with additional overexpression of serA, serC, and serB and double mutant of tnaA and sdaA in LH03) produced 620.9 mg L -1 of L-cysteine with yield of 6.0% g/g glucose, which increased the production by 12 times and the yield by 14 times more than those of LH03 in the original condition. In fed-batch fermentation performed in a 5-L reactor, the concentration of L-cysteine achieved 5.1 g L -1 in 32 hr. This work demonstrates that the combination of rational metabolic engineering and module strategy is a promising approach for increasing the L-cysteine production in E. coli. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combinatorial analysis of enzymatic bottlenecks of L-tyrosine pathway by p-coumaric acid production in Saccharomyces cerevisiae.

PubMed

Mao, Jiwei; Liu, Quanli; Song, Xiaofei; Wang, Hesuiyuan; Feng, Hui; Xu, Haijin; Qiao, Mingqiang

2017-07-01

To identify new enzymatic bottlenecks of L-tyrosine pathway for further improving the production of L-tyrosine and its derivatives. When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l -1 , respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l -1 ) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain. Combinatorial metabolic engineering provides a new strategy for further improvement of L-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

Biosynthesis of levan, a bacterial extracellular polysaccharide, in the yeast Saccharomyces cerevisiae.

PubMed

Franken, Jaco; Brandt, Bianca A; Tai, Siew L; Bauer, Florian F

2013-01-01

Levans are fructose polymers synthesized by a broad range of micro-organisms and a limited number of plant species as non-structural storage carbohydrates. In microbes, these polymers contribute to the formation of the extracellular polysaccharide (EPS) matrix and play a role in microbial biofilm formation. Levans belong to a larger group of commercially important polymers, referred to as fructans, which are used as a source of prebiotic fibre. For levan, specifically, this market remains untapped, since no viable production strategy has been established. Synthesis of levan is catalysed by a group of enzymes, referred to as levansucrases, using sucrose as substrate. Heterologous expression of levansucrases has been notoriously difficult to achieve in Saccharomyces cerevisiae. As a strategy, this study used an invertase (Δsuc2) null mutant and two separate, engineered, sucrose accumulating yeast strains as hosts for the expression of the levansucrase M1FT, previously cloned from Leuconostoc mesenteroides. Intracellular sucrose accumulation was achieved either by expression of a sucrose synthase (Susy) from potato or the spinach sucrose transporter (SUT). The data indicate that in both Δsuc2 and the sucrose accumulating strains, the M1FT was able to catalyse fructose polymerisation. In the absence of the predicted M1FT secretion signal, intracellular levan accumulation was significantly enhanced for both sucrose accumulation strains, when grown on minimal media. Interestingly, co-expression of M1FT and SUT resulted in hyper-production and extracellular build-up of levan when grown in rich medium containing sucrose. This study presents the first report of levan production in S. cerevisiae and opens potential avenues for the production of levan using this well established industrial microbe. Furthermore, the work provides interesting perspectives when considering the heterologous expression of sugar polymerizing enzymes in yeast.

Deviation of the neurosporaxanthin pathway towards beta-carotene biosynthesis in Fusarium fujikuroi by a point mutation in the phytoene desaturase gene.

PubMed

Prado-Cabrero, Alfonso; Schaub, Patrick; Díaz-Sánchez, Violeta; Estrada, Alejandro F; Al-Babili, Salim; Avalos, Javier

2009-08-01

Carotenoids are widespread terpenoid pigments with applications in the food and feed industries. Upon illumination, the gibberellin-producing fungus Fusarium fujikuroi (Gibberella fujikuroi mating population C) develops an orange pigmentation caused by an accumulation of the carboxylic apocarotenoid neurosporaxanthin. The synthesis of this xanthophyll includes five desaturation steps presumed to be catalysed by the carB-encoded phytoene desaturase. In this study, we identified a yellow mutant (SF21) by mutagenesis of a carotenoid-overproducing strain. HPLC analyses indicated a specific impairment in the ability of SF21-CarB to perform the fifth desaturation, as implied by the accumulation of gamma-carotene and beta-carotene, which arise through four-step desaturation. Sequencing of the SF21 carB allele revealed a single mutation resulting in an exchange of a residue conserved in other five-step desaturases. Targeted carB allele replacement proved that this single mutation is the cause of the SF21 carotenoid pattern. In support, expression of SF21 CarB in engineered carotene-producing Escherichia coli strains demonstrated its reduced ability to catalyse the fifth desaturation step on both monocyclic and acyclic substrates. Further mutagenesis of SF21 led to the isolation of two mutants, SF73 and SF98, showing low desaturase activities, which mediated only two desaturation steps, resulting in accumulation of the intermediate zeta-carotene at low levels. Both strains contained an additional mutation affecting a CarB domain tentatively associated with carotenoid binding. SF21 exhibited higher carotenoid amounts than its precursor strain or the SF73 and SF98 mutants, although carotenogenic mRNA levels were similar in the four strains.

Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

PubMed Central

Sauge-Merle, Sandrine; Cuiné, Stéphan; Carrier, Patrick; Lecomte-Pradines, Catherine; Luu, Doan-Trung; Peltier, Gilles

2003-01-01

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes. PMID:12514032

Improving the productivity of S-adenosyl-l-methionine by metabolic engineering in an industrial Saccharomyces cerevisiae strain.

PubMed

Zhao, Weijun; Hang, Baojian; Zhu, Xiangcheng; Wang, Ri; Shen, Minjie; Huang, Lei; Xu, Zhinan

2016-10-20

S-Adenosyl-l-methionine (SAM) is an important metabolite having prominent roles in treating various diseases. In order to improve the production of SAM, the regulation of three metabolic pathways involved in SAM biosynthesis were investigated in an industrial yeast strain ZJU001. GLC3 encoded glycogen-branching enzyme (GBE), SPE2 encoded SAM decarboxylase, as well as ERG4 and ERG6 encoded key enzymes in ergosterol biosynthesis, were knocked out in ZJU001 accordingly. The results indicated that blocking of either glycogen pathway or SAM decarboxylation pathway could improve the SAM accumulation significantly in ZJU001, while single disruption of either ERG4 or ERG6 gene had no obvious effect on SAM production. Moreover, the double mutant ZJU001-GS with deletion of both GLC3 and SPE2 genes was also constructed, which showed further improvement of SAM accumulation. Finally, SAM2 was overexpressed in ZJU001-GS to give the best SAM-producing recombinant strain ZJU001-GS-SAM2, in which 12.47g/L SAM was produced by following our developed pseudo-exponential fed-batch cultivation strategy, about 81.0% increase comparing to its parent strain ZJU001. The present work laid a solid base for large-scale SAM production with the industrial Saccharomyces cerevisiae strain. Copyright © 2016 Elsevier B.V. All rights reserved.

The comprehensive profile of fermentation products during in situ CO2 recycling by Rubisco-based engineered Escherichia coli.

PubMed

Yang, Cheng-Han; Liu, En-Jung; Chen, Yi-Ling; Ou-Yang, Fan-Yu; Li, Si-Yu

2016-08-02

In our previous study, the feasibility of Rubisco-based engineered E. coli (that contains heterologous phosphoribulokinase (PrkA) and Rubisco) for in situ CO2 recycling during the fermentation of pentoses or hexoses was demonstrated. Nevertheless, it is perplexing to see that only roughly 70 % of the carbon fed to the bacterial culture could be accounted for in the standard metabolic products. This low carbon recovery during fermentation occurred even though CO2 emission was effectively reduced by Rubisco-based engineered pathway. In this study, the heterologous expression of form I Rubisco was found to enhance the accumulation of pyruvate in Escherichia coli MZLF [E. coli BL21(DE3) Δzwf, Δldh, Δfrd]. This may be attributed to the enhanced glycolytic reaction supported by the increased biomass and the ethanol/acetate ratio. Besides, it was found that the transcription of arcA (encodes the redox-dependent transcriptional activators ArcA that positively regulates the transcription of pyruvate formate-lyase) was down-regulated in the presence of Rubisco. The enhanced accumulation of pyruvate also occurs when PrkA is co-expressed with Rubisco in E. coli MZLF. Furthermore, E. coli containing Rubisco-based engineered pathway has a distinct profile of the fermentation products, indicating CO2 was converted into fermentation products. By analyzing the ratio of total C-2 (2-carbon fermentation products) to total C-1 (1-carbon fermentation product) of MZLFB (MZLF containing Rubisco-based engineered pathway), it is estimated that 9 % of carbon is directed into Rubisco-based engineered pathway. Here, we report for the first time the complete profile of fermentation products using E. coli MZLF and its derived strains. It has been shown that the expression of Rubisco alone in MZLF enhances the accumulation of pyruvate. By including the contribution of pyruvate accumulation, the perplexing problem of low carbon recovery during fermentation by E. coli containing Rubisco-based engineered pathway has been solved. 9 % of glucose consumption is directed from glycolysis to Rubisco-based engineered pathway in MZLFB. The principle characteristics of mixotroph MZLFB are the high bacterial growth and the low CO2 emission.

Engineering E. coli for triglyceride accumulation through native and heterologous metabolic reactions.

PubMed

Rucker, Joanna; Paul, Julie; Pfeifer, Blaine A; Lee, Kyongbum

2013-03-01

Triglycerides, traditionally sourced from plant oils, are heavily used in both industrial and healthcare applications. Commercially significant products produced from triglycerides include biodiesel, lubricants, moisturizers, and oils for cooking and dietary supplements. The need to rely upon plant-based production, however, raises concerns of increasing demand and sustainability. The reliance on crop yields and a strong demand for triglycerides provides motivation to engineer production from a robust microbial platform. In this study, Escherichia coli was engineered to synthesize and accumulate triglycerides. Triglycerides were produced from cell wall phospholipid precursors through engineered expression of two enzymes, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). A liquid chromatography-mass spectrometry (LC-MS) method was developed to analyze the production of triglycerides by the engineered E. coli strains. This proof-of-concept study demonstrated a yield of 1.1 mg/L triglycerides (2 g/L dry cell weight) in lysogeny broth medium containing 5 g/L glucose at 8 h following induction of PAP and DGAT expression. LC-MS results also demonstrated that the intracellular triglyceride composition of E. coli was highly conserved. Triglycerides containing the fatty acid distributions 16:0/16:0/16:1, 16:0/16:0/18:1, and 18:1/16:0/16:1 were found in highest concentrations and represent ∼70 % of triglycerides observed.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger.

PubMed

Hu, Wei; Li, Wenjian; Chen, Hao; Liu, Jing; Wang, Shuyang; Chen, Jihong

2017-01-01

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research.

Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation.

PubMed

Agrimi, Gennaro; Mena, Maria C; Izumi, Kazuki; Pisano, Isabella; Germinario, Lucrezia; Fukuzaki, Hisashi; Palmieri, Luigi; Blank, Lars M; Kitagaki, Hiroshi

2014-03-01

Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose.

PubMed

Nichols, Nancy N; Saha, Badal C

2016-05-01

In conversion of biomass to fuels or chemicals, inhibitory compounds arising from physical-chemical pretreatment of the feedstock can interfere with fermentation of the sugars to product. Fungal strain Coniochaeta ligniaria NRRL30616 metabolizes the furan aldehydes furfural and 5-hydroxymethylfurfural, as well as a number of aromatic and aliphatic acids and aldehydes. Use of NRRL30616 to condition biomass sugars by metabolizing the inhibitors improves their fermentability. Wild-type C. ligniaria has the ability to grow on xylose as sole source of carbon and energy, with no accumulation of xylitol. Mutants of C. ligniaria unable to grow on xylose were constructed. Xylose reductase and xylitol dehydrogenase activities were reduced by approximately two thirds in mutant C8100. The mutant retained ability to metabolize inhibitors in biomass hydrolysates. Although C. ligniaria C8100 did not grow on xylose, the strain converted a portion of xylose to xylitol, producing 0.59 g xylitol/g xylose in rich medium and 0.48 g xylitol/g xylose in corn stover dilute acid hydrolysate. 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016 © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:606-612, 2016. © 2016 American Institute of Chemical Engineers.

Progress in Metabolic Engineering of Saccharomyces cerevisiae

PubMed Central

Nevoigt, Elke

2008-01-01

Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate. PMID:18772282

Physiologically Distributed Loading Patterns Drive the Formation of Zonally Organized Collagen Structures in Tissue-Engineered Meniscus.

PubMed

Puetzer, Jennifer L; Bonassar, Lawrence J

2016-07-01

The meniscus is a dense fibrocartilage tissue that withstands the complex loads of the knee via a unique organization of collagen fibers. Attempts to condition engineered menisci with compression or tensile loading alone have failed to reproduce complex structure on the microscale or anatomic scale. Here we show that axial loading of anatomically shaped tissue-engineered meniscus constructs produced spatial distributions of local strain similar to those seen in the meniscus when the knee is loaded at full extension. Such loading drove formation of tissue with large organized collagen fibers, levels of mechanical anisotropy, and compressive moduli that match native tissue. Loading accelerated the development of native-sized and aligned circumferential and radial collagen fibers. These loading patterns contained both tensile and compressive components that enhanced the major biochemical and functional properties of the meniscus, with loading significantly improved glycosaminoglycan (GAG) accumulation 200-250%, collagen accumulation 40-55%, equilibrium modulus 1000-1800%, and tensile moduli 500-1200% (radial and circumferential). Furthermore, this study demonstrates local changes in mechanical environment drive heterogeneous tissue development and organization within individual constructs, highlighting the importance of recapitulating native loading environments. Loaded menisci developed cartilage-like tissue with rounded cells, a dense collagen matrix, and increased GAG accumulation in the more compressively loaded horns, and fibrous collagen-rich tissue in the more tensile loaded outer 2/3, similar to native menisci. Loaded constructs reached a level of organization not seen in any previous engineered menisci and demonstrate great promise as meniscal replacements.

An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

PubMed Central

2012-01-01

Background Pyruvate-decarboxylase negative (Pdc-) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc- strains cannot grow on high glucose concentrations and require C2-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Results Genetic analysis of a Pdc- strain previously evolved to overcome these deficiencies revealed a 225bp in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc- strain enabled growth on 20 g l-1 glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h-1) similar to that of the evolved Pdc- strain (0.23 h-1). Furthermore, the reverse engineered Pdc- strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h-1) than the evolved strain (0.20 h-1). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc-S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc- strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C2-compound auxotrophy. Conclusions In this study we have discovered and characterised a mutation in MTH1 enabling Pdc- strains to grow on glucose as the sole carbon source. This successful example of reverse engineering not only increases the understanding of the glucose tolerance of evolved Pdc-S. cerevisiae, but also allows introduction of this portable genetic element into various industrial yeast strains, thereby simplifying metabolic engineering strategies. PMID:22978798

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx

PubMed Central

Suárez, Gabriel A.; Renda, Brian A.; Dasgupta, Aurko

2017-01-01

ABSTRACT The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting “clean-genome” ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. PMID:28667117

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

PubMed

Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

2017-09-01

The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting "clean-genome" ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. Copyright © 2017 American Society for Microbiology.

Release of radiogenic noble gases as a new signal of rock deformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bauer, Stephen J.; Gardner, W. Payton; Lee, Hyunwoo

In this paper we investigate the release of radiogenic noble gas isotopes during mechanical deformation. We developed an analytical system for dynamic mass spectrometry of noble gas composition and helium release rate of gas produced during mechanical deformation of rocks. Our results indicate that rocks release accumulated radiogenic helium and argon from mineral grains as they undergo deformation. We found that the release of accumulated 4He and 40Ar from rocks follows a reproducible pattern and can provide insight into the deformation process. Increased gas release can be observed before dilation, and macroscopic failure is observed during high-pressure triaxial rock deformationmore » experiments. Accumulated radiogenic noble gases can be released due to fracturing of mineral grains during small-scale strain in Earth materials. Helium and argon are highly mobile, conservative species and could be used to provide information on changes in the state of stress and strain in Earth materials, and as an early warning signal of macroscopic failure. These results pave the way for the use of noble gases to trace and monitor rock deformation for earthquake prediction and a variety of other subsurface engineering projects.« less

Release of radiogenic noble gases as a new signal of rock deformation

DOE PAGES

Bauer, Stephen J.; Gardner, W. Payton; Lee, Hyunwoo

2016-10-09

In this paper we investigate the release of radiogenic noble gas isotopes during mechanical deformation. We developed an analytical system for dynamic mass spectrometry of noble gas composition and helium release rate of gas produced during mechanical deformation of rocks. Our results indicate that rocks release accumulated radiogenic helium and argon from mineral grains as they undergo deformation. We found that the release of accumulated 4He and 40Ar from rocks follows a reproducible pattern and can provide insight into the deformation process. Increased gas release can be observed before dilation, and macroscopic failure is observed during high-pressure triaxial rock deformationmore » experiments. Accumulated radiogenic noble gases can be released due to fracturing of mineral grains during small-scale strain in Earth materials. Helium and argon are highly mobile, conservative species and could be used to provide information on changes in the state of stress and strain in Earth materials, and as an early warning signal of macroscopic failure. These results pave the way for the use of noble gases to trace and monitor rock deformation for earthquake prediction and a variety of other subsurface engineering projects.« less

A new regulatory mechanism controlling carotenogenesis in the fungus Mucor circinelloides as a target to generate β-carotene over-producing strains by genetic engineering.

PubMed

Zhang, Yingtong; Navarro, Eusebio; Cánovas-Márquez, José T; Almagro, Lorena; Chen, Haiqin; Chen, Yong Q; Zhang, Hao; Torres-Martínez, Santiago; Chen, Wei; Garre, Victoriano

2016-06-07

Carotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. They also have important industrial applications as food colourants, animal feed additives and nutraceuticals. Some of them, such as β-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. The mucoral Blakeslea trispora is used by the industry to produce β-carotene, although optimisation of production by molecular genetic engineering is unfeasible. However, the phylogenetically closely related Mucor circinelloides, which is also able to accumulate β-carotene, possesses a vast collection of genetic tools with which to manipulate its genome. This work combines classical forward and modern reverse genetic techniques to deepen the regulation of carotenoid synthesis and generate candidate strains for biotechnological production of β-carotene. Mutagenesis followed by screening for mutants with altered colour in the dark and/or in light led to the isolation of 26 mutants that, together with eight previously isolated mutants, have been analysed in this work. Although most of the mutants harboured mutations in known structural and regulatory carotenogenic genes, eight of them lacked mutations in those genes. Whole-genome sequencing of six of these strains revealed the presence of many mutations throughout their genomes, which makes identification of the mutation that produced the phenotype difficult. However, deletion of the crgA gene, a well-known repressor of carotenoid biosynthesis in M. circinelloides, in two mutants (MU206 and MU218) with high levels of β-carotene resulted in a further increase in β-carotene content to differing extents with respect to the crgA single-null strain; in particular, one strain derived from MU218 was able to accumulate up to 4 mg/g of β-carotene. The additive effect of crgA deletion and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA. The use of a mucoral model such as M. circinelloides can allow the identification of the regulatory mechanisms that control carotenoid biosynthesis, which can then be manipulated to generate tailored strains of biotechnological interest. Mutants in the repressor crgA and in the newly identified regulatory mechanism generated in this work accumulate high levels of β-carotene and are candidates for further improvements in biotechnological β-carotene production.

Systems analysis of ethanol production in the genetically engineered cyanobacterium Synechococcus sp. PCC 7002.

PubMed

Kopka, Joachim; Schmidt, Stefanie; Dethloff, Frederik; Pade, Nadin; Berendt, Susanne; Schottkowski, Marco; Martin, Nico; Dühring, Ulf; Kuchmina, Ekaterina; Enke, Heike; Kramer, Dan; Wilde, Annegret; Hagemann, Martin; Friedrich, Alexandra

2017-01-01

Future sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms, which are engineered to synthesize valuable products directly from CO 2 and sunlight. As cyanobacteria can be cultivated in large scale on non-arable land, these phototrophic bacteria have become attractive organisms for production of biofuels. Synechococcus sp. PCC 7002, one of the cyanobacterial model organisms, provides many attractive properties for biofuel production such as tolerance of seawater and high light intensities. Here, we performed a systems analysis of an engineered ethanol-producing strain of the cyanobacterium Synechococcus sp. PCC 7002, which was grown in artificial seawater medium over 30 days applying a 12:12 h day-night cycle. Biosynthesis of ethanol resulted in a final accumulation of 0.25% (v/v) ethanol, including ethanol lost due to evaporation. The cultivation experiment revealed three production phases. The highest production rate was observed in the initial phase when cells were actively growing. In phase II growth of the producer strain stopped, but ethanol production rate was still high. Phase III was characterized by a decrease of both ethanol production and optical density of the culture. Metabolomics revealed that the carbon drain due to ethanol diffusion from the cell resulted in the expected reduction of pyruvate-based intermediates. Carbon-saving strategies successfully compensated the decrease of central intermediates of carbon metabolism during the first phase of fermentation. However, during long-term ethanol production the producer strain showed clear indications of intracellular carbon limitation. Despite the decreased levels of glycolytic and tricarboxylic acid cycle intermediates, soluble sugars and even glycogen accumulated in the producer strain. The changes in carbon assimilation patterns are partly supported by proteome analysis, which detected decreased levels of many enzymes and also revealed the stress phenotype of ethanol-producing cells. Strategies towards improved ethanol production are discussed. Systems analysis of ethanol production in Synechococcus sp. PCC 7002 revealed initial compensation followed by increasing metabolic limitation due to excessive carbon drain from primary metabolism.

Gene Transfer in Leptolyngbya sp. Strain BL0902, a Cyanobacterium Suitable for Production of Biomass and Bioproducts

PubMed Central

Taton, Arnaud; Lis, Ewa; Adin, Dawn M.; Dong, Guogang; Cookson, Scott; Kay, Steve A.; Golden, Susan S.; Golden, James W.

2012-01-01

Current cyanobacterial model organisms were not selected for their growth traits or potential for the production of renewable biomass, biofuels, or other products. The cyanobacterium strain BL0902 emerged from a search for strains with superior growth traits. Morphology and 16S rRNA sequence placed strain BL0902 in the genus Leptolyngbya. Leptolyngbya sp. strain BL0902 (hereafter Leptolyngbya BL0902) showed robust growth at temperatures from 22°C to 40°C and tolerated up to 0.5 M NaCl, 32 mM urea, high pH, and high solar irradiance. Its growth rate under outdoor conditions rivaled Arthrospira (“pirulina” strains. Leptolyngbya BL0902 accumulated higher lipid content and a higher proportion of monounsaturated fatty acids than Arthrospira strains. In addition to these desirable qualities, Leptolyngbya BL0902 is amenable to genetic engineering that is reliable, efficient, and stable. We demonstrated conjugal transfer from Escherichia coli of a plasmid based on RSF1010 and expression of spectinomycin/streptomycin resistance and yemGFP reporter transgenes. Conjugation efficiency was investigated in biparental and triparental matings with and without a “elper”plasmid that carries DNA methyltransferase genes, and with two different conjugal plasmids. We also showed that Leptolyngbya BL0902 is amenable to transposon mutagenesis with a Tn5 derivative. To facilitate genetic manipulation of Leptolyngbya BL0902, a conjugal plasmid vector was engineered to carry a trc promoter upstream of a Gateway recombination cassette. These growth properties and genetic tools position Leptolyngbya BL0902 as a model cyanobacterial production strain. PMID:22292073

Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

PubMed Central

Hetzler, Stephan; Bröker, Daniel

2013-01-01

The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step. PMID:23793636

Ergot Alkaloid Biosynthesis in the Maize (Zea mays) Ergot Fungus Claviceps gigantea.

PubMed

Bragg, Paige E; Maust, Matthew D; Panaccione, Daniel G

2017-12-13

Biosynthesis of the dihydrogenated forms of ergot alkaloids is of interest because many of the ergot alkaloids used as pharmaceuticals may be derived from dihydrolysergic acid (DHLA) or its precursor dihydrolysergol. The maize (Zea mays) ergot pathogen Claviceps gigantea has been reported to produce dihydrolysergol, a hydroxylated derivative of the common ergot alkaloid festuclavine. We hypothesized expression of C. gigantea cloA in a festuclavine-accumulating mutant of the fungus Neosartorya fumigata would yield dihydrolysergol because the P450 monooxygenase CloA from other fungi performs similar oxidation reactions. We engineered such a strain, and high performance liquid chromatography and liquid chromatography-mass spectrometry analyses demonstrated the modified strain produced DHLA, the fully oxidized product of dihydrolysergol. Accumulation of high concentrations of DHLA in field-collected C. gigantea sclerotia and discovery of a mutation in the gene lpsA, downstream from DHLA formation, supported our finding that DHLA rather than dihydrolysergol is the end product of the C. gigantea pathway.

Formation of norisoprenoid flavor compounds in carrot (Daucus carota L.) roots: characterization of a cyclic-specific carotenoid cleavage dioxygenase 1 gene.

PubMed

Yahyaa, Mosaab; Bar, Einat; Dubey, Neeraj Kumar; Meir, Ayala; Davidovich-Rikanati, Rachel; Hirschberg, Joseph; Aly, Radi; Tholl, Dorothea; Simon, Philipp W; Tadmor, Yaakov; Lewinsohn, Efraim; Ibdah, Mwafaq

2013-12-18

Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and β-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and β-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.

Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production.

PubMed

Xie, Dongming; Jackson, Ethel N; Zhu, Quinn

2015-02-01

The omega-3 fatty acids, cis-5, 8, 11, 14, and 17-eicosapentaenoic acid (C20:5; EPA) and cis-4, 7, 10, 13, 16, and 19-docosahexaenoic acid (C22:6; DHA), have wide-ranging benefits in improving heart health, immune function, mental health, and infant cognitive development. Currently, the major source for EPA and DHA is from fish oil, and a minor source of DHA is from microalgae. With the increased demand for EPA and DHA, DuPont has developed a clean and sustainable source of the omega-3 fatty acid EPA through fermentation using metabolically engineered strains of Yarrowia lipolytica. In this mini-review, we will focus on DuPont's technology for EPA production. Specifically, EPA biosynthetic and supporting pathways have been introduced into the oleaginous yeast to synthesize and accumulate EPA under fermentation conditions. This Yarrowia platform can also produce tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acid mixtures in the cellular lipid profiles. Fundamental research such as metabolic engineering for strain construction, high-throughput screening for strain selection, fermentation process development, and process scale-up were all needed to achieve the high levels of EPA titer, rate, and yield required for commercial application. Here, we summarize how we have combined the fundamental bioscience and the industrial engineering skills to achieve large-scale production of Yarrowia biomass containing high amounts of EPA, which led to two commercial products, New Harvest™ EPA oil and Verlasso® salmon.

Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

PubMed

Radek, Andreas; Müller, Moritz-Fabian; Gätgens, Jochem; Eggeling, Lothar; Krumbach, Karin; Marienhagen, Jan; Noack, Stephan

2016-08-10

Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization. Copyright © 2016 Elsevier B.V. All rights reserved.

Systems metabolic engineering of Escherichia coli for production of the antitumor drugs violacein and deoxyviolacein.

PubMed

Rodrigues, André L; Trachtmann, Nathalie; Becker, Judith; Lohanatha, Ananta F; Blotenberg, Jana; Bolten, Christoph J; Korneli, Claudia; de Souza Lima, André O; Porto, Luismar M; Sprenger, Georg A; Wittmann, Christoph

2013-11-01

Violacein and deoxyviolacein are interesting therapeutics against pathogenic bacteria and viruses as well as tumor cells. In the present work, systems-wide metabolic engineering was applied to target Escherichia coli, a widely accepted recombinant host in pharmaceutical biotechnology, for production of these high-value products. The basic producer, E. coli dVio-1, that expressed the vioABCE cluster from Chromobacterium violaceum under control of the inducible araC system, accumulated 180 mg L(-1) of deoxyviolacein. Targeted intracellular metabolite analysis then identified bottlenecks in tryptophan supporting pathways, the major product building block. This was used for comprehensive engineering of serine, chorismate and tryptophan biosynthesis and the non-oxidative pentose-phosphate pathway. The final strain, E. coli dVio-6, accumulated 320 mg L(-1) deoxyviolacein in shake flask cultures. The created chassis of a high-flux tryptophan pathway was complemented by genomic integration of the vioD gene of Janthinobacterium lividum, which enabled exclusive production of violacein. In a fed-batch process, the resulting producer E. coli Vio-4 accumulated 710 mg L(-1) of the desired product. With straightforward broth extraction and subsequent crystallization, violacein could be obtained with 99.8% purity. This demonstrates the potential of E. coli as a platform for production of tryptophan based therapeutics. Copyright © 2013 Elsevier Inc. All rights reserved.

Optimization of odd chain fatty acid production by Yarrowia lipolytica.

PubMed

Park, Young-Kyoung; Dulermo, Thierry; Ledesma-Amaro, Rodrigo; Nicaud, Jean-Marc

2018-01-01

Odd chain fatty acids (odd FAs) have a wide range of applications in therapeutic and nutritional industries, as well as in chemical industries including biofuel. Yarrowia lipolytica is an oleaginous yeast considered a preferred microorganism for the production of lipid-derived biofuels and chemicals. However, it naturally produces negligible amounts of odd chain fatty acids. The possibility of producing odd FAs using Y. lipolytica was investigated. Y. lipolytica wild-type strain was shown able to grow on weak acids; acetate, lactate, and propionate. Maximal growth rate on propionate reached 0.24 ± 0.01 h -1 at 2 g/L, and growth inhibition occurred at concentration above 10 g/L. Wild-type strain accumulated lipids ranging from 7.39 to 8.14% (w/w DCW) depending on the carbon source composition, and odd FAs represented only 0.01-0.12 g/L. We here proved that the deletion of the PHD1 gene improved odd FAs production, which reached a ratio of 46.82% to total lipids. When this modification was transferred to an obese strain, engineered for improving lipid accumulation, further increase odd FAs production reaching a total of 0.57 g/L was shown. Finally, a fed-batch co-feeding strategy was optimized for further increase odd FAs production, which generated 0.75 g/L, the best production described so far in Y. lipolytica . A Y. lipolytica strain able to accumulate high level of odd chain fatty acids, mainly heptadecenoic acid, has been successfully developed. In addition, a fed-batch co-feeding strategy was optimized to further improve lipid accumulation and odd chain fatty acid content. These lipids enriched in odd chain fatty acid can (1) improve the properties of the biodiesel generated from Y. lipolytica lipids and (2) be used as renewable source of odd chain fatty acid for industrial applications. This work paves the way for further improvements in odd chain fatty acids and fatty acid-derived compound production.

Synergic effects of tactolimus and azole antifungal agents against azole-resistant Candida albican strains.

PubMed

Maesaki, S; Marichal, P; Hossain, M A; Sanglard, D; Vanden Bossche, H; Kohno, S

1998-12-01

We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans.

New approaches for improving the production of the 1st and 2nd generation ethanol by yeast.

PubMed

Kurylenko, Olena; Semkiv, Marta; Ruchala, Justyna; Hryniv, Orest; Kshanovska, Barbara; Abbas, Charles; Dmytruk, Kostyantyn; Sibirny, Andriy

2016-01-01

Increase in the production of 1st generation ethanol from glucose is possible by the reduction in the production of ethanol co-products, especially biomass. We have developed a method to reduce biomass accumulation of Saccharomyces cerevisiae by the manipulation of the intracellular ATP level due to overexpression of genes of alkaline phosphatase, apyrase or enzymes involved in futile cycles. The strains constructed accumulated up to 10% more ethanol on a cornmeal hydrolysate medium. Similar increase in ethanol accumulation was observed in the mutants resistant to the toxic inhibitors of glycolysis like 3-bromopyruvate and others. Substantial increase in fuel ethanol production will be obtained by the development of new strains of yeasts that ferment sugars of the abundant lignocellulosic feedstocks, especially xylose, a pentose sugar. We have found that xylose can be fermented under elevated temperatures by the thermotolerant yeast, Hansenula polymorpha. We combined protein engineering of the gene coding for xylose reductase (XYL1) along with overexpression of the other two genes responsible for xylose metabolism in yeast (XYL2, XYL3) and the deletion of the global transcriptional activator CAT8, with the selection of mutants defective in utilizing ethanol as a carbon source using the anticancer drug, 3-bromopyruvate. Resulted strains accumulated 20-25 times more ethanol from xylose at the elevated temperature of 45°C with up to 12.5 g L(-1) produced. Increase in ethanol yield and productivity from xylose was also achieved by overexpression of genes coding for the peroxisomal enzymes: transketolase (DAS1) and transaldolase (TAL2), and deletion of the ATG13 gene.

Pathway engineering of Enterobacter aerogenes to improve acetoin production by reducing by-products formation.

PubMed

Jang, Ji-Woong; Jung, Hwi-Min; Im, Dae-Kyun; Jung, Moo-Young; Oh, Min-Kyu

2017-11-01

Enterobacter aerogenes was metabolically engineered for acetoin production. To remove the pathway enzymes that catalyzed the formation of by-products, the three genes encoding a lactate dehydrogenase (ldhA) and two 2,3-butanediol dehydrogenases (budC, and dhaD), respectively, were deleted from the genome. The acetoin production was higher under highly aerobic conditions. However, an extracellular glucose oxidative pathway in E. aerogenes was activated under the aerobic conditions, resulting in the accumulation of 2-ketogluconate. To decrease the accumulation of this by-product, the gene encoding a glucose dehydrogenase (gcd) was also deleted. The resulting strain did not produce 2-ketogluconate but produced significant amounts of acetoin, with concentration reaching 71.7g/L with 2.87g/L/h productivity in fed-batch fermentation. This result demonstrated the importance of blocking the glucose oxidative pathway under highly aerobic conditions for acetoin production using E. aerogenes. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoneda, Aki; Henson, William R.; Goldner, Nicholas K.

Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showedmore » higher phenol consumption rates (~20 mg/l/h) and ~2-fold higher lipid production from phenol than the wild-type strain.Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products.« less

Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630

DOE PAGES

Yoneda, Aki; Henson, William R.; Goldner, Nicholas K.; ...

2016-02-02

Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showedmore » higher phenol consumption rates (~20 mg/l/h) and ~2-fold higher lipid production from phenol than the wild-type strain.Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products.« less

Identification of FadAB Complexes Involved in Fatty Acid β-Oxidation in Streptomyces coelicolor and Construction of a Triacylglycerol Overproducing strain

PubMed Central

Menendez-Bravo, Simón; Paganini, Julián; Avignone-Rossa, Claudio; Gramajo, Hugo; Arabolaza, Ana

2017-01-01

Oleaginous microorganisms represent possible platforms for the sustainable production of oleochemicals and biofuels due to their metabolic robustness and the possibility to be engineered. Streptomyces coelicolor is among the narrow group of prokaryotes capable of accumulating triacylglycerol (TAG) as carbon and energy reserve. Although the pathways for TAG biosynthesis in this organism have been widely addressed, the set of genes required for their breakdown have remained elusive so far. Here, we identified and characterized three gene clusters involved in the β-oxidation of fatty acids (FA). The role of each of the three different S. coelicolor FadAB proteins in FA catabolism was confirmed by complementation of an Escherichia coliΔfadBA mutant strain deficient in β-oxidation. In S. coelicolor, the expression profile of the three gene clusters showed variation related with the stage of growth and the presence of FA in media. Flux balance analyses using a corrected version of the current S. coelicolor metabolic model containing detailed TAG biosynthesis reactions suggested the relevance of the identified fadAB genes in the accumulation of TAG. Thus, through the construction and analysis of fadAB knockout mutant strains, we obtained an S. coelicolor mutant that showed a 4.3-fold increase in the TAG content compared to the wild type strain grown under the same culture conditions. PMID:28824562

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger

PubMed Central

Li, Wenjian; Chen, Hao; Liu, Jing; Wang, Shuyang; Chen, Jihong

2017-01-01

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research. PMID:28650980

Accumulation of 10 Fluoroquinolones by Wild-Type or Efflux Mutant Streptococcus pneumoniae

PubMed Central

Piddock, Laura J. V.; Johnson, M. M.

2002-01-01

A method for measuring fluoroquinolone accumulation by Streptococcus pneumoniae was rigorously examined. The accumulation of ciprofloxacin, clinafloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, sitafloxacin, and trovafloxacin in the presence and absence of either carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or reserpine was determined for two wild-type fluoroquinolone-susceptible capsulated S. pneumoniae strains (M3 and M4) and the noncapsulated strain R6. Two efflux mutants, R6N (which overexpresses PmrA) and a mutant of M4, M22 (no expression of PmrA), were also examined. Essentially, the fluoroquinolones fell into two groups. (i) One group consisting of ciprofloxacin, grepafloxacin, and norfloxacin accumulated to 72 to 92 ng/mg (dry weight) of cells in all strains. (ii) The remainder of the agents accumulated to 3 to 30 ng/mg (dry weight) of cells. With a decrease in hydrophobicity, there was a decrease in the concentration accumulated. With an increase in the molecular weight of the free form of each agent, there was also a decrease in the concentration accumulated. The strains differed in their responses to reserpine and CCCP. For the three fluoroquinolone-susceptible strains, only reserpine had a significant effect upon accumulation of moxifloxacin and clinafloxacin by M3 and showed no effect for the other agents and strains. For M3 and M4, CCCP enhanced the concentration of ciprofloxacin and norfloxacin accumulated, whereas for R6, the effect was only statistically significant for ofloxacin. Efflux mutant M22 accumulated less ciprofloxacin, gatifloxacin, and ofloxacin than M4 did. M22 accumulated more norfloxacin than M4 did. Reserpine and CCCP had variable effects as for the other strains. Differences in the accumulation of fluoroquinolones by R6 and R6N were highly dependent upon growth phase, and only for norfloxacin was there a significant difference between two strains. PMID:11850266

Performance Evaluation and Modeling of Erosion Resistant Turbine Engine Thermal Barrier Coatings

NASA Technical Reports Server (NTRS)

Miller, Robert A.; Zhu, Dongming; Kuczmarski, Maria

2008-01-01

The erosion resistant turbine thermal barrier coating system is critical to the rotorcraft engine performance and durability. The objective of this work was to determine erosion resistance of advanced thermal barrier coating systems under simulated engine erosion and thermal gradient environments, thus validating a new thermal barrier coating turbine blade technology for future rotorcraft applications. A high velocity burner rig based erosion test approach was established and a new series of rare earth oxide- and TiO2/Ta2O5- alloyed, ZrO2-based low conductivity thermal barrier coatings were designed and processed. The low conductivity thermal barrier coating systems demonstrated significant improvements in the erosion resistance. A comprehensive model based on accumulated strain damage low cycle fatigue is formulated for blade erosion life prediction. The work is currently aiming at the simulated engine erosion testing of advanced thermal barrier coated turbine blades to establish and validate the coating life prediction models.

Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production

PubMed Central

2013-01-01

Background There has been a great deal of interest in fuel productions from lignocellulosic biomass to minimize the conflict between food and fuel use. The bioconversion of xylose, which is the second most abundant sugar present after glucose in lignocellulosic biomass, is important for the development of cost effective bioprocesses to fuels. Rhodococcus opacus PD630, an oleaginous bacterium, accumulates large amounts of triacylglycerols (TAGs), which can be processed into advanced liquid fuels. However, R. opacus PD630 does not metabolize xylose. Results We generated DNA libraries from a Streptomyces bacterium capable of utilizing xylose and introduced them into R. opacus PD630. Xsp8, one of the engineered strains, was capable of growing on up to 180 g L-1 of xylose. Xsp8 grown in batch-cultures derived from unbleached kraft hardwood pulp hydrolysate containing 70 g L-1 total sugars was able to completely and simultaneously utilize xylose and glucose present in the lignocellulosic feedstock, and yielded 11.0 g L-1 of TAGs as fatty acids, corresponding to 45.8% of the cell dry weight. The yield of total fatty acids per gram of sugars consumed was 0.178 g, which consisted primarily of palmitic acid and oleic acid. The engineered strain Xsp8 was introduced with two heterologous genes from Streptomyces: xylA, encoding xylose isomerase, and xylB, encoding xylulokinase. We further demonstrated that in addition to the introduction and the concomitant expression of heterologous xylA and xylB genes, there is another molecular target in the R. opacus genome which fully enables the functionality of xylA and xylB genes to generate the robust xylose-fermenting strain capable of efficiently producing TAGs at high xylose concentrations. Conclusion We successfully engineered a R. opacus strain that is capable of completely utilizing high concentrations of xylose or mixed xylose/glucose simultaneously, and substantiated its suitability for TAG production. This study demonstrates that the engineered strain possesses a key trait of converters for lipid-based fuels production from lignocellulosic biomass. PMID:24041310

Investigation of relationship between 2,3-butanediol toxicity and production during growth of Paenibacillus polymyxa.

PubMed

Okonkwo, Christopher Chukwudi; Ujor, Victor; Ezeji, Thaddeus Chukwuemeka

2017-01-25

Understanding the capacity of Paenibacillus polymyxa DSM 365 to tolerate increasing concentrations of 2,3-butanediol (2,3-BD) is critical to engineering a 2,3-BD-overproducing strain. Hence, we investigated the response of P. polymyxa to high 2,3-BD concentrations. In fed-batch cultures (6-L bioreactor) 2,3-BD was accumulated to a maximum concentration of 47g/L despite the presence of residual 13g/L glucose in the medium. Concomitantly, accumulation of acetoin, the precursor of 2,3-BD increased after maximum 2,3-BD concentration was reached, suggesting that 2,3-BD was reconverted to acetoin after the concentration tolerance threshold of 2,3-BD was exceeded. Cultures of P. polymyxa were then challenged with levo-2,3-BD (20, 40 and 60g/L) at 0h in a glucose medium, and a concentration dependent growth inhibition response to levo-2,3-BD was observed. The growth of P. polymyxa was completely inhibited by 60g/L levo-2,3-BD. Furthermore, P. polymyxa was challenged with incremental 2,3-BD concentrations (20, 40 and 60g/L at 12, 24 and 36h, respectively) to mimic 2,3-BD accumulation during fermentation. Interestingly, 2,3-BD was reconverted to acetoin when its concentration reached 60g/L, possibly to alleviate 2,3-BD toxicity. Collectively, our findings indicate that 2,3-BD-mediated toxicity is a major metabolic impediment to 2,3-BD overproduction, thus, making it an important metabolic engineering target towards rational design of a 2,3-BD-overproducing strain. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 on ruminal pH and the lactic acid concentration of simulated rumen acidosis in vitro.

PubMed

Long, M; Feng, W J; Li, P; Zhang, Y; He, R X; Yu, L H; He, J B; Jing, W Y; Li, Y M; Wang, Z; Liu, G W

2014-02-01

The aim of this study was to examine the effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 (M. elsdenii H6F32) on ruminal pH and the lactic acid concentrations in simulated rumen acidosis conditions in vitro. A mixed culture of ruminal bacteria, buffer, and primarily degradable substrates was inoculated with equal numbers of M. elsdenii H6 or M. elsdenii H6F32. The pH and lactic acid concentrations in the mixed culture were determined at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h of incubation. Acid-tolerant M. elsdenii H6F32 reduced the accumulation of lactic acid and increased the pH value. These results indicate that acid-tolerant M. elsdenii H6F32 could be a potential candidate for preventing rumen acidosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

L-Methionine Production.

PubMed

Shim, Jihyun; Shin, Yonguk; Lee, Imsang; Kim, So Young

L-Methionine has been used in various industrial applications such as the production of feed and food additives and has been used as a raw material for medical supplies and drugs. It functions not only as an essential amino acid but also as a physiological effector, for example, by inhibiting fat accumulation and enhancing immune response. Producing methionine from fermentation is beneficial in that microorganisms can produce L-methionine selectively using eco-sustainable processes. Nevertheless, the fermentative method has not been used on an industrial scale because it is not competitive economically compared with chemical synthesis methods. Presented are efforts to develop suitable strains, engineered enzymes, and alternative process of producing L-methionine that overcomes problems of conventional fermentation methods. One of the alternative processes is a two-step process in which the L-methionine precursor is produced by fermentation and then converted to L-methionine by enzymes. Directed efforts toward strain development and enhanced enzyme engineering will advance industrial production of L-methionine based on fermentation.

Proline accumulation in baker's yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough.

PubMed

Sasano, Yu; Haitani, Yutaka; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

2012-01-03

During bread-making processes, yeast cells are exposed to various baking-associated stresses. High-sucrose concentrations exert severe osmotic stress that seriously damages cellular components by generation of reactive oxygen species (ROS). Previously, we found that the accumulation of proline conferred freeze-thaw stress tolerance and the baker's yeast strain that accumulated proline retained higher-level fermentation abilities in frozen doughs than the wild-type strain. In this study, we constructed self-cloning diploid baker's yeast strains that accumulate proline. These resultant strains showed higher cell viability and lower intracellular oxidation levels than that observed in the wild-type strain under high-sucrose stress condition. Proline accumulation also enhanced the fermentation ability in high-sucrose-containing dough. These results demonstrate the usefulness of proline-accumulating baker's yeast for sweet dough baking. Copyright © 2011 Elsevier B.V. All rights reserved.

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering. PMID:21219616

Bioaccumulation of Vanadium by Vanadium-Resistant Bacteria Isolated from the Intestine of Ascidia sydneiensis samea.

PubMed

Romaidi; Ueki, Tatsuya

2016-06-01

Isolation of naturally occurring bacterial strains from metal-rich environments has gained popularity due to the growing need for bioremediation technologies. In this study, we found that the vanadium concentration in the intestine of the vanadium-rich ascidian Ascidia sydneiensis samea could reach 0.67 mM, and thus, we isolated vanadium-resistant bacteria from the intestinal contents and determined the ability of each bacterial strain to accumulate vanadium and other heavy metals. Nine strains of vanadium-resistant bacteria were successfully isolated, of which two strains, V-RA-4 and S-RA-6, accumulated vanadium at a higher rate than did the other strains. The maximum vanadium absorption by these bacteria was achieved at pH 3, and intracellular accumulation was the predominant mechanism. Each strain strongly accumulated copper and cobalt ions, but accumulation of nickel and molybdate ions was relatively low. These bacterial strains can be applied to protocols for bioremediation of vanadium and heavy metal toxicity.

Balanced activation of IspG and IspH to eliminate MEP intermediate accumulation and improve isoprenoids production in Escherichia coli.

PubMed

Li, Qingyan; Fan, Feiyu; Gao, Xiang; Yang, Chen; Bi, Changhao; Tang, Jinlei; Liu, Tao; Zhang, Xueli

2017-11-01

The MEP pathway genes were modulated to investigate whether there were new rate-limiting steps and toxic intermediates in this pathway. Activating IspG led to significant decrease of cell growth and β-carotene production. It was found that ispG overexpression led to accumulation of intermediate HMBPP, which seriously interfered with synthesis machinery of nucleotide and protein in Escherichia coli. Activation of the downstream enzyme IspH could solve HMBPP accumulation problem and eliminate the negative effects of ispG overexpression. In addition, intermediate MECPP accumulated in the starting strain, while balanced activation of IspG and IspH could push the carbon flux away from MECPP and led to 73% and 77% increase of β-carotene and lycopene titer respectively. Our work for the first time identified HMBPP to be a cytotoxic intermediate in MEP pathway and demonstrated that balanced activation of IspG and IspH could eliminate accumulation of HMBPP and MECPP and improve isoprenoids production. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Accumulated financial strain and women's health over three decades.

PubMed

Shippee, Tetyana Pylypiv; Wilkinson, Lindsay R; Ferraro, Kenneth F

2012-09-01

Drawing from cumulative inequality theory, this research examines how accumulated financial strain affects women's self-rated health in middle and later life. Using data from the National Longitudinal Survey of Mature Women (1967-2003), we employ random-coefficient growth curve models to examine whether recurring financial strain influences women's health, above and beyond several measures of objective social status. Predicted probabilities of poor health were estimated by the frequency of financial strain. Financial strain is associated with rapid declines in women's health during middle and later life, especially for those women who reported recurrent strain. Changes in household income and household wealth were also associated with women's health but did not eliminate the effects due to accumulated financial strain. Accumulated financial strain has long-term effects on women's health during middle and later life. The findings demonstrate the importance of measuring life course exposure to stressors in studies of health trajectories.

Using strain rates to forecast seismic hazards

USGS Publications Warehouse

Evans, Eileen

2017-01-01

One essential component in forecasting seismic hazards is observing the gradual accumulation of tectonic strain accumulation along faults before this strain is suddenly released as earthquakes. Typically, seismic hazard models are based on geologic estimates of slip rates along faults and historical records of seismic activity, neither of which records actively accumulating strain. But this strain can be estimated by geodesy: the precise measurement of tiny position changes of Earth’s surface, obtained from GPS, interferometric synthetic aperture radar (InSAR), or a variety of other instruments.

Characterization of genome-reduced Bacillus subtilis strains and their application for the production of guanosine and thymidine.

PubMed

Li, Yang; Zhu, Xujun; Zhang, Xueyu; Fu, Jing; Wang, Zhiwen; Chen, Tao; Zhao, Xueming

2016-06-03

Genome streamlining has emerged as an effective strategy to boost the production efficiency of bio-based products. Many efforts have been made to construct desirable chassis cells by reducing the genome size of microbes. It has been reported that the genome-reduced Bacillus subtilis strain MBG874 showed clear advantages for the production of several heterologous enzymes including alkaline cellulase and protease. In addition to enzymes, B. subtilis is also used for the production of chemicals. To our best knowledge, it is still unknown whether genome reduction could be used to optimize the production of chemicals such as nucleoside products. In this study, we constructed a series of genome-reduced strains by deleting non-essential regions in the chromosome of B. subtilis 168. These strains with genome reductions ranging in size from 581.9 to 814.4 kb displayed markedly decreased growth rates, sporulation ratios, transformation efficiencies and maintenance coefficients, as well as increased cell yields. We re-engineered the genome-reduced strains to produce guanosine and thymidine, respectively. The strain BSK814G2, in which purA was knocked out, and prs, purF and guaB were co-overexpressed, produced 115.2 mg/L of guanosine, which was 4.4-fold higher compared to the control strain constructed by introducing the same gene modifications into the parental strain. We also constructed a thymidine producer by deleting the tdk gene and overexpressing the prs, ushA, thyA, dut, and ndk genes from Escherichia coli in strain BSK756, and the resulting strain BSK756T3 accumulated 151.2 mg/L thymidine, showing a 5.2-fold increase compared to the corresponding control strain. Genome-scale genetic manipulation has a variety of effects on the physiological characteristics and cell metabolism of B. subtilis. By introducing specific gene modifications related to guanosine and thymidine accumulation, respectively, we demonstrated that genome-reduced strains had greatly improved properties compared to the wild-type strain as chassis cells for the production of these two products. These strains also have great potential for the production of other nucleosides and similar derived chemicals.

Fermentative Production of the Diamine Putrescine: System Metabolic Engineering of Corynebacterium Glutamicum

PubMed Central

Nguyen, Anh Q. D.; Schneider, Jens; Reddy, Gajendar Komati; Wendisch, Volker F.

2015-01-01

Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g−1·h−1 and a yield on glucose of 0.26 g·g−1. PMID:25919117

Modulation of medium-chain fatty acid synthesis in Synechococcus sp. PCC 7002 by replacing FabH with a Chaetoceros Ketoacyl-ACP synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novomore » assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase Ill increased MCFA synthesis up to fivefold. In conclusion, the level of increase is dependent on promoter strength and culturing conditions.« less

Modulation of medium-chain fatty acid synthesis in Synechococcus sp. PCC 7002 by replacing FabH with a Chaetoceros Ketoacyl-ACP synthase

DOE PAGES

Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.; ...

2016-05-26

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novomore » assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase Ill increased MCFA synthesis up to fivefold. In conclusion, the level of increase is dependent on promoter strength and culturing conditions.« less

Relationship of compressive stress-strain response of engineering materials obtained at constant engineering and true strain rates

DOE PAGES

Song, Bo; Sanborn, Brett

2018-05-07

In this paper, a Johnson–Cook model was used as an example to analyze the relationship of compressive stress-strain response of engineering materials experimentally obtained at constant engineering and true strain rates. There was a minimal deviation between the stress-strain curves obtained at the same constant engineering and true strain rates. The stress-strain curves obtained at either constant engineering or true strain rates could be converted from one to the other, which both represented the intrinsic material response. There is no need to specify the testing requirement of constant engineering or true strain rates for material property characterization, provided that eithermore » constant engineering or constant true strain rate is attained during the experiment.« less

Relationship of compressive stress-strain response of engineering materials obtained at constant engineering and true strain rates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Bo; Sanborn, Brett

In this paper, a Johnson–Cook model was used as an example to analyze the relationship of compressive stress-strain response of engineering materials experimentally obtained at constant engineering and true strain rates. There was a minimal deviation between the stress-strain curves obtained at the same constant engineering and true strain rates. The stress-strain curves obtained at either constant engineering or true strain rates could be converted from one to the other, which both represented the intrinsic material response. There is no need to specify the testing requirement of constant engineering or true strain rates for material property characterization, provided that eithermore » constant engineering or constant true strain rate is attained during the experiment.« less

Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid.

PubMed

Ro, Dae-Kyun; Ouellet, Mario; Paradise, Eric M; Burd, Helcio; Eng, Diana; Paddon, Chris J; Newman, Jack D; Keasling, Jay D

2008-11-04

Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 microg mL(-1) in shake-flask cultures and 1 g L(-1) in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast. The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.

Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

PubMed

Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

2017-07-01

NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering. Copyright © 2017 American Society for Microbiology.

Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane.

PubMed

Nguyen, Anh Duc; Hwang, In Yeub; Lee, Ok Kyung; Kim, Donghyuk; Kalyuzhnaya, Marina G; Mariyana, Rina; Hadiyati, Susila; Kim, Min Sik; Lee, Eun Yeol

2018-04-16

Methane is considered a next-generation feedstock, and methanotrophic cell-based biorefinery is attractive for production of a variety of high-value compounds from methane. In this work, we have metabolically engineered Methylomicrobium alcaliphilum 20Z for 2,3-butanediol (2,3-BDO) production from methane. The engineered strain 20Z/pBudK.p, harboring the 2,3-BDO synthesis gene cluster (budABC) from Klebsiella pneumoniae, accumulated 2,3-BDO in methane-fed shake flask cultures with a titer of 35.66 mg/L. Expression of the most efficient gene cluster was optimized using selection of promoters, translation initiation rates (TIR), and the combination of 2,3-BDO synthesis genes from different sources. A higher 2,3-BDO titer of 57.7 mg/L was measured in the 20Z/pNBM-Re strain with budA of K. pneumoniae and budB of Bacillus subtilis under the control of the Tac promoter. The genome-scale metabolic network reconstruction of M. alcaliphilum 20Z enabled in silico gene knockout predictions using an evolutionary programming method to couple growth and 2,3-BDO production. The ldh, ack, and mdh genes in M. alcaliphilum 20Z were identified as potential knockout targets. Pursuing these targets, a triple-mutant strain ∆ldh ∆ack ∆mdh was constructed, resulting in a further increase of the 2,3-BDO titer to 68.8 mg/L. The productivity of this optimized strain was then tested in a fed-batch stirred tank bioreactor, where final product concentrations of up to 86.2 mg/L with a yield of 0.0318 g-(2,3-BDO) /g-CH 4 were obtained under O 2 -limited conditions. This study first demonstrates the strategy of in silico simulation-guided metabolic engineering and represents a proof-of-concept for the production of value-added compounds using systematic approaches from engineered methanotrophs. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

The use of heterodyne speckle photogrammetry to measure high-temperature strain distributions

NASA Technical Reports Server (NTRS)

Stetson, K. A.

1983-01-01

Thermal and mechanical strains have been measured on samples of a common material used in jet engine burner liners, which were heated from room temperature to 870 C and cooled back to 220 C, in a laboratory furnace. The physical geometry of the sample surface was recorded to select temperatures by means of a set of twelve single-exposure specklegrams. Sequential pairs of specklegrams were compared in a heterodyne interferometer which allowed high-precision measurement of differential displacements. Good speckle correlation was observed between the first and last specklegrams also, which showed the durability of the surface microstructure, and permitted a check on accumulated errors. Agreement with calculated thermal expansion was to within a few hundred microstrain over a range of fourteen thousand.

Engineering three-dimensional cell mechanical microenvironment with hydrogels.

PubMed

Huang, Guoyou; Wang, Lin; Wang, Shuqi; Han, Yulong; Wu, Jinhui; Zhang, Qiancheng; Xu, Feng; Lu, Tian Jian

2012-12-01

Cell mechanical microenvironment (CMM) significantly affects cell behaviors such as spreading, migration, proliferation and differentiation. However, most studies on cell response to mechanical stimulation are based on two-dimensional (2D) planar substrates, which cannot mimic native three-dimensional (3D) CMM. Accumulating evidence has shown that there is a significant difference in cell behavior in 2D and 3D microenvironments. Among the materials used for engineering 3D CMM, hydrogels have gained increasing attention due to their tunable properties (e.g. chemical and mechanical properties). In this paper, we provide an overview of recent advances in engineering hydrogel-based 3D CMM. Effects of mechanical cues (e.g. hydrogel stiffness and externally induced stress/strain in hydrogels) on cell behaviors are described. A variety of approaches to load mechanical stimuli in 3D hydrogel-based constructs are also discussed.

Metabolic Design of Corynebacterium glutamicum for Production of l-Cysteine with Consideration of Sulfur-Supplemented Animal Feed.

PubMed

Joo, Young-Chul; Hyeon, Jeong Eun; Han, Sung Ok

2017-06-14

l-Cysteine is a valuable sulfur-containing amino acid widely used as a nutrition supplement in industrial food production, agriculture, and animal feed. However, this amino acid is mostly produced by acid hydrolysis and extraction from human or animal hairs. In this study, we constructed recombinant Corynebacterium glutamicum strains that overexpress combinatorial genes for l-cysteine production. The aims of this work were to investigate the effect of the combined overexpression of serine acetyltransferase (CysE), O-acetylserine sulfhydrylase (CysK), and the transcriptional regulator CysR on l-cysteine production. The CysR-overexpressing strain accumulated approximately 2.7-fold more intracellular sulfide than the control strain (empty pMT-tac vector). Moreover, in the resulting CysEKR recombinant strain, combinatorial overexpression of genes involved in l-cysteine production successfully enhanced its production by approximately 3.0-fold relative to that in the control strain. This study demonstrates a biotechnological model for the production of animal feed supplements such as l-cysteine using metabolically engineered C. glutamicum.

Production of ethanol from thin stillage by metabolically engineered Escherichia coli.

PubMed

Gonzalez, Ramon; Campbell, Paul; Wong, Matthew

2010-03-01

Thin stillage is a by-product generated in large amounts during the production of ethanol that is rich in carbon sources like glycerol, glucose and maltose. Unfortunately, the fermentation of thin stillage results in a mixture of organic acids and ethanol and minimum utilization of glycerol, the latter a compound that can represent up to 80% of the available substrates in this stream. We report here the efficient production of ethanol from thin stillage by a metabolically engineered strain of Escherichia coli. Simultaneous utilization of glycerol and sugars was achieved by overexpressing either the fermentative or the respiratory glycerol-utilization pathway. However, amplification of the fermentative pathway (encoded by gldA and dhaKLM) led to more efficient consumption of glycerol and promoted the synthesis of reduced products, including ethanol. A previously constructed strain, EH05, containing mutations that prevented the accumulation of competing by-products (i.e. lactate, acetate, and succinate) and overexpressing the fermentative pathway for glycerol utilization [i.e. strain EH05 (pZSKLMgldA)], efficiently converted thin stillage supplemented with only mineral salts to ethanol at yields close to 85% of the theoretical maximum. Ethanol accounted for about 90% (w/w) of the product mixture. These results, along with the comparable performance of strain EH05 (pZSKLMgldA) in 0.5 and 5 l fermenters, indicate a great potential for the adoption of this process by the biofuels industry.

Arsenic accumulation by the aquatic fern Azolla: comparison of arsenate uptake, speciation and efflux by A. caroliniana and A. filiculoides.

PubMed

Zhang, Xin; Lin, Ai-Jun; Zhao, Fang-Jie; Xu, Guo-Zhong; Duan, Gui-Lan; Zhu, Yong-Guan

2008-12-01

This study investigates As accumulation and tolerance of the aquatic fern Azolla. Fifty strains of Azolla showed a large variation in As accumulation. The highest- and lowest-accumulating ferns among the 50 strains were chosen for further investigations. Azolla caroliniana accumulated two times more As than Azolla filiculoides owing to a higher influx velocity for arsenate. A. filiculoides was more resistant to external arsenate due to a lower uptake. Both strains showed a similar degree of tolerance to internal As. Arsenate and arsenite were the dominant As species in both Azolla strains, with methylated As species accounting for <5% of the total As. A. filiculoides had a higher proportion of arsenite than A. caroliniana. Both strains effluxed more arsenate than arsenite, and the amount of As efflux was proportional to the amount of As accumulation. The potential of growing Azolla in paddy fields to reduce As transfer from soil and water to rice should be further evaluated.

Constant strain accumulation rate between major earthquakes on the North Anatolian Fault.

PubMed

Hussain, Ekbal; Wright, Tim J; Walters, Richard J; Bekaert, David P S; Lloyd, Ryan; Hooper, Andrew

2018-04-11

Earthquakes are caused by the release of tectonic strain accumulated between events. Recent advances in satellite geodesy mean we can now measure this interseismic strain accumulation with a high degree of accuracy. But it remains unclear how to interpret short-term geodetic observations, measured over decades, when estimating the seismic hazard of faults accumulating strain over centuries. Here, we show that strain accumulation rates calculated from geodetic measurements around a major transform fault are constant for its entire 250-year interseismic period, except in the ~10 years following an earthquake. The shear strain rate history requires a weak fault zone embedded within a strong lower crust with viscosity greater than ~10 20  Pa s. The results support the notion that short-term geodetic observations can directly contribute to long-term seismic hazard assessment and suggest that lower-crustal viscosities derived from postseismic studies are not representative of the lower crust at all spatial and temporal scales.

Sugar versus fat: elimination of glycogen storage improves lipid accumulation in Yarrowia lipolytica.

PubMed

Bhutada, Govindprasad; Kavšcek, Martin; Ledesma-Amaro, Rodrigo; Thomas, Stéphane; Rechberger, Gerald N; Nicaud, Jean-Marc; Natter, Klaus

2017-05-01

Triacylglycerol (TAG) and glycogen are the two major metabolites for carbon storage in most eukaryotic organisms. We investigated the glycogen metabolism of the oleaginous Yarrowia lipolytica and found that this yeast accumulates up to 16% glycogen in its biomass. Assuming that elimination of glycogen synthesis would result in an improvement of lipid accumulation, we characterized and deleted the single gene coding for glycogen synthase, YlGSY1. The mutant was grown under lipogenic conditions with glucose and glycerol as substrates and we obtained up to 60% improvement in TAG accumulation compared to the wild-type strain. Additionally, YlGSY1 was deleted in a background that was already engineered for high lipid accumulation. In this obese background, TAG accumulation was also further increased. The highest lipid content of 52% was found after 3 days of cultivation in nitrogen-limited glycerol medium. Furthermore, we constructed mutants of Y. lipolytica and Saccharomyces cerevisiae that are deleted for both glycogen and TAG synthesis, demonstrating that the ability to store carbon is not essential. Overall, this work showed that glycogen synthesis is a competing pathway for TAG accumulation in oleaginous yeasts and that deletion of the glycogen synthase has beneficial effects on neutral lipid storage. © FEMS 2017.

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation.

PubMed

Stefan, Alessandra; Hochkoeppler, Alejandro; Ugolini, Luisa; Lazzeri, Luca; Conte, Emanuele

2016-01-01

The conversion of industrial by-products into high-value added compounds is a challenging issue. Crude glycerol, a by-product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol-based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ∼5 g L(-1) of biomass and 0.8 g L(-1) of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L(-1) of urea or ammonium sulfate and 20 g L(-1) of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium-chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers.

Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode

PubMed Central

2012-01-01

Background Plasmid DNA (pDNA) is a promising molecule for therapeutic applications. pDNA is produced by Escherichia coli in high cell-density cultivations (HCDC) using fed-batch mode. The typical limitations of such cultivations, including metabolic deviations like aerobic acetate production due to the existence of substrate gradients in large-scale bioreactors, remain as serious challenges for fast and effective pDNA production. We have previously demonstrated that the substitution of the phosphotransferase system by the over-expressed galactose permease for glucose uptake in E. coli (strain VH33) allows efficient growth, while strongly decreases acetate production. In the present work, additional genetic modifications were made to VH33 to further improve pDNA production. Several genes were deleted from strain VH33: the recA, deoR, nupG and endA genes were inactivated independently and in combination. The performance of the mutant strains was evaluated in shake flasks for the production of a 6.1 kb plasmid bearing an antigen gene against mumps. The best producer strain was cultivated in lab-scale bioreactors using 100 g/L of glucose to achieve HCDC in batch mode. For comparison, the widely used commercial strain DH5α, carrying the same plasmid, was also cultivated under the same conditions. Results The various mutations tested had different effects on the specific growth rate, glucose uptake rate, and pDNA yields (YP/X). The triple mutant VH33 Δ (recA deoR nupG) accumulated low amounts of acetate and resulted in the best YP/X (4.22 mg/g), whereas YP/X of strain VH33 only reached 1.16 mg/g. When cultivated at high glucose concentrations, the triple mutant strain produced 186 mg/L of pDNA, 40 g/L of biomass and only 2.2 g/L of acetate. In contrast, DH5α produced only 70 mg/L of pDNA and accumulated 9.5 g/L of acetate. Furthermore, the supercoiled fraction of the pDNA produced by the triple mutant was nearly constant throughout the cultivation. Conclusion The pDNA concentration obtained with the engineered strain VH33 Δ (recA deoR nupG) is, to the best of our knowledge, the highest reported for a batch cultivation, and its supercoiled fraction remained close to 80%. Strain VH33 Δ (recA deoR nupG) and its cultivation using elevated glucose concentrations represent an attractive technology for fast and efficient pDNA production and a valuable alternative to fed-batch cultivations of commercial strains. PMID:22992433

Accumulation of intra-cellular polyphosphate in Chlorella vulgaris cells is related to indole-3-acetic acid produced by Azospirillum brasilense.

PubMed

Meza, Beatriz; de-Bashan, Luz E; Hernandez, Juan-Pablo; Bashan, Yoav

2015-06-01

Accumulation of intra-cellular phosphate, as polyphosphate, was measured when the microalga Chlorella vulgaris was immobilized in alginate with either of two wild-type strains of the microalgae growth-promoting bacterium Azospirillum brasilense or their corresponding IAA-attenuated mutants. Wild type strains of A. brasilense induced higher amounts of intra-cellular phosphate in Chlorella than their respective mutants. Calculations comparing intra-cellular phosphate accumulation by culture or net accumulation by the cell and the amount of IAA that was produced by each of these strains revealed that higher IAA was linked to higher accumulations of intra-cellular phosphate. Application of four levels of exogenous IAA reported for A. brasilense and their IAA-attenuated mutants to cultures of C. vulgaris enhanced accumulation of intra-cellular phosphate; the higher the content of IAA per culture or per single cell, the higher was the amount of accumulated phosphate. When an IAA-attenuated mutant was complemented with exogenous IAA, accumulation of intra-cellular phosphate at the culture level was even higher than phosphate accumulation with the respective wild type strains. When calculating the net accumulation of intra-cellular phosphate in the complementation experiment, net intra-cellular phosphate induced by the IAA-attenuated mutant was completely restored and was similar to the wild strains. We propose that IAA produced by A. brasilense is linked to polyphosphate accumulation in C. vulgaris. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics.

PubMed

Gold, Nicholas D; Gowen, Christopher M; Lussier, Francois-Xavier; Cautha, Sarat C; Mahadevan, Radhakrishnan; Martin, Vincent J J

2015-05-28

L-tyrosine is a common precursor for a wide range of valuable secondary metabolites, including benzylisoquinoline alkaloids (BIAs) and many polyketides. An industrially tractable yeast strain optimized for production of L-tyrosine could serve as a platform for the development of BIA and polyketide cell factories. This study applied a targeted metabolomics approach to evaluate metabolic engineering strategies to increase the availability of intracellular L-tyrosine in the yeast Saccharomyces cerevisiae CEN.PK. Our engineering strategies combined localized pathway engineering with global engineering of central metabolism, facilitated by genome-scale steady-state modelling. Addition of a tyrosine feedback resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase Aro4 from S. cerevisiae was combined with overexpression of either a tyrosine feedback resistant yeast chorismate mutase Aro7, the native pentafunctional arom protein Aro1, native prephenate dehydrogenase Tyr1 or cyclohexadienyl dehydrogenase TyrC from Zymomonas mobilis. Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. The TAL gene from Rhodobacter sphaeroides was used to produce coumarate as a simple test case of a heterologous by-product of tyrosine. Additionally, multiple strategies for engineering global metabolism to promote tyrosine production were evaluated using metabolic modelling. The T21E mutant of pyruvate kinase Cdc19 was hypothesized to slow the conversion of phosphoenolpyruvate to pyruvate and accumulate the former as precursor to the shikimate pathway. The ZWF1 gene coding for glucose-6-phosphate dehydrogenase was deleted to create an NADPH deficiency designed to force the cell to couple its growth to tyrosine production via overexpressed NADP(+)-dependent prephenate dehydrogenase Tyr1. Our engineered Zwf1(-) strain expressing TYRC ARO4(FBR) and grown in the presence of methionine achieved an intracellular L-tyrosine accumulation up to 520 μmol/g DCW or 192 mM in the cytosol, but sustained flux through this pathway was found to depend on the complete elimination of feedback inhibition and degradation pathways. Our targeted metabolomics approach confirmed a likely regulatory site at DAHP synthase and identified another possible cofactor limitation at prephenate dehydrogenase. Additionally, the genome-scale metabolic model identified design strategies that have the potential to improve availability of erythrose 4-phosphate for DAHP synthase and cofactor availability for prephenate dehydrogenase. We evaluated these strategies and provide recommendations for further improvement of aromatic amino acid biosynthesis in S. cerevisiae.

Improving Xylose Utilization of Saccharomyces cerevisiae by Expressing the MIG1 Mutant from the Self-Flocculating Yeast SPSC01.

PubMed

Xu, Jian-Ren; Zhao, Xin-Qing; Liu, Chen-Guang; Bai, Feng-Wu

2018-01-01

The major carbohydrate components of lignocellulosic biomass are cellulose and hemicelluloses. Saccharomyces cerevisiae cannot efficiently utilize xylose derived upon the hydrolysis of hemicelluloses. Although engineering the yeast with xylose metabolic pathway has been intensively studied, challenges are still ahead for developing robust strains for lignocellulosic bioethanol production. The main objective of this study was to reveal the role of the MIG1 mutant isolated from the self-flocculating S. cerevisiae SPSC01 in xylose utilization, glucose repression and ethanol fermentation by S. cerevisiae. The MIG1 mutant was amplified from S. cerevisiae SPSC01 by PCR and MIG1- overexpression-cassette was transformed into S. cerevisiae S288c and xylose-metabolizing strain YB-2625-T through homologous recombination. Yeast growth was measured by colony assay on plates with or without xylose supplementation. Then xylose utilization and ethanol production were further evaluated through flask fermentation when mixed sugars of glucose and xylose at 3:1 and 2:1, respectively, were supplied. Fermentation products were detected by HPLC, and activities of xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) were also measured. The transcription of genes regulated by the expression of the MIG1 mutant was analyzed by RTqPCR. Evolutionary relationship of various MIG1s was developed by gene sequencing and sequence alignment. No difference was observed for S288c growing with xylose when it was engineered with the overexpression or deletion of its native MIG1, but its growth was enhanced when overexpressing the MIG1 mutant from SPSC01. The submerged culture of YB-2625-T MIG1-SPSC engineered with xylose-metabolic pathway and the MIG1 mutant indicated that xylitol accumulation was decreased, and consequently, more biomass was accumulated. Furthermore, improved activities of the key enzymes such as XR, XDH and XK were detected in YB-2625-T MIG1-SPSC. Evolutionary analysis of MIG1s amplified from S. cerevisiae strains commonly used for ethanol production revealed a close relationship of SPSC01 and YB-2625. Our results demonstrated the effect of the overexpression of the MIG1 mutant from SPSC01 on xylose utilization of S. cerevisiae. This study could be an alternative strategy for engineering S. cerevisiae with improved xylose utilization. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Deletion of FPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

PubMed Central

Wei, Na; Xu, Haiqing; Kim, Soo Rin

2013-01-01

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD+/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation. PMID:23475614

Physiological diversity and trehalose accumulation in Schizosaccharomyces pombe strains isolated from spontaneous fermentations during the production of the artisanal Brazilian cachaça.

PubMed

Gomes, Fátima C O; Pataro, Carla; Guerra, Juliana B; Neves, Maria J; Corrêa, Soraya R; Moreira, Elizabeth S A; Rosa, Carlos A

2002-05-01

Twenty-seven Schizosaccharomyces pombe isolates from seven cachaça distilleries were tested for maximum temperature of growth and fermentation, osmotolerance, ethanol resistance, invertase production, and trehalose accumulation. Two isolates were selected for studies of trehalose accumulation under heat shock and ethanol stress. The S. pombe isolates were also characterized by RAPD-PCR. The isolates were able to grow and ferment at 41 degrees C, resisted concentrations of 10% ethanol, and grew on 50% glucose medium. Four isolates yielded invertase activity of more than 100 micromol of reducing sugar x mg(-1) x min(-1). The S. pombe isolates were able to accumulate trehalose during stationary phase. Two isolates, strains UFMG-A533 and UFMG-A1000, submitted to a 15 min heat shock, were able to accumulate high trehalose levels. Strain UFMG-A533 had a marked reduction in viability during heat shock, but strain UFMG-A1000 preserved a viability rate of almost 20% after 15 min at 48 degrees C. No clear correlation was observed between trehalose accumulation and cell survival during ethanol stress. Strain UFMG-A1000 had higher trehalose accumulation levels than strain UFMG-A533 under conditions of combined heat treatment and ethanol stress. Molecular analysis showed that some strains are maintained during the whole cachaça production period; using the RAPD-PCR profiles, it was possible to group the isolates according to their isolation sites.

Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation.

PubMed

Weber, Nora; Gorwa-Grauslund, Marie; Carlquist, Magnus

2017-01-03

Whole-cell biocatalysis based on metabolically active baker's yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels. Herein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5'-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using L-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions. Altogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion.

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling ▿ †

PubMed Central

Gaspar, Paula; Neves, Ana Rute; Gasson, Michael J.; Shearman, Claire A.; Santos, Helena

2011-01-01

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds. PMID:21841021

Metabolic regulation of triacylglycerol accumulation in the green algae: identification of potential targets for engineering to improve oil yield.

PubMed

Goncalves, Elton C; Wilkie, Ann C; Kirst, Matias; Rathinasabapathi, Bala

2016-08-01

The great need for more sustainable alternatives to fossil fuels has increased our research interests in algal biofuels. Microalgal cells, characterized by high photosynthetic efficiency and rapid cell division, are an excellent source of neutral lipids as potential fuel stocks. Various stress factors, especially nutrient-starvation conditions, induce an increased formation of lipid bodies filled with triacylglycerol in these cells. Here we review our knowledge base on glycerolipid synthesis in the green algae with an emphasis on recent studies on carbon flux, redistribution of lipids under nutrient-limiting conditions and its regulation. We discuss the contributions and limitations of classical and novel approaches used to elucidate the algal triacylglycerol biosynthetic pathway and its regulatory network in green algae. Also discussed are gaps in knowledge and suggestions for much needed research both on the biology of triacylglycerol accumulation and possible avenues to engineer improved algal strains. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Starch and lipid accumulation in eight strains of six Chlorella species under comparatively high light intensity and aeration culture conditions.

PubMed

Takeshita, Tsuyoshi; Ota, Shuhei; Yamazaki, Tomokazu; Hirata, Aiko; Zachleder, Vilém; Kawano, Shigeyuki

2014-04-01

The microalgae family Chlorella species are known to accumulate starch and lipids. Although nitrogen or phosphorous deficiencies promote starch and lipids formation in many microalgae, these deficiencies also limit their growth and productivity. Therefore, the Chlorellaceae strains were attempted to increase starch and lipids productivity under high-light-intensity conditions (600-μmol photons m(-2)s(-1)). The 12:12-h light-dark (LD) cycle conditions elicited more stable growth than the continuous light (LL) conditions, whereas the starch and lipids yields increased in LL conditions. The amount of starch and lipids per cell increased in Chlorella viscosa and Chlorella vulgaris in sulfur-deficient medium, and long-chain fatty acids with 20 or more carbon atoms accumulated in cells grown in sulfur-deficient medium. Accumulation of starch and lipids was investigated in eight strains. The accumulation was strain-dependent, and varied according to the medium and light conditions. Five of the eight Chlorella strains exhibited similar accumulation patterns. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genome-scale model-driven strain design for dicarboxylic acid production in Yarrowia lipolytica.

PubMed

Mishra, Pranjul; Lee, Na-Rae; Lakshmanan, Meiyappan; Kim, Minsuk; Kim, Byung-Gee; Lee, Dong-Yup

2018-03-19

Recently, there have been several attempts to produce long-chain dicarboxylic acids (DCAs) in various microbial hosts. Of these, Yarrowia lipolytica has great potential due to its oleaginous characteristics and unique ability to utilize hydrophobic substrates. However, Y. lipolytica should be further engineered to make it more competitive: the current approaches are mostly intuitive and cumbersome, thus limiting its industrial application. In this study, we proposed model-guided metabolic engineering strategies for enhanced production of DCAs in Y. lipolytica. At the outset, we reconstructed genome-scale metabolic model (GSMM) of Y. lipolytica (iYLI647) by substantially expanding the previous models. Subsequently, the model was validated using three sets of published culture experiment data. It was finally exploited to identify genetic engineering targets for overexpression, knockout, and cofactor modification by applying several in silico strain design methods, which potentially give rise to high yield production of the industrially relevant long-chain DCAs, e.g., dodecanedioic acid (DDDA). The resultant targets include (1) malate dehydrogenase and malic enzyme genes and (2) glutamate dehydrogenase gene, in silico overexpression of which generated additional NADPH required for fatty acid synthesis, leading to the increased DDDA fluxes by 48% and 22% higher, respectively, compared to wild-type. We further investigated the effect of supplying branched-chain amino acids on the acetyl-CoA turn-over rate which is key metabolite for fatty acid synthesis, suggesting their significance for production of DDDA in Y. lipolytica. In silico model-based strain design strategies allowed us to identify several metabolic engineering targets for overproducing DCAs in lipid accumulating yeast, Y. lipolytica. Thus, the current study can provide a methodological framework that is applicable to other oleaginous yeasts for value-added biochemical production.

Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects

PubMed Central

Lennen, Rebecca M.; Nilsson Wallin, Annika I.; Pedersen, Margit; Bonde, Mads; Luo, Hao; Herrgård, Markus J.; Sommer, Morten O. A.

2016-01-01

Homologous recombination of single-stranded oligonucleotides is a highly efficient process for introducing precise mutations into the genome of E. coli and other organisms when mismatch repair (MMR) is disabled. This can result in the rapid accumulation of off-target mutations that can mask desired phenotypes, especially when selections need to be employed following the generation of combinatorial libraries. While the use of inducible mutator phenotypes or other MMR evasion tactics have proven useful, reported methods either require non-mobile genetic modifications or costly oligonucleotides that also result in reduced efficiencies of replacement. Therefore a new system was developed, Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE), that solves problems encountered in other methods for oligonucleotide-mediated recombination. TM-MAGE enables nearly equivalent efficiencies of allelic replacement to the use of strains with fully disabled MMR and with an approximately 12- to 33-fold lower off-target mutation rate. Furthermore, growth temperatures are not restricted and a version of the plasmid can be readily removed by sucrose counterselection. TM-MAGE was used to combinatorially reconstruct mutations found in evolved salt-tolerant strains, enabling the identification of causative mutations and isolation of strains with up to 75% increases in growth rate and greatly reduced lag times in 0.6 M NaCl. PMID:26496947

Factors influencing the accumulation of ciprofloxacin in Pseudomonas aeruginosa.

PubMed Central

Celesk, R A; Robillard, N J

1989-01-01

Ciprofloxacin accumulation in Pseudomonas aeruginosa was measured by a bioassay. Drug accumulation in strain PAO2 was compared with that of three spontaneous ciprofloxacin-resistant mutants selected with 0.5 micrograms of ciprofloxacin per ml. PAO4701 cfxA2 contains a mutation in the gyrA gene, PAO4742 cfxB5 may represent a permeability mutant based on pleiotropic drug resistance, and PAO4700 cfxA1 cfxB1 contains both types of mutations. In all strains, drug accumulation was similar, reaching steady state during the first minute of exposure. Drug accumulation was unsaturable over a range of 5 to 80 micrograms/ml, suggesting that ciprofloxacin accumulates by diffusion in P. aeruginosa. Although all four strains accumulated two- to sevenfold more ciprofloxacin in the presence of the inhibitor carbonyl cyanide m-chlorophenylhydrazone, the cfxB mutants accumulated two- to fourfold less drug than either PAO2 or the cfxA2 mutant. Polyacrylamide gel analysis revealed a protein common to cfxB mutants only, while all strains had similar lipopolysaccharide profiles. The results suggest that ciprofloxacin accumulation in P. aeruginosa is a complex phenomenon that may be affected by both an energy-dependent drug efflux process and outer envelope composition. Images PMID:2514623

An Analysis of Strain Accumulation in the Western Part of Black Sea Region in Turkey

NASA Astrophysics Data System (ADS)

Deniz, I.; Avsar, N. B.; Deniz, R.; Mekik, C.; Kutoglu, S.

2014-12-01

Turkish National Horizontal Control Network (TNHCN) based on the European Datum 1950 (ED50) was used as the principal geodetic network until 2005 in Turkey. Since 2005, Turkish Large Scale Map and Map Information Production Regulation have required that that all the densification points have been produced within the same datum of Turkish National Fundamental GPS Network (TNFGN) put into practise in 2002 and based on International Terrestrial Reference Frame (ITRF). Hence, the common points were produced in both European Datum 1950 (ED50), and TNFGN.It is known that the geological and geophysical information about the network area can be obtained by the evaluation of the coordinate and scale variations in a geodetic network. For one such evaluation, the coordinate variations and velocities of network points, and also the strains are investigated. However, the principal problem in derivation of velocities arises from two different datums. In this context, the computation of velocities using the coordinate data of the ED50 and TNFGN is not accurate and reliable. Likewise, the analysis of strain from the coordinate differences is not reliable. However, due to the fact that the scale of a geodetic network is independent from datum, the strains can be derived from scale variations accurately and reliably.In this study, a test area limited 39.5°-42.0° northern latitudes and 31.0°-37.0° eastern longitudes was chosen. The benchmarks in this test area are composed of 30 geodetic control points derived with the aim of cadastral and engineering applications. We used data mining to investigate the common benchmarks in both reference systems for this area. Accordingly, the ED50 and TNFGN coordinates refer 1954 and 2005, respectively. Thus, it has been investigated the strain accumulation of 51 years in this region. It should be also noted that since 1954, the earthquakes have not registered greater than magnitude 6.0 in the test area. It is a considerable situation for this evaluation. The finite element analysis is used in order to derive the strain accumulation and rates in the test area (Figure 1). The results have been indicated that the minimum and maximum strains are 17μs and 3041μs, respectively.

Overexpression of malic enzyme (ME) of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis.

PubMed

Li, Zhi; Sun, Hanxiao; Mo, Xuemei; Li, Xiuying; Xu, Bo; Tian, Peng

2013-06-01

The oleaginous yeast Rhodotorula glutinis has been known to be a potential feedstock for lipid production. In the present study, we investigated the enhancement of expression of malic enzyme (ME; NADP(+) dependent; EC 1.1.1.40) from Mucor circinelloides as a strategy to improve lipid content inside the yeast cells. The 26S rDNA and 5.8S rDNA gene fragments isolated from Rhodotorula glutinis were used for homologous integration of ME gene into R. glutinis chromosome under the control of the constitutively highly expressed gene phosphoglycerate kinase 1 to achieve stable expression. We demonstrated that by increasing the expression of the foreign ME gene in R. glutinis, we successfully improved the lipid content by more than twofold. At the end of lipid accumulation phrase (96 h) in the transformants, activity of ME was increased by twofold and lipid content of the yeast cells was increased from 18.74 % of the biomass to 39.35 %. Simultaneously, there were no significant differences in fatty acid profiles between the wild-type strain and the recombinant strain. Over 94 % of total fatty acids were C16:0, C18:0, C16:1, C18:1, and C18:2. Our results indicated that heterologous expression of NADP(+)-dependent ME involved in fatty acid biosynthesis indeed increased the lipid accumulation in the oleaginous yeast R. glutinis.

Genome sequence of a microbial lipid producing fungus Cryptococcus albidus NT2002.

PubMed

Yong, Xiaoyu; Yan, Zhiying; Xu, Lin; Zhou, Jun; Wu, Xiayuan; Wu, Yuandong; Li, Yang; Chen, Zugeng; Zhou, Hua; Wei, Ping; Jia, Honghua

2016-04-10

Cryptococcus albidus NT2002, isolated from the soil in Xinjiang, China, appeared to have the ability to accumulate microbial lipid by utilizing various carbon sources. The predominant properties make it as a potential bio-platform for biodiesel production. Here, we report the complete genome sequence of C. albidus NT2002, which might provide a basis for further elucidation of the genetic background of this promising strain for developing metabolic engineering strategies to produce biodiesel in a green and sustainable manner. Copyright © 2016 Elsevier B.V. All rights reserved.

Biosynthesis of adipic acid via microaerobic hydrogenation of cis,cis-muconic acid by oxygen-sensitive enoate reductase.

PubMed

Sun, Jing; Raza, Muslim; Sun, Xinxiao; Yuan, Qipeng

2018-06-06

Adipic acid (AA) is an important dicarboxylic acid used for the manufacture of nylon and polyurethane plastics. In this study, a novel adipic acid biosynthetic pathway was designed by extending the cis,cis-muconic acid (MA) biosynthesis through biohydrogenation. Enoate reductase from Clostridium acetobutylicum (CaER), an oxygen-sensitive reductase, was demonstrated to have in vivo enzyme activity of converting cis,cis-muconic acid to adipic acid under microaerobic condition. Engineered Escherichia coli strains were constructed to express the whole pathway and accumulated 5.8 ± 0.9 mg/L adipic acid from simple carbon sources. Considering the different oxygen demands between cis,cis-muconic acid biosynthesis and its degradation, a co-culture system was constructed. To improve production, T7 promoter instead of lac promoter was used for higher level expression of the key enzyme CaER and the titer of adipic acid increased to 18.3 ± 0.6 mg/L. To decrease the oxygen supply to downstream strains expressing CaER, Vitreoscilla hemoglobin (VHb) was introduced to upstream strains for its ability on oxygen obtaining. This attempt further improved the production of this novel pathway and 27.6 ± 1.3 mg/L adipic acid was accumulated under microaerobic condition. Copyright © 2018. Published by Elsevier B.V.

Construction of Escherichia coli strains producing L-serine from glucose.

PubMed

Li, Yu; Chen, Gu-Kui; Tong, Xin-Wei; Zhang, Hui-Tu; Liu, Xiao-Guang; Liu, Yi-Han; Lu, Fu-Ping

2012-08-01

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.

Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum

PubMed Central

Heinrich, Daniel; Raberg, Matthias; Fricke, Philipp; Kenny, Shane T.; Morales-Gamez, Laura; Babu, Ramesh P.; O'Connor, Kevin E.

2016-01-01

ABSTRACT The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO2-containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter PcooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum. P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHAMCL), enhanced gene expression through the PcooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the Plac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHAMCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition. IMPORTANCE Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs. PMID:27520812

Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum.

PubMed

Heinrich, Daniel; Raberg, Matthias; Fricke, Philipp; Kenny, Shane T; Morales-Gamez, Laura; Babu, Ramesh P; O'Connor, Kevin E; Steinbüchel, Alexander

2016-10-15

The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO 2 -containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter P cooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHA MCL ), enhanced gene expression through the P cooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the P lac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHA MCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition. Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Cellular and Molecular Responses of Dunaliella tertiolecta by Expression of a Plant Medium Chain Length Fatty Acid Specific Acyl-ACP Thioesterase

PubMed Central

Lin, Huixin; Shen, Hui; Lee, Yuan K.

2018-01-01

Metabolic engineering of microalgae to accumulate high levels of medium chain length fatty acids (MCFAs) has met with limited success. Traditional approaches employ single introduction of MCFA specific acyl-ACP thioesterases (TEs), but our current research in transgenic Dunaliella tertiolecta line has highlighted that, there is no single rate-limiting approach that can effectively increase MCFA levels. Here, we explore the accumulation of MCFAs in D. tertiolecta after transgenic expression of myristic acid biased TE (C14TE). We observe that the MCFA levels were negatively correlated to the fatty acid (FA) synthesis genes, ketoacyl-ACP synthase II (KASII), stearoyl-CoA-9-desaturase (Δ9D), and oleoyl-CoA-12-desaturase (Δ12D). To further examine the molecular mechanism of MCFA accumulation in microalgae, we investigate the transcriptomic dynamics of the MCFA producing strain of D. tertiolecta. At the transcript level, enhanced MCFA accumulation primarily involved up-regulation of photosynthetic genes and down-regulation of genes from central carbon metabolic processes, resulting in an overall decrease in carbon precursors for FA synthesis. We additionally observe that MCFA specific peroxisomal β-oxidation gene (ACX3) was greatly enhanced to prevent excessive build-up of unusual MCFA levels. Besides, long chain acyl-CoA synthetase gene (LACS) was down-regulated, likely in attempt to control fatty acyl supply flux to FA synthesis cycle. This article provides a spatial regulation model of unusual FA accumulation in microalgae and a platform for additional metabolic engineering targeting pathways from FA synthesis, FA transport, and peroxisomal β-oxidation to achieve microalgae oils with higher levels of MCFAs. PMID:29670594

Ratcheting fatigue behavior of Zircaloy-2 at room temperature

NASA Astrophysics Data System (ADS)

Rajpurohit, R. S.; Sudhakar Rao, G.; Chattopadhyay, K.; Santhi Srinivas, N. C.; Singh, Vakil

2016-08-01

Nuclear core components of zirconium alloys experience asymmetric stress or strain cycling during service which leads to plastic strain accumulation and drastic reduction in fatigue life as well as dimensional instability of the component. Variables like loading rate, mean stress, and stress amplitude affect the influence of asymmetric loading. In the present investigation asymmetric stress controlled fatigue tests were conducted with mean stress from 80 to 150 MPa, stress amplitude from 270 to 340 MPa and stress rate from 30 to 750 MPa/s to study the process of plastic strain accumulation and its effect on fatigue life of Zircaloy-2 at room temperature. It was observed that with increase in mean stress and stress amplitude accumulation of ratcheting strain was increased and fatigue life was reduced. However, increase in stress rate led to improvement in fatigue life due to less accumulation of ratcheting strain.

Systems biology for understanding and engineering of heterotrophic oleaginous microorganisms.

PubMed

Park, Beom Gi; Kim, Minsuk; Kim, Joonwon; Yoo, Heewang; Kim, Byung-Gee

2017-01-01

Heterotrophic oleaginous microorganisms continue to draw interest as they can accumulate a large amount of lipids which is a promising feedstock for the production of biofuels and oleochemicals. Nutrient limitation, especially nitrogen limitation, is known to effectively trigger the lipid production in these microorganisms. For the aim of developing improved strains, the mechanisms behind the lipid production have been studied for a long time. Nowadays, system-level understanding of their metabolism and associated metabolic switches is attainable with modern systems biology tools. This work reviews the systems biology studies, based on (i) top-down, large-scale 'omics' tools, and (ii) bottom-up, mathematical modeling methods, on the heterotrophic oleaginous microorganisms with an emphasis on further application to metabolic engineering. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced degradation of 1-naphthol in landfill leachate using Arthrobacter sp.

PubMed

Hu, Wenyong; Min, Xiaobo; Li, Xinyu; Liu, Jingyi; Yu, Haibin; Yang, Yuan; Zhang, Jiachao; Luo, Lin; Chai, Liyuan; Zhou, Yaoyu

2017-12-06

Arthrobacter sp. named as JY5-1 isolated from contaminated soil of a coking plant can degrade 1-naphthol as the sole carbon source. Through identification of species, analysis of the optimal degradation condition and kinetic equation, the degradation characteristic of Arthrobacter sp. JY5-1 was obtained. Later, the acclimated strain was added into the bio-reactor to observe treatment performance of landfill leachate. The results showed that the optimal conditions for strain JY5-1 biodegradation in the study were pH 7.0 and 30 o C. The bio-reactor operation experiment declared that Arthrobacter sp. JY5-1 had a strengthened effect on COD removal of landfill leachate. Moreover, the efficiency of COD removal could be high and stable when JY5-1 was accumulated as a biofilm together with active sludge. These results demonstrate that adding 1-naphthol-degrading strain JY5-1 is a feasible technique for the enhanced treatment of sanitary landfill leachate, providing theoretical support for engineering utilization.

Self-cloning baker's yeasts that accumulate proline enhance freeze tolerance in doughs.

PubMed

Kaino, Tomohiro; Tateiwa, Tetsuya; Mizukami-Murata, Satomi; Shima, Jun; Takagi, Hiroshi

2008-09-01

We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding gamma-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.

Tyrosine decarboxylase activity of enterococci grown in media with different nutritional potential: tyramine and 2-phenylethylamine accumulation and tyrDC gene expression.

PubMed

Bargossi, Eleonora; Tabanelli, Giulia; Montanari, Chiara; Lanciotti, Rosalba; Gatto, Veronica; Gardini, Fausto; Torriani, Sandra

2015-01-01

The ability to accumulate tyramine and 2-phenylethylamine by two strains of Enterococcus faecalis and two strains Enterococcus faecium was evaluated in two cultural media added or not with tyrosine. All the enterococcal strains possessed a tyrosine decarboxylase (tyrDC) which determined tyramine accumulation in all the conditions tested, independently on the addition of high concentration of free tyrosine. Enterococci differed in rate and level of biogenic amines accumulation. E. faecalis EF37 and E. faecium FC12 produced tyramine in high amount since the exponential growth phase, while 2-phenylethylamine was accumulated when tyrosine was depleted. E. faecium FC12 and E. faecalis ATCC 29212 showed a slower tyraminogenic activity which took place mainly in the stationary phase up to 72 h of incubation. Moreover, E. faecalis ATCC 29212 produced 2-phenylethylamine only in the media without tyrosine added. In BHI added or not with tyrosine the tyrDC gene expression level differed considerably depending on the strains and the growth phase. In particular, the tyrDC gene expression was high during the exponential phase in rich medium for all the strains and subsequently decreased except for E. faecium FC12. Even if tyrDC presence is common among enterococci, this study underlines the extremely variable decarboxylating potential of strains belonging to the same species, suggesting strain-dependent implications in food safety.

Energy-Dependent Accumulation of Fluoroquinolones in Quinolone-Resistant Klebsiella pneumoniae Strains

PubMed Central

Martínez-Martínez, Luis; García, Isabel; Ballesta, Sofía; Benedí, Vicente Javier; Hernández-Allés, Santiago; Pascual, Alvaro

1998-01-01

The intracellular accumulation of norfloxacin and pefloxacin in Klebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents. PMID:9661034

Interactions of Saprophytic Yeasts with a nor Mutant of Aspergillus flavus

PubMed Central

Hua, Sui-Sheng T.; Baker, James L.; Flores-Espiritu, Melanie

1999-01-01

The nor mutant of Aspergillus flavus has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the nor mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the nor mutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of A. flavus. These yeasts may be useful for reducing aflatoxin contamination of food commodities. PMID:10347069

High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production.

PubMed

Back, Alexandre; Rossignol, Tristan; Krier, François; Nicaud, Jean-Marc; Dhulster, Pascal

2016-08-23

Because the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment. To address this issue, small cultivation systems have been developed that mimic the conditions in benchtop bioreactors. In this work, we used one such microbioreactor system, the BioLector, to develop high-throughput fermentation procedures that optimize growth and lipid accumulation in Y. lipolytica. Using this system, we were able to monitor lipid and biomass production in real time throughout the culture duration. The BioLector can monitor the growth of Y. lipolytica in real time by evaluating scattered light; this produced accurate measurements until cultures reached an equivalent of OD600nm = 115 and a cell dry weight of 100 g L(-1). In addition, a lipid-specific fluorescent probe was applied which reliably monitored lipid production up to a concentration of 12 g L(-1). Through screening various growing conditions, we determined that a carbon/nitrogen ratio of 35 was the most efficient for lipid production. Further screening showed that ammonium chloride and glycerol were the most valuable nitrogen and carbon sources, respectively, for growth and lipid production. Moreover, a carbon concentration above 1 M appeared to impair growth and lipid accumulation. Finally, we used these optimized conditions to screen engineered strains of Y. lipolytica with high lipid-accumulation capability. The growth and lipid content of the strains cultivated in the BioLector were compared to those grown in benchtop bioreactors. To our knowledge, this is the first time that the BioLector has been used to track lipid production in real time and to monitor the growth of Y. lipolytica. The present study also showed the efficacy of the BioLector in screening growing conditions and engineered strains prior to scale-up. The method described here could be applied to other oleaginous microorganisms.

Biosynthesis of rare ketoses through constructing a recombination pathway in an engineered Corynebacterium glutamicum.

PubMed

Yang, Jiangang; Zhu, Yueming; Li, Jitao; Men, Yan; Sun, Yuanxia; Ma, Yanhe

2015-01-01

Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars. © 2014 Wiley Periodicals, Inc.

Determining Recoverable and Irrecoverable Contributions to Accumulated Strain in a NiTiPd High-Temperature Shape Memory Alloy During Thermomechanical Cycling

NASA Technical Reports Server (NTRS)

Monroe, J. A.; Karaman, I.; Lagoudas, D. C.; Bigelow, G.; Noebe, R. D.; Padula, S., II

2011-01-01

When Ni(29.5)Ti(50.5)Pd30 shape memory alloy is thermally cycled under stress, significant strain can accumulate due to elasticity, remnant oriented martensite and plasticity. The strain due to remnant martensite can be recovered by further thermal cycling under 0 MPa until the original transformation-induced volume change and martensite coefficient of thermal expansion are obtained. Using this technique, it was determined that the 8.15% total accumulated strain after cycling under 200 MPa consisted of 0.38%, 3.97% and 3.87% for elasticity, remnant oriented martensite and creep/plasticity, respectively.

Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer.

PubMed

Fukui, Toshiaki; Abe, Hideki; Doi, Yoshiharu

2002-01-01

Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rod, M.L. Alam, K.Y.; Cunningham, P.R.; Clark, D.P.

When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium. The sugar was identified as trehalose. Strains of E. coli K-12 could be divided into two major classes with respect of osmoregulation. Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline. Most class A strains carried suppressor mutations which arose during their derivation from the wildmore » type, whereas the osmodefective strains of class B were suppressor free. When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup{sup o} osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity. It appears that the original K-12 strain of E. coli carries an amber mutation in a gene affecting osmoregulation. Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity. Trehalose synthesis was repressed by exogenous glycine betaine but not by proline.« less

Streptomyces sp. ASBV-1 reduces aflatoxin accumulation by Aspergillus parasiticus in peanut grains.

PubMed

Zucchi, T D; de Moraes, L A B; de Melo, I S

2008-12-01

To evaluate the ability of Streptomyces sp. (strain ASBV-1) to restrict aflatoxin accumulation in peanut grains. In the control of many phytopathogenic fungi the Streptomyces sp. ASBV-1 strain showed promise. An inhibitory test using this strain and A. parasiticus was conducted in peanut grains to evaluate the effects of this interaction on spore viability and aflatoxin accumulation. In some treatments the Streptomyces sp ASBV-1 strain reduced the viability of A. parasiticus spores by c. 85%, and inhibited aflatoxin accumulation in peanut grains. The values of these reductions ranged from 63 to 98% and from 67% to 96% for aflatoxins B(1) and G(1), respectively. It was demonstrated that Streptomyces sp. ASBV-1 is able to colonize peanut grains and thus inhibit the spore viability of A. parasiticus, as well as reducing aflatoxin production. The positive finding for aflatoxin accumulation reduction in peanut grains seems promising and suggests a wider use of this actinobacteria in biological control programmes.

Mercury bioremediation by mercury accumulating Enterobacter sp. cells and its alginate immobilized application.

PubMed

Sinha, Arvind; Khare, Sunil Kumar

2012-02-01

The effective microbial remediation of the mercury necessitates the mercury to be trapped within the cells without being recycled back to the environment. The study describes a mercury bioaccumulating strain of Enterobacter sp., which remediated mercury from the medium simultaneous to its growth. The transmission electron micrographs and electron dispersive X-ray analysis revealed the accumulation of remediated mercury as nano-size particles in the cytoplasm as well as on the cell wall. The Enterobacter sp. in the present work was able to accumulate mercury, without being engineered in its native form. The possibility of recovering the accumulated mercury from the cells is also indicated. The applicability of the alginate immobilized cells in removing mercury from synthetic and complex industrial effluent in a batch mode was amply demonstrated. The initial load of 7.3 mg l(-1) mercury in the industrial effluent was completely removed in 72 h. The cells immobilized in calcium alginate were similarly effective in the complete removal of 5 mg l(-1) HgCl(2) of mercury from the synthetic effluent in less than 72 h. The immobilized cells could be reused for multiple cycles.

High-oleate yeast oil without polyunsaturated fatty acids.

PubMed

Tsakraklides, Vasiliki; Kamineni, Annapurna; Consiglio, Andrew L; MacEwen, Kyle; Friedlander, Jonathan; Blitzblau, Hannah G; Hamilton, Maureen A; Crabtree, Donald V; Su, Austin; Afshar, Jonathan; Sullivan, John E; LaTouf, W Greg; South, Colin R; Greenhagen, Emily H; Shaw, A Joe; Brevnova, Elena E

2018-01-01

Oleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements. We sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content. High-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.

Draft genome sequence of the docosahexaenoic acid producing thraustochytrid Aurantiochytrium sp. T66.

PubMed

Liu, Bin; Ertesvåg, Helga; Aasen, Inga Marie; Vadstein, Olav; Brautaset, Trygve; Heggeset, Tonje Marita Bjerkan

2016-06-01

Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids.

Strain accumulation and rotation in the Eastern California Shear Zone

USGS Publications Warehouse

Savage, J.C.; Gan, Weijun; Svarc, J.L.

2001-01-01

Although the Eastern California Shear Zone (ECSZ) (strike ???N25??W) does not quite coincide with a small circle drawn about the Pacific-North America pole of rotation, trilateration and GPS measurements demonstrate that the motion within the zone corresponds to right-lateral simple shear across a vertical plane (strike N33??W??5??) roughly parallel to the tangent to that local small circle (strike ???N40??W). If the simple shear is released by slip on faults subparallel to the shear zone, the accumulated rotation is also released, leaving no secular rotation. South of the Garlock fault the principal faults (e.g., Calico-Blackwater fault) strike ???N40??W, close enough to the strike of the vertical plane across which maximum right-lateral shear accumulates to almost wholly accommodate that accumulation of both strain and rotation by right-lateral slip. North of the Garlock fault dip slip as well as strike slip on the principal faults (strike ???N20??W) is required to accommodate the simple shear accumulation. In both cases the accumulated rotation is released with the shear strain. The Garlock fault, which transects the ECSZ, is not offset by north-northwest striking faults nor, despite geological evidence for long-term left-lateral slip, does it appear at the present time to be accumulating left-lateral simple shear strain across the fault due to slip at depth. Rather the motion is explained by right-lateral simple shear across the orthogonal ECSZ. Left-lateral slip on the Garlock fault will release the shear strain accumulating there but would augment the accumulating rotation, resulting in a secular clockwise rotation rate ???80 nrad yr-1 (4.6?? Myr-1).

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli.

PubMed

Kawaguchi, Hideo; Katsuyama, Yohei; Danyao, Du; Kahar, Prihardi; Nakamura-Tsuruta, Sachiko; Teramura, Hiroshi; Wakai, Keiko; Yoshihara, Kumiko; Minami, Hiromichi; Ogino, Chiaki; Ohnishi, Yasuo; Kondo, Ahikiko

2017-07-01

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

Effect of l-Proline on Sake Brewing and Ethanol Stress in Saccharomyces cerevisiae

PubMed Central

Takagi, Hiroshi; Takaoka, Miki; Kawaguchi, Akari; Kubo, Yoshito

2005-01-01

During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. l-Proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular l-proline in cells of S. cerevisiae grown under ethanol stress. An l-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1D154N, which encodes the Asp154Asn mutant γ-glutamyl kinase. This mutation increases the activity of γ-glutamyl kinase and γ-glutamyl phosphate reductase, which catalyze the first two steps of l-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of the l-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation of l-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting the PUT1 gene, which is required for l-proline utilization, and replacing the wild-type PRO1 allele with the pro1D154N allele. The resultant strain accumulated l-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulate l-proline to brew sake containing five times more l-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles. PMID:16332860

Structural and electronic properties of in-plane phase engineered WSe2: A DFT study

NASA Astrophysics Data System (ADS)

Bhart, Ankush; Kapoor, Pooja; Sharma, Munish; Sharma, Raman; Ahluwalia, P. K.

2018-04-01

We present first principal investigations on structural and electronic properties of in-plane phase engineered WSe2 with armchair type interface. The 2H and 1T phases of WSe2, joined along x-direction is a natural metal-semiconductor heterostructure and therefore shows potential for applications in 2D electronics and opto-electronics. The electronic properties transit towards metallic 1T region. No inflections across interface shows negligible mismatch strain which is unlike what has been reported for MoS2. Charge density analysis shows charge accumulation on 1T domain. This can lead to reduction of Schottky barrier heights at the metal-semiconductor junction. STM analysis confirms transition of 1T phase towards distorted 1T' structure. The present results provide essential insights for nano-devices using 2D hybrid materials.

New and improved tools and methods for enhanced biosynthesis of natural products in microorganisms.

PubMed

Wang, Zhiqing; Cirino, Patrick C

2016-12-01

Engineering efficient biosynthesis of natural products in microorganisms requires optimizing gene expression levels to balance metabolite flux distributions and to minimize accumulation of toxic intermediates. Such metabolic optimization is challenged with identifying the right gene targets, and then determining and achieving appropriate gene expression levels. After decades of having a relatively limited set of gene regulation tools available, metabolic engineers are recently enjoying an ever-growing repertoire of more precise and tunable gene expression platforms. Here we review recent applications of natural and designed transcriptional and translational regulatory machinery for engineering biosynthesis of natural products in microorganisms. Customized trans-acting RNAs (sgRNA, asRNA and sRNA), along with appropriate accessory proteins, are allowing for unparalleled tuning of gene expression. Meanwhile metabolite-responsive transcription factors and riboswitches have been implemented in strain screening and evolution, and in dynamic gene regulation. Further refinements and expansions on these platform technologies will circumvent many long-term obstacles in natural products biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Improvement of SAM Accumulation by Integrating the Endogenous Methionine Adenosyltransferase Gene SAM2 in Genome of the Industrial Saccharomyces cerevisiae Strain.

PubMed

Zhao, Weijun; Shi, Feng; Hang, Baojian; Huang, Lei; Cai, Jin; Xu, Zhinan

2016-03-01

S-Adenosyl-L-methionine (SAM) plays important roles in trans-methylation, trans-sulfuration, and polyamine synthesis in all living cells, and it is also an effective cure for liver disease, depressive syndromes, and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, a multiple-copy integrative plasmid pYMIKP-SAM2 was introduced into the chromosome of wild-type Saccharomyces cerevisiae strain ZJU001 to construct the recombined strain R1-ZJU001. Further studies showed that the recombinant yeast exhibited higher enzymatic activity of methionine adenosyltransferase and improved its SAM biosynthesis. With a three-phase fed-batch strategy in 15-liter bench-top fermentor, 8.81 g/L SAM was achieved after 52 h cultivation of R1-ZJU001, about 27.1 % increase over its parent strain ZJU001, whereas the SAM content was also improved from 64.6 mg/g DCW to 91.0 mg/g DCW. Our results shall provide insights into the metabolic engineering of SAM pathway in yeast for improved productivity of SAM and subsequent industrial applications.

Metabolic engineering of microorganisms for the production of L-arginine and its derivatives.

PubMed

Shin, Jae Ho; Lee, Sang Yup

2014-12-03

L-arginine (ARG) is an important amino acid for both medicinal and industrial applications. For almost six decades, the research has been going on for its improved industrial level production using different microorganisms. While the initial approaches involved random mutagenesis for increased tolerance to ARG and consequently higher ARG titer, it is laborious and often leads to unwanted phenotypes, such as retarded growth. Discovery of L-glutamate (GLU) overproducing strains and using them as base strains for ARG production led to improved ARG production titer. Continued effort to unveil molecular mechanisms led to the accumulation of detailed knowledge on amino acid metabolism, which has contributed to better understanding of ARG biosynthesis and its regulation. Moreover, systems metabolic engineering now enables scientists and engineers to efficiently construct genetically defined microorganisms for ARG overproduction in a more rational and system-wide manner. Despite such effort, ARG biosynthesis is still not fully understood and many of the genes in the pathway are mislabeled. Here, we review the major metabolic pathways and its regulation involved in ARG biosynthesis in different prokaryotes including recent discoveries. Also, various strategies for metabolic engineering of bacteria for the overproduction of ARG are described. Furthermore, metabolic engineering approaches for producing ARG derivatives such as L-ornithine (ORN), putrescine and cyanophycin are described. ORN is used in medical applications, while putrescine can be used as a bio-based precursor for the synthesis of nylon-4,6 and nylon-4,10. Cyanophycin is also an important compound for the production of polyaspartate, another important bio-based polymer. Strategies outlined here will serve as a general guideline for rationally designing of cell-factories for overproduction of ARG and related compounds that are industrially valuable.

Disease-Associated Prion Protein in Neural and Lymphoid Tissues of Mink (Mustela vison) Inoculated with Transmissible Mink Encephalopathy

PubMed Central

Schneider, D. A.; Harrington, R. D.; Zhuang, D.; Yan, H.; Truscott, T. C.; Dassanayake, R. P.; O'Rourke, K. I.

2012-01-01

Summary Transmissible spongiform encephalopathies (TSEs) are diagnosed by immunodetection of disease-associated prion protein (PrPd). The distribution of PrPd within the body varies with the time-course of infection and between species, during interspecies transmission, as well as with prion strain. Mink are susceptible to a form of TSE known as transmissible mink encephalopathy (TME), presumed to arise due to consumption of feed contaminated with a single prion strain of ruminant origin. After extended passage of TME isolates in hamsters, two strains emerge, HY and DY, each of which is associated with unique structural isoforms of PrPTME and of which only the HY strain is associated with accumulation of PrPTME in lymphoid tissues. Information on the structural nature and lymphoid accumulation of PrPTME in mink is limited. In this study, 13 mink were challenged by intracerebral inoculation using late passage TME inoculum after which brain and lymphoid tissues were collected at preclinical and clinical time points. The distribution and molecular nature of PrPTME was investigated by techniques including blotting of paraffin wax-embedded tissue and epitope mapping by western blotting. PrPTME was detected readily in the brain and retropharyngeal lymph node during preclinical infection with delayed progression of accumulation within other lymphoid tissues. For comparison, three mink were inoculated by the oral route and examined during clinical disease. Accumulation of PrPTME in these mink was greater and more widespread, including follicles of rectoanal mucosa-associated lymphoid tissue. Western blot analyses revealed that PrPTME accumulating in the brain of mink is structurally most similar to that accumulating in the brain of hamsters infected with the DY strain. Collectively, the results of extended passage in mink are consistent with the presence of only a single strain of TME, the DY strain, capable of inducing accumulation of PrPTME in the lymphoid tissues of mink but not in hamsters. Thus mink are a relevant animal model for further study of this unique strain, which ultimately may have been introduced through consumption of a TSE of ruminant origin. PMID:22595634

Research on the resurrection evolution mechanism of Gendakan ancient landslide in the upstream on Lancang River, China

NASA Astrophysics Data System (ADS)

H, D.

2017-12-01

The Gendakan ancient landslide is located on the West bank of the upstream on Lancang River and about 4 km downstream from the Gushui hydropower station dam site. The ancient landslide is 850 m long and 700 m wide, the drill cores show that the maximum thickness of the landslide body is 107 m, with a mean thickness of approximately 80 m. Thus, the overall volume is about 3000×104m3. At present, the landslide has obvious deformation and failure signs, the leading edge is collapsing step by step. Once the landslide is unstable, it will affect the construction and operation of the Gushui hydropower station. In this paper, the development characteristics of the landslide accumulation body and the characteristics of the resurrection deformation are summarized in detail from the regional geological environment of the Gandakan landslide accumulation body. The three-dimensional geological model is established to analyze the stress and strain, displacement change and deformation failure characteristics and further evaluate its resurrection evolution trend , Combined with the developmental characteristics of the typical rock mass in the nearshore slope of the engineering area, analyzes the process of the resurrection and evolution of the rooted landslide accumulation. The FLAC-3D finite difference software was used to analyze the shear strain increment, displacement and plastic zone of the landslide accumulation body under natural conditions and rainfall conditions. The results show: the Gendakan landslide is stable in the natural state, and its deformation and failure are mainly caused by the tensile and shearing of the surface, under the rainfall condition, the local deformation and failure of the landslide accumulation body is obvious and the resurrection deformation Intensified. The resurrection evolutionary process of Gendakan ancient landslide includes three steps below. 1) The landslide body trailing edge creep cracking, leading edge shear deformation. 2) Sliding surface, accelerate the decline. 3)Disintegration of collision and impact into the dam.

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation.

PubMed

Satomura, Atsushi; Katsuyama, Yoshiaki; Miura, Natsuko; Kuroda, Kouichi; Tomio, Ayako; Bamba, Takeshi; Fukusaki, Eiichiro; Ueda, Mitsuyoshi

2013-01-01

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60-1, was bred from a parental strain, MT8-1, via stepwise adaptation. YK60-1 grew at 40°C, a temperature at which MT8-1 could not grow at all. YK60-1 exhibited faster growth than MT8-1 at 30°C. To investigate the mechanisms how MT8-1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress-responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60-1. Furthermore, nontargeting metabolome analysis showed that YK60-1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8-1. In conclusion, S. cerevisiae MT8-1 acquired thermotolerance by induction of specific stress-responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers.

High-Yield Secretion of Multiple Client Proteins in Aspergillus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segato, F.; Damasio, A. R. L.; Goncalves, T. A.

2012-07-15

Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degradingmore » enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.« less

Transcriptomic Analysis of Carboxylic Acid Challenge in Escherichia coli: Beyond Membrane Damage

PubMed Central

Royce, Liam A.; Boggess, Erin; Fu, Yao; Liu, Ping; Shanks, Jacqueline V.; Dickerson, Julie; Jarboe, Laura R.

2014-01-01

Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0 to probe mechanisms of toxicity. This analysis highlights the intracellular acidification and membrane damage caused by C8 challenge. Network component analysis identified transcription factors with altered activity including GadE, the activator of the glutamate-dependent acid resistance system (AR2) and Lrp, the amino acid biosynthesis regulator. The intracellular acidification was quantified during exogenous challenge, but was not observed in a carboxylic acid producing strain, though this may be due to lower titers than those used in our exogenous challenge studies. We developed a framework for predicting the proton motive force during adaptation to strong inorganic acids and carboxylic acids. This model predicts that inorganic acid challenge is mitigated by cation accumulation, but that carboxylic acid challenge inverts the proton motive force and requires anion accumulation. Utilization of native acid resistance systems was not useful in terms of supporting growth or alleviating intracellular acidification. AR2 was found to be non-functional, possibly due to membrane damage. We proposed that interaction of Lrp and C8 resulted in repression of amino acid biosynthesis. However, this hypothesis was not supported by perturbation of lrp expression or amino acid supplementation. E. coli strains were also engineered for altered cyclopropane fatty acid content in the membrane, which had a dramatic effect on membrane properties, though C8 tolerance was not increased. We conclude that achieving higher production titers requires circumventing the membrane damage. As higher titers are achieved, acidification may become problematic. PMID:24586888

Genetic engineering of industrial strains of Saccharomyces cerevisiae.

PubMed

Le Borgne, Sylvie

2012-01-01

Genetic engineering has been successfully applied to Saccharomyces cerevisiae laboratory strains for different purposes: extension of substrate range, improvement of productivity and yield, elimination of by-products, improvement of process performance and cellular properties, and extension of product range. The potential of genetically engineered yeasts for the massive production of biofuels as bioethanol and other nonfuel products from renewable resources as lignocellulosic biomass hydrolysates has been recognized. For such applications, robust industrial strains of S. cerevisiae have to be used. Here, some relevant genetic and genomic characteristics of industrial strains are discussed in relation to the problematic of the genetic engineering of such strains. General molecular tools applicable to the manipulation of S. cerevisiae industrial strains are presented and examples of genetically engineered industrial strains developed for the production of bioethanol from lignocellulosic biomass are given.

Investigation of Creep Processes and Microdamages in 10Kh9V2MFBR-Sh High-Chromium Steel

NASA Astrophysics Data System (ADS)

Grin', E. A.; Pchelintsev, A. V.

2018-01-01

During the modernization and the new construction of power units at TPPs in Russia, high-chromium martensitic steels with higher heat-resistant properties than the traditional perlite steels are increasingly used as structural materials. High-chromium steels have a necessary regulatory support for their use in domestic power engineering. However, up to the present time, the issue of assessing the quality of these steels at the analysis of their state during long-term operation remains open. The article proposed is one of the first attempts to create a system of quality criteria for martensitic steels based on their microdamage parameters. Tests were carried out on the long-term strength and creep of samples from 10Kh9V2MFBR-Sh steel at high temperatures with the construction of creep curves in relative coordinates "deformation related to the deformation of fracture, current time related to time to failure." For some samples, the tests were interrupted and the metal was subjected to metallographic studies consisting of the analysis of microdamage with reference to the accumulated creep strain. It has been shown experimentally that the deformation curve of high-chromium steel differs from the analogous curve of pearlitic steel by a longer and flat section of steady creep and by a sharper transition to the third accelerated creep stage, which has a very short time period (approximately 10% of the total durability). The tendency to the increase in the microdamage of the structure of steel as the accumulated creep strain increases with time was confirmed. The beginning of transition to the final creep phase is characterized by the formation of contours of future pore chains and by the appearance of individual large pores of up to 6 μm in size, the presence of which in the microstructure of the martensitic steel indicates a very significant accumulation of creep strain, and corresponds to the predestruction stage of metal. It is necessary to continue the research to obtain quantitative indicators on the accumulation of microdamage in high-chromium steel in a conjunction with the development of a metal resource under creep conditions.

Hydrolase BioH knockout in E. coli enables efficient fatty acid methyl ester bioprocessing.

PubMed

Kadisch, Marvin; Schmid, Andreas; Bühler, Bruno

2017-03-01

Fatty acid methyl esters (FAMEs) originating from plant oils are most interesting renewable feedstocks for biofuels and bio-based materials. FAMEs can also be produced and/or functionalized by engineered microbes to give access to, e.g., polymer building blocks. Yet, they are often subject to hydrolysis yielding free fatty acids, which typically are degraded by microbes. We identified BioH as the key enzyme responsible for the hydrolysis of medium-chain length FAME derivatives in different E. coli K-12 strains. E. coli ΔbioH strains showed up to 22-fold reduced FAME hydrolysis rates in comparison with respective wild-type strains. Knockout strains showed, beside the expected biotin auxotrophy, unchanged growth behavior and biocatalytic activity. Thus, high specific rates (~80 U g CDW -1 ) for terminal FAME oxyfunctionalization catalyzed by a recombinant alkane monooxygenase could be combined with reduced hydrolysis. Biotransformations in process-relevant two-liquid phase systems profited from reduced fatty acid accumulation and/or reduced substrate loss via free fatty acid metabolization. The BioH knockout strategy was beneficial in all tested strains, although its effect was found to differ according to specific strain properties, such as FAME hydrolysis and FFA degradation activities. BioH or functional analogs can be found in virtually all microorganisms, making bioH deletion a broadly applicable strategy for efficient microbial bioprocessing involving FAMEs.

Engineering tobacco to remove mercury from polluted soil.

PubMed

Chang, S; Wei, F; Yang, Y; Wang, A; Jin, Z; Li, J; He, Y; Shu, H

2015-04-01

Tobacco is an ideal plant for modification to remove mercury from soil. Although several transgenic tobacco strains have been developed, they either release elemental mercury directly into the air or are only capable of accumulating small quantities of mercury. In this study, we constructed two transgenic tobacco lines: Ntk-7 (a tobacco plant transformed with merT-merP-merB1-merB2-ppk) and Ntp-36 (tobacco transformed with merT-merP-merB1-merB2-pcs1). The genes merT, merP, merB1, and merB2 were obtained from the well-known mercury-resistant bacterium Pseudomonas K-62. Ppk is a gene that encodes polyphosphate kinase, a key enzyme for synthesizing polyphosphate in Enterobacter aerogenes. Pcs1 is a tobacco gene that encodes phytochelatin synthase, which is the key enzyme for phytochelatin synthesis. The genes were linked with LP4/2A, a sequence that encodes a well-known linker peptide. The results demonstrate that all foreign genes can be abundantly expressed. The mercury resistance of Ntk-7 and Ntp-36 was much higher than that of the wild type whether tested with organic mercury or with mercuric ions. The transformed plants can accumulate significantly more mercury than the wild type, and Ntp-36 can accumulate more mercury from soil than Ntk-7. In mercury-polluted soil, the mercury content in Ntp-36's root can reach up to 251 μg/g. This is the first report to indicate that engineered tobacco can not only accumulate mercury from soil but also retain this mercury within the plant. Ntp-36 has good prospects for application in bioremediation for mercury pollution.

The synchronous TAG production with the growth by the expression of chloroplast transit peptide-fused ScPDAT in Chlamydomonas reinhardtii.

PubMed

Zhu, Zhen; Yuan, Guangze; Fan, Xuran; Fan, Yan; Yang, Miao; Yin, Yalei; Liu, Jiao; Liu, Yang; Cao, Xupeng; Tian, Jing; Xue, Song

2018-01-01

The synchronous triacylglycerol (TAG) production with the growth is a key step to lower the cost of the microalgae-based biofuel production. Phospholipid: diacylglycerol acyltransferase (PDAT) has been identified recently and catalyzes the phospholipid contributing acyl group to diacylglycerol to synthesize TAG, and is considered as the important source of TAG in Chlamydomonas reinhardtii . Using a chimeric Hsp70A-RbcS2 promoter, exogenous PDAT form Saccharomyces cerevisiae fused with a chloroplast transit peptide was expressed in C. reinhardtii CC-137. Proved by western blot, the expression of ScPDAT showed a synchronous trend to the growth in the exponential phase. Compared to the wild type, the strain of Scpdat achieved 22% increase in the content of total fatty acids and 32% increase in TAG content. In addition, the fluctuation of C16 series fatty acid in monogalactosyldiacylglycerol, diacylglyceryltrimethylhomoserine and TAG indicated an enhancement in the TAG accumulation pathway. The TAG production was enhanced in the regular cultivation without the nutrient stress by strengthening the conversion of polar lipid to TAG in C. reinhardtii and the findings provide a candidate strategy for rational engineered strain to overcome the decline in the growth during the TAG accumulation triggered by nitrogen starvation.

Rapid intraplate strain accumulation in the New Madrid seismic zone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, L.; Zoback, M.D.; Segall, P.

1992-09-01

Remeasurement of a triangulation network in the southern part of the New Madrid seismic zone with the Global Positioning System has revealed rapid crustal strain accumulation since the 1950s. This area experienced three large (moment magnitudes greater than 8) earthquakes in 1811 to 1812. The orientation and sense of shear is consistent with right-lateral strike slip motion along a northeast-trending fault zone (as indicated by current seismicity). Detection of crustal strain accumulation may be a useful discriminant for identifying areas where potentially damaging intraplate earthquakes may occur despite the absence of large earthquakes during historic time. 34 refs.

Incorporating comparative genomics into the design-test-learn cycle of microbial strain engineering.

PubMed

Sardi, Maria; Gasch, Audrey P

2017-08-01

Engineering microbes with new properties is an important goal in industrial engineering, to establish biological factories for production of biofuels, commodity chemicals and pharmaceutics. But engineering microbes to produce new compounds with high yield remains a major challenge toward economically viable production. Incorporating several modern approaches, including synthetic and systems biology, metabolic modeling and regulatory rewiring, has proven to significantly advance industrial strain engineering. This review highlights how comparative genomics can also facilitate strain engineering, by identifying novel genes and pathways, regulatory mechanisms and genetic background effects for engineering. We discuss how incorporating comparative genomics into the design-test-learn cycle of strain engineering can provide novel information that complements other engineering strategies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440

DOE PAGES

Johnson, Christopher W.; Abraham, Paul E.; Linger, Jeffrey G.; ...

2017-05-31

Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect ofmore » carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36 h was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72 h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24 h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72 h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). In conclusion, these results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals.« less

Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Christopher W.; Abraham, Paul E.; Linger, Jeffrey G.

Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect ofmore » carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36 h was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72 h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24 h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72 h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). In conclusion, these results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals.« less

FY 1987 Aquatic Species Program: Annual report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, D.A.; Sprague, S.

The goal of the Department of Energy/Solar Energy Research Institute Aquatic Species Program is to develop the technology base to produce liquid fuels from microalagae at prices competitive with conventional alternatives. Microalgae are unusual plants that can accumulate large quantities of oil and can thrive in high-salinity water, which currently has no competing uses. The algal oils, in turn, are readily converted into gasoline and diesel fuels. The best site for successful microalgae production was determined to be the US desert Southwest, with potential applications to other warm areas. Aggressive research is needed, but the improvements required are attainable. Themore » four prime research areas in the development of this technology are growth and production, engineering design, harvesting, and conversion. Algae are selected for three criteria: tolerance to environmental fluctuations, high growth rates, and high lipid production. From 1982 to 1986, the program collected more than 3000 strains of microalgae that are more than twice as tolerant to temperature and salinity fluctuation than the initial strains. Productivity has been increased by a factor of two in outdoor culture systems since 1982, and lipid content has also been increased from 20% of body weight in 1982 to greater than 66% of body weight in 1987. Research programs are ongoing in lipid biochemistry and genetic engineering so that ultimately strains can be modified and improved to combine their best characteristics. An outdoor test facility is being built in Roswell, New Mexico.« less

Strain accumulation in bituminous binders under repeated creep-recovery loading predicted from small-amplitude oscillatory shear (SAOS) experiments

NASA Astrophysics Data System (ADS)

Laukkanen, Olli-Ville; Winter, H. Henning

2017-11-01

The creep-recovery (CR) test starts out with a period of shearing at constant stress (creep) and is followed by a period of zero-shear stress where some of the accumulated shear strain gets reversed. Linear viscoelasticity (LVE) allows one to predict the strain response to repeated creep-recovery (RCR) loading from measured small-amplitude oscillatory shear (SAOS) data. Only the relaxation and retardation time spectra of a material need to be known and these can be determined from SAOS data. In an application of the Boltzmann superposition principle (BSP), the strain response to RCR loading can be obtained as a linear superposition of the strain response to many single creep-recovery tests. SAOS and RCR data were collected for several unmodified and modified bituminous binders, and the measured and predicted RCR responses were compared. Generally good agreement was found between the measured and predicted strain accumulation under RCR loading. However, in the case of modified binders, the strain accumulation was slightly overestimated (≤20% relative error) due to the insufficient SAOS information at long relaxation times. Our analysis also demonstrates that the evolution in the strain response under RCR loading, caused by incomplete recovery, can be reasonably well predicted by the presented methodology. It was also shown that the outlined modeling framework can be used, as a first approximation, to estimate the rutting resistance of bituminous binders by predicting the values of the Multiple Stress Creep Recovery (MSCR) test parameters.

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10).

PubMed

Cluis, Corinne P; Ekins, Andrew; Narcross, Lauren; Jiang, Heng; Gold, Nicholas D; Burja, Adam M; Martin, Vincent J J

2011-11-01

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q(10) (CoQ(10)), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ(10) precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ(10) was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ(10)-producing E. coli strain resulted in an increase in CoQ(10) content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ(10) content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB. Copyright © 2011 Elsevier Inc. All rights reserved.

Thermomechanical and Environmental Durability of Environmental Barrier Coated Ceramic Matrix Composites Under Thermal Gradients

NASA Technical Reports Server (NTRS)

Zhu, Dongming; Bhatt, Ramakrishna T.; Harder, Bryan

2016-01-01

This paper presents the developments of thermo-mechanical testing approaches and durability performance of environmental barrier coatings (EBCs) and EBC coated SiCSiC ceramic matrix composites (CMCs). Critical testing aspects of the CMCs will be described, including state of the art instrumentations such as temperature, thermal gradient, and full field strain measurements; materials thermal conductivity evolutions and thermal stress resistance; NDE methods; thermo-mechanical stress and environment interactions associated damage accumulations. Examples are also given for testing ceramic matrix composite sub-elements and small airfoils to help better understand the critical and complex CMC and EBC properties in engine relevant testing environments.

Inactivation of dhaD and dhaK abolishes by-product accumulation during 1,3-propanediol production in Klebsiella pneumoniae.

PubMed

Horng, Yu-Tze; Chang, Kai-Chih; Chou, Ta-Chung; Yu, Chung-Jen; Chien, Chih-Ching; Wei, Yu-Hong; Soo, Po-Chi

2010-07-01

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P(BAD) promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.

Metal Accumulation and Vanadium-Induced Multidrug Resistance by Environmental Isolates of Escherichia hermannii and Enterobacter cloacae

PubMed Central

Hernández, Alicia; Mellado, Rafael P.; Martínez, José L.

1998-01-01

Contaminated soils from an oil refinery were screened for the presence of microorganisms capable of accumulating either nickel, vanadium, or both metals. Three strains of bacteria that belonged to the family Enterobacteriaceae were selected. Two of them were Escherichia hermannii strains, and outer membrane profile (OMP) analysis showed that they were similar to a strain of clinical origin; the other one was an Enterobacter cloacae strain that differed from clinical isolates. The selected bacteria accumulated both nickel and vanadium. Growth in the presence of vanadium induced multidrug resistance phenotypes in E. hermannii and E. cloacae. Incubation with this metal changed the OMP profile of E. hermannii but did not produce variations in the expression of the major OMPs of E. cloacae. PMID:9797283

Characterization of cadmium-resistant bacteria and their potential for reducing accumulation of cadmium in rice grains.

PubMed

Lin, Xiaoyan; Mou, Renxiang; Cao, Zhaoyun; Xu, Ping; Wu, Xiaoliang; Zhu, Zhiwei; Chen, Mingxue

2016-11-01

Cadmium (Cd) pollution is a serious widespread environmental problem that not only destroys the microbial ecology of soil and decreases crop production, but also poses a serious risk to human health. Many methods have been used for the remediation of Cd pollution but none of these is totally satisfactory. Microbial remediation strategies have attracted increasing interest since they are environmentally friendly and cost-effective. In the present study, three Cd-resistant bacteria were isolated and evaluated for potential application in Cd bioremediation. Based on their morphological, physiological and biochemical characteristics, together with 16S rDNA gene sequence analyses, bacteria were identified as Stenotrophomonas acidaminiphila (2#), Pseudomonas aeruginosa (9#) and Delftia tsuruhatensis (12#). Pseudomonas aeruginosa showed very high tolerance to metals, especially Cd (2200mg/L), Zn (1800mg/L) and Pb (1200mg/L), and is thought to be a multi-metal-resistant bacterium. Pseudomonas aeruginosa was also sensitive to 13 different antibiotics. The effects of the bacterial strains on the growth of rice plants and their ability to reduce Cd accumulation from Cd-contaminated soils in pot experiments were also evaluated. For Oryza sativa L. A grown in contaminated soil (3mg/kg Cd), the accumulation of Cd was decreased by 31.2 and 25.5% in brown rice and polished rice, respectively, by strain 9#; Pseudomonas aeruginosa was more effective in reducing Cd accumulation in rice grains than a mixture of strains. For Oryza sativa L. B, a mixture of strains acting synergistically was more effective than a single strain in reducing Cd accumulation; treatment with mixed strains (strains+3mg/kg Cd) resulted in 41.3, 35.9, and 32.6% reductions in Cd accumulation in unhulled rice, brown rice and polished rice, respectively. Although different results were obtained for two rice varieties, it can still be concluded that Cd-resistant bacteria are suitable for reducing Cd accumulation in rice grains and show potential for bioremediation of Cd-contaminated soils. Copyright © 2016 Elsevier B.V. All rights reserved.

40 CFR 90.118 - Certification procedure-service accumulation and usage of deterioration factors.

Code of Federal Regulations, 2010 CFR

2010-07-01

... recommended lubrication and filter changes, may be performed during service accumulation without the... on the test engine(s). (e) For purposes of establishing whether Phase 2 engines comply with...

Effect of Heat Input on the Tensile Damage Evolution in Pulsed Laser Welded Ti6Al4V Titanium Sheets

NASA Astrophysics Data System (ADS)

Liu, Jing; Gao, Xiaolong; Zhang, Jianxun

2016-11-01

The present paper is focused on studying the effect of heat input on the tensile damage evolution of pulsed Nd:YAG laser welding of Ti6Al4V alloy under monotonic loading. To analyze the reasons that the tensile fracture site of the pulsed-laser-welded Ti6Al4V sheet joints changes with the heat input under monotonic loading, the microstructure of the sample with different nominal strain values was investigated by in situ observation. Experiment results show that the tensile ductility and fatigue life of welded joints with low heat input are higher than that of welded joints with high heat input. Under tensile loads, the critical engineering strain for crack initiation is much lower in the welded joint with high heat input than in the welded joints with low and medium heat input. And the microstructural damage accumulation is much faster in the fusion zone than in the base metal for the welded joints with high input, whereas the microstructural damage accumulation is much faster in the base metal than in the fusion zone for the welded joints with low input. Consequently, the welded joints fractured in the fusion zone for the welds with high heat input, whereas the welded joints ruptured in the base metal for the welds with low heat input. It is proved that the fine grain microstructure produced by low heat input can improve the critical nominal strain for crack initiation and the resistance ability of microstructural damage.

Episodic strain accumulation in southern california.

PubMed

Thatcher, W

1976-11-12

Reexamination of horizontal geodetic data in the region of recently discovered aseismic uplift has demonstrated that equally unusual horizontal crustal deformation accompanied the development of the uplift. During this time interval compressive strains were oriented roughly normal to the San Andreas fault, suggesting that the uplift produced little shear strain accumulation across this fault. On the other hand, the orientation of the anomalous shear straining is consistent with strain accumulation across northdipping range-front thrusts like the San Fernando fault. Accordingly, the horizontal and vertical crustal deformation disclosed by geodetic observation is interpreted as a short epoch of rapid strain accumulation on these frontal faults. If this interpretation is correct, thrust-type earthquakes will eventually release the accumulated strains, but the geodetic data examined here cannot be used to estimate when these events might occur. However, observation of an unusual sequence of tilts prior to 1971 on a level line lying to the north of the magnitude 6.4 San Fernando earthquake offers some promise for precursor monitoring. The data are adequately explained by a simple model of up-dip aseismic slip propagation toward the 1971 epicentral region. These observations and the simple model that accounts for them suggest a conceptually straightforward monitoring scheme to search for similar uplift and tilt precursors within the uplifted region. Such premonitory effects could be detected by a combination of frequenlty repeated short (30 to 70 km in length) level line measurements, precise gravity traverses, and continuously recording gravimeters sited to the north of the active frontal thrust faults. Once identified, such precursors could be closely followed in space and time, and might then provide effective warnings of impending potentially destructive earth-quakes.

A new strain of Claviceps purpurea accumulating tetracyclic clavine alkaloids.

PubMed

Schumann, B; Erge, D; Maier, W; Gröger, D

1982-05-01

A new strain of Claviceps was isolated from a blokked mutant of Claviceps purpurea. This strain accumulates substantial amounts of clavine alkaloids (2 g/l). The alkaloid fraction is composed of chanoclavine-I ( approximately 10%) and a mixture of agroclavine/elymoclavine (90%). Most suitable for alkaloid production in submerged culture is an ammoncitrate/sucrose medium. The genealogy of the new strain, designated Pepty 695/ch-I is the following one: Pepty 695/S (ergotoxine producer) --> Pepty 695/ch (secoergoline producer) --> Pepty 695/ch-I (tetracyclic clavine producer).

Real-time, high-resolution study of nanocrystallization and fatigue cracking in a cyclically strained metallic glass.

PubMed

Wang, Cheng-Cai; Mao, Yun-Wei; Shan, Zhi-Wei; Dao, Ming; Li, Ju; Sun, Jun; Ma, Evan; Suresh, Subra

2013-12-03

Metallic glasses (MGs) exhibit greater elastic limit and stronger resistance to plastic deformation than their crystalline metal counterparts. Their capacity to withstand plastic straining is further enhanced at submicrometer length scales. For a range of microelectromechanical applications, the resistance of MGs to damage and cracking from thermal and mechanical stress or strain cycling under partial or complete constraint is of considerable scientific and technological interest. However, to our knowledge, no real-time, high-resolution transmission electron microscopy observations are available of crystallization, damage, and failure from the controlled imposition of cyclic strains or displacements in any metallic glass. Here we present the results of a unique in situ study, inside a high-resolution transmission electron microscope, of glass-to-crystal formation and fatigue of an Al-based MG. We demonstrate that cyclic straining progressively leads to nanoscale surface roughening in the highly deformed region of the starter notch, causing crack nucleation and formation of nanocrystals. The growth of these nanograins during cyclic straining impedes subsequent crack growth by bridging the crack. In distinct contrast to this fatigue behavior, only distributed nucleation of smaller nanocrystals is observed with no surface roughening under monotonic deformation. We further show through molecular dynamics simulation that these findings can be rationalized by the accumulation of strain-induced nonaffine atomic rearrangements that effectively enhances diffusion through random walk during repeated strain cycling. The present results thus provide unique insights into fundamental mechanisms of fatigue of MGs that would help shape strategies for material design and engineering applications.

Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae.

PubMed

Zhou, Pingping; Xie, Wenping; Li, Aipeng; Wang, Fan; Yao, Zhen; Bian, Qi; Zhu, Yongqiang; Yu, Hongwei; Ye, Lidan

2017-05-01

Highly efficient biosynthesis of the commercially valuable carotenoid astaxanthin by microbial cells is an attractive alternative to chemical synthesis and microalgae extraction. With the goal of enhancing heterologous astaxanthin production in Saccharomyces cerevisiae, metabolic engineering and protein engineering were integrated to improve both the expression and activity of rate-limiting enzymes. Firstly, to increase the supply of β-carotene as a key precursor for astaxanthin, a positive mutant of GGPP synthase (CrtE03M) was overexpressed together with three other rate-limiting enzymes tHMG1, CrtI and CrtYB. Subsequently, to accelerate the conversion of β-carotene to astaxanthin, a color screening system was developed and adopted for directed evolution of β-carotene ketolase (OBKT), generating a triple mutant OBKTM (H165R/V264D/F298Y) with 2.4-fold improved activity. After adjusting copy numbers of the above-mentioned rate-limiting enzymes to further balance the metabolic flux, a diploid strain YastD-01 was generated by mating two astaxanthin-producing haploid strains carrying the same carotenogenic pathway. Finally, further overexpression of OCrtZ and OBKTM in YastD-01 resulted in accumulation of 8.10mg/g DCW (47.18mg/l) of (3S, 3'S)-astaxanthin in shake-flask cultures. This combinatorial strategy might be also applicable for alleviation of metabolic bottleneck in biosynthesis of other value-added products, especially colored metabolites. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering a Synthetic Microbial Consortium for Comprehensive Conversion of Algae Biomass into Terpenes for Advanced Biofuels and Bioproducts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Weihua; Wu, Benjamin Chiau-Pin; Davis, Ryan Wesley

Recent strategies for algae-based biofuels have primarily focused on biodiesel production by exploiting high algal lipid yields under nutrient stress conditions. However, under conditions supporting robust algal biomass accumulation, carbohydrate and proteins typically comprise up to ~80% of the ash-free dry weight of algae biomass. Therefore, comprehensive utilization of algal biomass for production of multipurpose intermediate- to high-value bio-based products will promote scale-up of algae production and processing to commodity volumes. Terpenes are hydrocarbon and hydrocarbon-like (C:O>10:1) compounds with high energy density, and are therefore potentially promising candidates for the next generation of value added bio-based chemicals and “drop-in” replacementsmore » for petroleum-based fuels. In this study, we demonstrated the feasibility of bioconversion of proteins into sesquiterpene compounds as well as comprehensive bioconversion of algal carbohydrates and proteins into biofuels. To achieve this, the mevalonate pathway was reconstructed into an E. coli chassis with six different terpene synthases (TSs). Strains containing the various TSs produced a spectrum of sesquiterpene compounds in minimal medium containing amino acids as the sole carbon source. The sesquiterpene production was optimized through three different regulation strategies using chamigrene synthase as an example. The highest total terpene titer reached 166 mg/L, and was achieved by applying a strategy to minimize mevalonate accumulation in vivo. The highest yields of total terpene were produced under reduced IPTG induction levels (0.25 mM), reduced induction temperature (25°C), and elevated substrate concentration (20 g/L amino acid mixture). A synthetic bioconversion consortium consisting of two engineering E. coli strains (DH1-TS and YH40-TS) with reconstructed terpene biosynthetic pathways was designed for comprehensive single-pot conversion of algal carbohydrates and proteins to sesquiterpenes. The consortium yielded the highest total terpene yields (187 mg/L) at an inoculum ratio 2:1 of strain YH40-TS: DH1-TS, corresponding to 31 mg fuel/g algae biomass ash free dry weight. This study therefore demonstrates a feasible process for comprehensive algal biofuel production.« less

77 FR 67263 - Airworthiness Directives; Airbus Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-09

... flight cycles. US Airways stated that the engine inlet cowl inspection should follow Airbus Mandatory... months after the engine air intake cowl has accumulated 5,000 total flight cycles. (2) For any engine air... the same airplane has accumulated 5,000 flight cycles or less since the engine air intake cowl was...

Metabolically engineered glucose-utilizing Shewanella strains under anaerobic conditions.

PubMed

Choi, Donggeon; Lee, Sae Bom; Kim, Sohyun; Min, Byoungnam; Choi, In-Geol; Chang, In Seop

2014-02-01

Comparative genome analysis of Shewanella strains predicted that the strains metabolize preferably two- and three-carbon carbohydrates as carbon/electron source because many Shewanella genomes are deficient of the key enzymes in glycolysis (e.g., glucokinase). In addition, all Shewanella genomes are known to have only one set of genes associated with the phosphotransferase system required to uptake sugars. To engineer Shewanella strains that can utilize five- and six-carbon carbohydrates, we constructed glucose-utilizing Shewanella oneidensis MR-1 by introducing the glucose facilitator (glf; ZMO0366) and glucokinase (glk; ZMO0369) genes of Zymomonas mobilis. The engineered MR-1 strain was able to grow on glucose as a sole carbon/electron source under anaerobic conditions. The glucose affinity (Ks) and glucokinase activity in the engineered MR-1 strain were 299.46 mM and 0.259 ± 0.034 U/g proteins. The engineered strain was successfully applied to a microbial fuel cell system and exhibited current generation using glucose as the electron source. Copyright © 2013 Elsevier Ltd. All rights reserved.

Photoautotrophic production of D-lactic acid in an engineered cyanobacterium

PubMed Central

2013-01-01

Background The world faces the challenge to develop sustainable technologies to replace thousands of products that have been generated from fossil fuels. Microbial cell factories serve as promising alternatives for the production of diverse commodity chemicals and biofuels from renewable resources. For example, polylactic acid (PLA) with its biodegradable properties is a sustainable, environmentally friendly alternative to polyethylene. At present, PLA microbial production is mainly dependent on food crops such as corn and sugarcane. Moreover, optically pure isomers of lactic acid are required for the production of PLA, where D-lactic acid controls the thermochemical and physical properties of PLA. Henceforth, production of D-lactic acid through a more sustainable source (CO2) is desirable. Results We have performed metabolic engineering on Synechocystis sp. PCC 6803 for the phototrophic synthesis of optically pure D-lactic acid from CO2. Synthesis of optically pure D-lactic acid was achieved by utilizing a recently discovered enzyme (i.e., a mutated glycerol dehydrogenase, GlyDH*). Significant improvements in D-lactic acid synthesis were achieved through codon optimization and by balancing the cofactor (NADH) availability through the heterologous expression of a soluble transhydrogenase. We have also discovered that addition of acetate to the cultures improved lactic acid production. More interestingly, 13C-pathway analysis revealed that acetate was not used for the synthesis of lactic acid, but was mainly used for synthesis of certain biomass building blocks (such as leucine and glutamate). Finally, the optimal strain was able to accumulate 1.14 g/L (photoautotrophic condition) and 2.17 g/L (phototrophic condition with acetate) of D-lactate in 24 days. Conclusions We have demonstrated the photoautotrophic production of D-lactic acid by engineering a cyanobacterium Synechocystis 6803. The engineered strain shows an excellent D-lactic acid productivity from CO2. In the late growth phase, the lactate production rate by the engineered strain reached a maximum of ~0.19 g D-lactate/L/day (in the presence of acetate). This study serves as a good complement to the recent metabolic engineering work done on Synechocystis 6803 for L-lactate production. Thereby, our study may facilitate future developments in the use of cyanobacterial cell factories for the commercial production of high quality PLA. PMID:24274114

Photoautotrophic production of D-lactic acid in an engineered cyanobacterium.

PubMed

Varman, Arul M; Yu, Yi; You, Le; Tang, Yinjie J

2013-11-25

The world faces the challenge to develop sustainable technologies to replace thousands of products that have been generated from fossil fuels. Microbial cell factories serve as promising alternatives for the production of diverse commodity chemicals and biofuels from renewable resources. For example, polylactic acid (PLA) with its biodegradable properties is a sustainable, environmentally friendly alternative to polyethylene. At present, PLA microbial production is mainly dependent on food crops such as corn and sugarcane. Moreover, optically pure isomers of lactic acid are required for the production of PLA, where D-lactic acid controls the thermochemical and physical properties of PLA. Henceforth, production of D-lactic acid through a more sustainable source (CO2) is desirable. We have performed metabolic engineering on Synechocystis sp. PCC 6803 for the phototrophic synthesis of optically pure D-lactic acid from CO2. Synthesis of optically pure D-lactic acid was achieved by utilizing a recently discovered enzyme (i.e., a mutated glycerol dehydrogenase, GlyDH*). Significant improvements in D-lactic acid synthesis were achieved through codon optimization and by balancing the cofactor (NADH) availability through the heterologous expression of a soluble transhydrogenase. We have also discovered that addition of acetate to the cultures improved lactic acid production. More interestingly, (13)C-pathway analysis revealed that acetate was not used for the synthesis of lactic acid, but was mainly used for synthesis of certain biomass building blocks (such as leucine and glutamate). Finally, the optimal strain was able to accumulate 1.14 g/L (photoautotrophic condition) and 2.17 g/L (phototrophic condition with acetate) of D-lactate in 24 days. We have demonstrated the photoautotrophic production of D-lactic acid by engineering a cyanobacterium Synechocystis 6803. The engineered strain shows an excellent D-lactic acid productivity from CO2. In the late growth phase, the lactate production rate by the engineered strain reached a maximum of ~0.19 g D-lactate/L/day (in the presence of acetate). This study serves as a good complement to the recent metabolic engineering work done on Synechocystis 6803 for L-lactate production. Thereby, our study may facilitate future developments in the use of cyanobacterial cell factories for the commercial production of high quality PLA.

Survival differences among freeze-dried genetically engineered and wild-type bacteria.

PubMed Central

Israeli, E; Shaffer, B T; Hoyt, J A; Lighthart, B; Ganio, L M

1993-01-01

Because the death mechanisms of freeze-dried and air-dried bacteria are thought to be similar, freeze-drying was used to investigate the survival differences between potentially airborne genetically engineered microorganisms and their wild types. To this end, engineered strains of Escherichia coli and Pseudomonas syringae were freeze-dried and exposed to air, visible light, or both. The death rates of all engineered strains were significantly higher than those of their parental strains. Light and air exposure were found to increase the death rates of all strains. Application of death rate models to freeze-dried engineered bacteria to be released into the environment is discussed. PMID:8434925

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli.

PubMed

Oesterle, Sabine; Gerngross, Daniel; Schmitt, Steven; Roberts, Tania Michelle; Panke, Sven

2017-09-26

Multiplexed gene expression optimization via modulation of gene translation efficiency through ribosome binding site (RBS) engineering is a valuable approach for optimizing artificial properties in bacteria, ranging from genetic circuits to production pathways. Established algorithms design smart RBS-libraries based on a single partially-degenerate sequence that efficiently samples the entire space of translation initiation rates. However, the sequence space that is accessible when integrating the library by CRISPR/Cas9-based genome editing is severely restricted by DNA mismatch repair (MMR) systems. MMR efficiency depends on the type and length of the mismatch and thus effectively removes potential library members from the pool. Rather than working in MMR-deficient strains, which accumulate off-target mutations, or depending on temporary MMR inactivation, which requires additional steps, we eliminate this limitation by developing a pre-selection rule of genome-library-optimized-sequences (GLOS) that enables introducing large functional diversity into MMR-proficient strains with sequences that are no longer subject to MMR-processing. We implement several GLOS-libraries in Escherichia coli and show that GLOS-libraries indeed retain diversity during genome editing and that such libraries can be used in complex genome editing operations such as concomitant deletions. We argue that this approach allows for stable and efficient fine tuning of chromosomal functions with minimal effort.

Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion.

PubMed

Hector, Ronald E; Dien, Bruce S; Cotta, Michael A; Qureshi, Nasib

2011-09-01

Saccharomyces' physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04-0.17 h(-1). Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17-0.31 g/l/h, with ethanol yields 0.18-0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13-17% more ethanol than the parental control strains because of their ability to ferment xylose.

Chromium resistance characteristics of Cr(VI) resistance genes ChrA and ChrB in Serratia sp. S2.

PubMed

He, Yuan; Dong, Lanlan; Zhou, Simin; Jia, Yan; Gu, Ruijia; Bai, Qunhua; Gao, Jieying; Li, Yingli; Xiao, Hong

2018-08-15

To find an efficient chromium (VI) resistance system, with a highly efficient, economical, safe, and environmentally friendly chromium-removing strain, ChrA, ChrB, and ChrAB fragments of the chromium (VI) resistance gene in Serratia sp. S2 were cloned, and their prokaryotic expression vectors were constructed and transformed into E. coli BL21. The anti-chromium (VI) capacity and characteristics of engineered bacteria, role of ChrA and ChrB genes in the anti-chromium (VI) processes, and the mechanism of chromium metabolism, were explored. The PCR technique was used to amplify ChrA, ChrB, and ChrAB genes from the Serratia sp. S2 genome. ChrA, ChrB, and ChrAB genes were connected to the prokaryotic expression vector pET-28a and transferred into E. coli BL21 for prokaryotic expression. Cr-absorption and Cr-efflux ability of the engineered strains were determined. The effects of respiratory inhibitors and oxygenated anions on Cr-efflux of ChrA and ChrB engineered strains were explored. ChrA, ChrB, and ChrAB engineered strains were constructed successfully; there was no significant difference between the control strain and the ChrB engineered strain for Cr-metabolism (P > 0.05). Cr-absorption and Cr-efflux of ChrA and ChrAB engineered strains were significantly stronger than the control strain (P < 0.05). Oxyanions (sulfate and molybdate) and inhibitors (valinomycin and CN - ) could significantly inhibit the Cr-efflux capacities of ChrA and ChrAB engineered strains (P < 0.05), while NADPH could significantly promote such capacities (P < 0.05). The Cr-transporter, encoded by ChrA gene, confer the ability to pump out intracellular Cr on ChrA and ChrAB engineered strains. The ChrB gene plays a positive regulatory role in ChrA gene regulation. The Cr-metabolism ability of the ChrAB engineered strain is stronger than the ChrA engineered strain. ChrA and ChrAB genes in the Cr-resistance system may involve a variety of mechanisms, such as sulfate ion channel and respiratory chain electron transfer. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of Glutathione on the Taste and Texture of Type I Sourdough Bread.

PubMed

Tang, Kai Xing; Zhao, Cindy J; Gänzle, Michael G

2017-05-31

Type I sourdough fermentations with Lactobacillus sanfranciscensis as predominant organism accumulate reduced glutathione through glutathione reductase (GshR) activity of L. sanfranciscensis. Reduced glutathione acts as chain terminator for gluten polymerization but is also kokumi-active and may thus enhance bread taste. This study implemented a type I model sourdough fermentations to quantitate glutathione accumulation sourdough, bread dough, and bread and to assess the effect of L. sanfranciscensis GshR on bread volume by comparison of L. sanfranciscensis and an isogenic strain devoid of GshR. L. sanfranciscensis sourdough accumulated the highest amount of reduced glutathione during proofing. Bread produced with the wild type strain had a lower volume when compared to the gshR deficient mutant. The accumulation of γ-glutamyl-cysteine was also higher in L. sanfranciscensis sourdoughs when compared to doughs fermented with the gshR mutant strain. The accumulation of reduced glutathione in L. sanfranciscensis bread did not enhance the saltiness of bread.

Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

PubMed

Kim, Soo Rin; Skerker, Jeffrey M; Kong, In Iok; Kim, Heejin; Maurer, Matthew J; Zhang, Guo-Chang; Peng, Dairong; Wei, Na; Arkin, Adam P; Jin, Yong-Su

2017-03-01

Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A) exhibited a 14% higher ethanol yield and 46% lower byproduct yield than the IIK1 strain from anaerobic fermentation of the Miscanthus hydrolysate. Our results demonstrate that industrial yeast strains can be engineered via haploid isolation. The isolated haploid strain (4124-S60) can be used for metabolic engineering to produce fuels and chemicals. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Creep fatigue life prediction for engine hot section materials (isotropic)

NASA Technical Reports Server (NTRS)

Moreno, Vito; Nissley, David; Lin, Li-Sen Jim

1985-01-01

The first two years of a two-phase program aimed at improving the high temperature crack initiation life prediction technology for gas turbine hot section components are discussed. In Phase 1 (baseline) effort, low cycle fatigue (LCF) models, using a data base generated for a cast nickel base gas turbine hot section alloy (B1900+Hf), were evaluated for their ability to predict the crack initiation life for relevant creep-fatigue loading conditions and to define data required for determination of model constants. The variables included strain range and rate, mean strain, strain hold times and temperature. None of the models predicted all of the life trends within reasonable data requirements. A Cycle Damage Accumulation (CDA) was therefore developed which follows an exhaustion of material ductility approach. Material ductility is estimated based on observed similarities of deformation structure between fatigue, tensile and creep tests. The cycle damage function is based on total strain range, maximum stress and stress amplitude and includes both time independent and time dependent components. The CDA model accurately predicts all of the trends in creep-fatigue life with loading conditions. In addition, all of the CDA model constants are determinable from rapid cycle, fully reversed fatigue tests and monotonic tensile and/or creep data.

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering

PubMed Central

Pierce, Emily C.; LaFayette, Peter R.; Ortega, María A.; Joyce, Blake L.; Kopsell, Dean A.; Parrott, Wayne A.

2015-01-01

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 μg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, β-carotene, phytoene, α-carotene, lycopene, and β-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a β-carotene hydroxylase in addition to a β-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds. PMID:26376481

Impacts of genetically engineered alterations in carbon sink pathways on photosynthetic performance

DOE PAGES

Holland, Steven C.; Artier, Juliana; Miller, Neil T.; ...

2016-10-05

Genetic engineering of photosynthetic organisms typically redirects native metabolism towards desirable products, which thereby represent new metabolic sinks. There is limited information on how these modifications impact the evolved mechanisms of photosynthetic energy metabolism and cellular growth. Two engineered strains of Synechocystis sp. PCC 6803 with altered carbon sink capacity were assayed for their photosynthetic and CO 2 concentrating mechanism properties in conditions of high and low inorganic carbon (Ci) availability. In the ΔglgC mutant, glycogen cannot be synthesized and a carbon sink pathway has been effectively removed. The JU547 strain has been engineered by integration of the Pseudomonas syringaemore » ethylene forming enzyme and provides a new sink. When cultured under high carbon conditions, ΔglgC displayed diminished photochemical efficiency, a more reduced NADPH pool, delayed initiation of the Calvin-Benson-Bassham cycle, and impairment of linear and cyclic electron flows. It also exhibited a large decrease in photochemical quenching indicative of the accumulation of Q A-, normally associated with a reduced PQ pool, but appears instead to be the result of an undefined dissipative mechanism to spill excess energy. In the case of carbon sink integration, JU547 displayed slightly more oxidized PQ and NADPH pools and increased rates of cyclic electron flow and an enhanced demand for inorganic carbon as suggested by increase in the expression of the bicarbonate transporter, SbtA. Overall, the results highlight the importance of the native regulatory network of autotrophic metabolism in governing photosynthetic performance and provide cogent examples of both predicable and difficult to predict phenotypic consequences upon installation of new pathways in autotrophs.« less

Bioconversion of Xylan to triglycerides by oil-rich yeasts. [Cryptococcus albidus; Cryptococcus terricoluus; Trichosporon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fall, R.; Phelps, P.; Spindler, D.

A series of lipid-accumulating yeasts was examined for their potential to saccharify xylan and accumulate triglyceride. Of the genera tested, including Candida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, and Trichosporon, only Crytococcus and Trichosporon isolates saccharified xylan. All of the strains could assimilate xylose and accumuate triglyceride under nitrogen-limiting conditions. Strains of Cryptococcus albidus were found to be especially useful for a one-step saccharification of xylan coupled to triglyceride synthesis. Crytococcus terricolus, a strain constitutive for lipid accumulation, lacked extracellular xylanase, but did assimilate xylose and xylobiose and was able to continuously convert xylan to triglyceride if the culture medium was supplementedmore » with xylanase. 22 references.« less

Enhanced production of para-hydroxybenzoic acid by genetically engineered Saccharomyces cerevisiae.

PubMed

Averesch, Nils J H; Prima, Alex; Krömer, Jens O

2017-08-01

Saccharomyces cerevisiae is a popular organism for metabolic engineering; however, studies aiming at over-production of bio-replacement precursors for the chemical industry often fail to overcome proof-of-concept stage. When intending to show real industrial attractiveness, the challenge is twofold: formation of the target compound must be increased, while minimizing the formation of side and by-products to maximize titer, rate and yield. To tackle these, the metabolism of the organism, as well as the parameters of the process, need to be optimized. Addressing both we show that S. cerevisiae is well-suited for over-production of aromatic compounds, which are valuable in chemical industry and are particularly useful in space technology. Specifically, a strain engineered to accumulate chorismate was optimized for formation of para-hydroxybenzoic acid. Then a fed-batch bioreactor process was developed, which delivered a final titer of 2.9 g/L, a maximum rate of 18.625 mg pHBA /(g CDW  × h) and carbon-yields of up to 3.1 mg pHBA /g glucose .

Fusarium proliferatum strains change fumonisin biosynthesis and accumulation when exposed to host plant extracts.

PubMed

Górna, Karolina; Pawłowicz, Izabela; Waśkiewicz, Agnieszka; Stępień, Łukasz

2016-01-01

Fumonisin concentrations in mycelia and media were studied in liquid Fusarium proliferatum cultures supplemented with host plant extracts. Furthermore, the kinetics of fumonisin accumulation in media and mycelia collected before and after extract addition was analysed as well as the changes in the expression of the FUM1 gene. Fumonisin content in culture media increased in almost all F. proliferatum strains shortly after plant extracts were added. The asparagus extract induced the highest FB level increase and the garlic extract was the second most effective inducer. Fumonisin level decreased constantly until 14th day of culturing, though for some strains also at day 8th an elevated FB level was observed. Pineapple extract induced the highest increase of fum1 transcript levels as well as fumonisin synthesis in many strains, and the peas extract inhibited fungal growth and fumonisin biosynthesis. Moreover, fumonisins were accumulated in mycelia of studied strains and in the respective media. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Characterization of a selenium-tolerant rhizosphere strain from a novel Se-hyperaccumulating plant Cardamine hupingshanesis.

PubMed

Tong, Xinzhao; Yuan, Linxi; Luo, Lei; Yin, Xuebin

2014-01-01

A novel selenium- (Se-) hyperaccumulating plant, Cardamine hupingshanesis, accumulating Se as a form of SeCys2, was discovered in Enshi, Hubei, China, which could not be explained by present selenocysteine methyltransferase (SMT) theory. However, it is interesting to investigate if rhizosphere bacteria play some roles during SeCys2 accumulation. Here, one Se-tolerant rhizosphere strain, Microbacterium oxydans, was isolated from C. hupingshanesis. Phylogenetic analysis and 16S rRNA gene sequences determined the strain as a kind of Gram positive bacillus and belonged to the family Brevibacterium frigoritolerans. Furthermore, Se tolerance test indicated the strain could grow in extreme high Se level of 15.0 mg Se L(-1). When exposed to 1.5 mg Se L(-1), SeCys2 was the predominant Se species in the bacteria, consistent with the Se species in C. hupingshanesis. This coincidence might reveal that this strain played some positive effect in SeCys2 accumulation of C. hupingshanesis. Moreover, when exposed to 1.5 mg Se L(-1) or 15.0 mg Se L(-1), As absorption diminished in the logarithmic phase. In contrast, As absorption increased when exposed to 7.5 mg Se L(-1), indicating As metabolism processes could be affected by Se on this strain. The present study provided a sight on the role of rhizosphere bacteria during Se accumulation for Se-hyperaccumulating plant.

The interpretation of crustal dynamics data in terms of plate motions and regional deformation near plate boundaries

NASA Technical Reports Server (NTRS)

Solomon, Sean C.

1989-01-01

A particularly detailed set of observations in the vicinity of an intraplate, thrust earthquake (M 7.4) in Argentina, indicate a cyclic pattern of deformation very similar to that reported previously for interplate earthquakes. This deformation cycle, which may be characteristic of many seismically active areas, consists of: (1) steady strain accumulation, possibly punctuated by strain reversals; (2) coseismic strain release; (3) a period of continued strain release due to afterslip (persisting for perhaps a year or so); and (4) rapid postseismic strain accumulation which decreases exponentially and grades into steady strain accumulation. Deformation associated with three earthquakes in the U.S. (1940, M7.1 Imperial Valley California; 1964, M8.4 Alaska; 1959, M7.5 Hebgen Lake, Montana) are interpreted in light of this general earthquake cycle and are used to investigate the mechanics of strain release for these events. These examples indicate that large postseismic movements can occur for strike-slip, thrust, and normal fault events, and both viscoelastic relaxation and postseismic after-slip must be incorporated in models of earthquake related deformation. The mechanics of the strain release process revealed by these examples has implications for estimating earthquake repeat times from geodetic observations near active faults.

Development of paratransgenic Artemia as a platform for control of infectious diseases in shrimp mariculture.

PubMed

Subhadra, B; Hurwitz, I; Fieck, A; Rao, D V S; Rao, G Subba; Durvasula, R

2010-03-01

To study the accumulation and retention of recombinant proteins in Artemia gut for optimizing paratransgenic disease control in shrimp aquaculture. Transgenic Escherichia coli expressing fluorescent marker proteins and the transgenic cyanobacterium Synechococcus bacillarus expressing a functional murine single chain antibody, DB3, were fed to Artemia franciscana. Stable expression and retention of several marker molecules (e.g. GFP, DS Red and DB3) up to 10 h after of feeding with E. coli were evident within the gut of Artemia. Engineered strains of S. bacillarus expressing DB3 accumulated within the gut of Artemia with detectable antibody activity for 8-10 h of feeding via ELISA, coincident with the time period of the highest density of transgenic S. bacillarus in the Artemia gut. Artemia fed transgenic bacteria or algae accumulated recombinant proteins for up to 10 h that retained biological activity. Co-delivery of multiple recombinant proteins simultaneously in the gut of Artemia was also demonstrated. Expression of molecules that target infectious agents of mariculture in shrimp via commonly deployed feed organisms such as Artemia could potentially offer powerful new tools in the ongoing global effort to increase food supply.

Correlation between genome reduction and bacterial growth.

PubMed

Kurokawa, Masaomi; Seno, Shigeto; Matsuda, Hideo; Ying, Bei-Wen

2016-12-01

Genome reduction by removing dispensable genomic sequences in bacteria is commonly used in both fundamental and applied studies to determine the minimal genetic requirements for a living system or to develop highly efficient bioreactors. Nevertheless, whether and how the accumulative loss of dispensable genomic sequences disturbs bacterial growth remains unclear. To investigate the relationship between genome reduction and growth, a series of Escherichia coli strains carrying genomes reduced in a stepwise manner were used. Intensive growth analyses revealed that the accumulation of multiple genomic deletions caused decreases in the exponential growth rate and the saturated cell density in a deletion-length-dependent manner as well as gradual changes in the patterns of growth dynamics, regardless of the growth media. Accordingly, a perspective growth model linking genome evolution to genome engineering was proposed. This study provides the first demonstration of a quantitative connection between genomic sequence and bacterial growth, indicating that growth rate is potentially associated with dispensable genomic sequences. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Topological responses from chiral anomaly in multi-Weyl semimetals

NASA Astrophysics Data System (ADS)

Huang, Ze-Min; Zhou, Jianhui; Shen, Shun-Qing

2017-08-01

Multi-Weyl semimetals are a kind of topological phase of matter with discrete Weyl nodes characterized by multiple monopole charges, in which the chiral anomaly, the anomalous nonconservation of an axial current, occurs in the presence of electric and magnetic fields. Electronic transport properties related to the chiral anomaly in the presence of both electromagnetic fields and axial electromagnetic fields in multi-Weyl semimetals are systematically studied. It has been found that the anomalous Hall conductivity has a modification linear in the axial vector potential from inhomogeneous strains. The axial electric field leads to an axial Hall current that is proportional to the distance of Weyl nodes in momentum space. This axial current may generate chirality accumulation of Weyl fermions through delicately engineering the axial electromagnetic fields even in the absence of external electromagnetic fields. Therefore this work provides a nonmagnetic mechanism of generation of chirality accumulation in Weyl semimetals and might shed new light on the application of Weyl semimetals in the emerging field of valleytronics.

Salton Trough regional deformation estimated from combined trilateration and survey-mode GPS data

USGS Publications Warehouse

Anderson, G.; Agnew, D.C.; Johnson, H.O.

2003-01-01

The Salton Trough in southeastern California, United States, has one of the highest seismicity and deformation rates in southern California, including 20 earthquakes M 6 or larger since 1892. From 1972 through 1987, the U.S. Geological Survey (USGS) measured a 41-station trilateration network in this region. We remeasured 37 of the USGS baselines using survey-mode Global Positioning System methods from 1995 through 1999. We estimate the Salton Trough deformation field over a nearly 30-year period through combined analysis of baseline length time series from these two datasets. Our primary result is that strain accumulation has been steady over our observation span, at a resolution of about 0.05 ??strain/yr at 95% confidence, with no evidence for significant long-term strain transients despite the occurrence of seven large regional earthquakes during our observation period. Similar to earlier studies, we find that the regional strain field is consistent with 0.5 ?? 0.03 ??strain/yr total engineering shear strain along an axis oriented 311.6?? ?? 23?? east of north, approximately parallel to the strike of the major regional faults, the San Andreas and San Jacinto (all uncertainties in the text and tables are standard deviations unless otherwise noted). We also find that (1) the shear strain rate near the San Jacinto fault is at least as high as it is near the San Andreas fault, (2) the areal dilatation near the southeastern Salton Sea is significant, and (3) one station near the southeastern Salton Sea moved anomalously during the period 1987.95-1995.11.

Metabolic Engineering of Isoflavonoid Biosynthesis in Alfalfa1[w

PubMed Central

Deavours, Bettina E.; Dixon, Richard A.

2005-01-01

The potential health benefits of dietary isoflavones have generated considerable interest in engineering the synthesis of these phytoestrogens into plants. Genistein glucoside production (up to 50 nmol g−1 fresh weight) was engineered in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A (4′-O-methylgenistein) and pratensein (3′-hydroxybiochanin A) also accumulated. Although MtIFS1 was highly expressed in all organs examined, genistein accumulation was limited to leaves. MtIFS1-expressing lines accumulated several additional isoflavones, including formononetin and daidzein, in response to UV-B or Phoma medicaginis, whereas the chalcone and flavanone precursors of these compounds accumulated in control lines. Enhanced accumulation of the phytoalexin medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing plants. Microarray profiling indicated that MtIFS1 expression does not significantly alter global gene expression in the leaves. Our results highlight some of the challenges associated with metabolic engineering of plant natural products, including tissue-specific accumulation, potential for further modification by endogenous enzyme activities (hydroxylation, methylation, and glycosylation), and the differential response of engineered plants to environmental factors. PMID:16006598

Dissecting and engineering of the TetR family regulator SACE_7301 for enhanced erythromycin production in Saccharopolyspora erythraea.

PubMed

Wu, Hang; Chen, Meng; Mao, Yongrong; Li, Weiwei; Liu, Jingtao; Huang, Xunduan; Zhou, Ying; Ye, Bang-Ce; Zhang, Lixin; Weaver, David T; Zhang, Buchang

2014-11-13

Saccharopolyspora erythraea was extensively utilized for the industrial-scale production of erythromycin A (Er-A), a macrolide antibiotic commonly used in human medicine. Yet, S. erythraea lacks regulatory genes in the erythromycin biosynthetic gene (ery) cluster, hampering efforts to enhance Er-A production via the engineering of regulatory genes. By the chromosome gene inactivation technique based on homologous recombination with linearized DNA fragments, we have inactivated a number of candidate TetR family transcriptional regulators (TFRs) and identified one TFR (SACE_7301) positively controlling erythromycin biosynthesis in S. erythraea A226. qRT-PCR and EMSA analyses demonstrated that SACE_7301 activated the transcription of erythromycin biosynthetic gene eryAI and the resistance gene ermE by interacting with their promoter regions with low affinities, similar to BldD (SACE_2077) previously identified to regulate erythromycin biosynthesis and morphological differentiation. Therefore, we designed a strategy for overexpressing SACE_7301 with 1 to 3 extra copies under the control of PermE* in A226. Following up-regulated transcriptional expression of SACE_7301, eryAI and ermE, the SACE_7301-overexpressed strains all increased Er-A production over A226 proportional to the number of copies. Likewise, when SACE_7301 was overexpressed in an industrial S. erythraea WB strain, Er-A yields of the mutants WB/7301, WB/2×7301 and WB/3×7301 were respectively increased by 17%, 29% and 42% relative to that of WB. In a 5 L fermentor, Er-A accumulation increased to 4,230 mg/L with the highest-yield strain WB/3×7301, an approximately 27% production improvement over WB (3,322 mg/L). We have identified and characterized a TFR, SACE_7301, in S. erythraea that positively regulated erythromycin biosynthesis, and overexpression of SACE_7301 in wild-type and industrial S. erythraea strains enhanced Er-A yields. This study markedly improves our understanding of the unusual regulatory mechanism of erythromycin biosynthesis, and provides a novel strategy towards Er-A overproduction by engineering transcriptional regulators of S. erythraea.

Cyclic axial-torsional deformation behavior of a cobalt-base superalloy

NASA Technical Reports Server (NTRS)

Bonacuse, Peter J.; Kalluri, Sreeramesh

1992-01-01

Multiaxial loading, especially at elevated temperature, can cause the inelastic response of a material to differ significantly from that predicted by simple flow rules, i.e., von Mises or Tresca. To quantify some of these differences, the cyclic high-temperature, deformation behavior of a wrought cobalt-based superalloy, Haynes 188, is investigated under combined axial and torsional loads. Haynes 188 is currently used in many aerospace gas turbine and rocket engine applications, e.g., the combustor liner for the T800 turboshaft engine for the RAH-66 Comanche helicopter and the liquid oxygen posts in the main injector of the space shuttle main engine. The deformation behavior of this material is assessed through the examination of hysteresis loops generated from a biaxial fatigue test program. A high-temperature axial, torsional, and combined axial-torsional fatigue data base has been generated on Haynes 188 at 760 C. Cyclic loading tests have been conducted on uniform gauge section tubular specimens in a servohydraulic axial-torsional test rig. Test control and data acquisition were accomplished with a minicomputer. In this paper, the cyclic hardening characteristics and typical hysteresis loops in the axial stress versus axial strain, shear stress versus engineering shear strain, axial strain versus engineering shear strain, and axial stress versus shear stress spaces are presented for cyclic, in-phase and out-of-phase, axial torsional tests. For in-phase tests three different values of the proportionality constant, lambda (ratio of engineering shear strain amplitude to axial strain amplitude), are examined, viz., 0.86, 1.73, and 3.46. In the out-of-phase tests, three different values of the phase angle, phi (between the axial and engineering shear strain waveforms), are studied, viz., 30, 60, and 90 deg with lambda = 1.73. The cyclic hardening behaviors of all the tests conducted on Haynes 188 at 760 C are evaluated using the von Mises equivalent stress-strain and the maximum shear stress-maximum engineering shear strain (Tresca) curves. Comparisons are also made between the hardening behaviors of cyclic axial, torsional, and combined in-phase and out-of-phase axial-torsional fatigue tests. These comparisons are accomplished through simple Ramberg-Osgood type stress-strain functions for cyclic, axial stress-strain and shear stress-engineering shear strain curves.

Characterization of halophilic C50 carotenoid-producing archaea isolated from solar saltworks in Bohai Bay, China

NASA Astrophysics Data System (ADS)

Sui, Liying; Liu, Liangsen; Deng, Yuangao

2014-11-01

Halophilic archaea comprise the majority of microorganisms found in hypersaline environments. C50 carotenoids accumulated in archaea cells are considered potential biotechnological products and possess a number of biological functions. Ten red colonies were isolated from brine water in a saltern crystallizer pond of the Hangu Saltworks, China. 16S rRNA gene sequence analysis showed that the colonies belonged to the extremely halophilic archaea genera Halobacterium and Halorubrum. Two representative strains, Halobacterium strain SP-2 and Halorubrum strain SP-4, were selected for further study on the phenotypic characteristics and effects of salinity and pH on accumulation and composition of pigments in their cells. The archaeal strains were isolated and grown in a culture medium prepared by dissolving yeast extract (10 g/L) and acid-hydrolyzed casein (7.5 g/L) into brine water obtained from a local salt pond. Their optimum salinity and pH for growth were 250 and 7, respectively, although pigment accumulation (OD490 / mL broth) was highest at pH 8. In addition, at 150-300 salinity, increasing salinity resulted in decreasing pigment accumulation. Analysis of the UV-Vis spectrum, TLC and HLPC chromatograms showed that C50 carotenoid bacterioruberin is the major pigment in both strains.

Prion Strain Differences in Accumulation of PrPSc on Neurons and Glia Are Associated with Similar Expression Profiles of Neuroinflammatory Genes: Comparison of Three Prion Strains

PubMed Central

Carroll, James A.; Striebel, James F.; Rangel, Alejandra; Woods, Tyson; Phillips, Katie; Peterson, Karin E.; Race, Brent; Chesebro, Bruce

2016-01-01

Misfolding and aggregation of host proteins are important features of the pathogenesis of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia and prion diseases. In all these diseases, the misfolded protein increases in amount by a mechanism involving seeded polymerization. In prion diseases, host prion protein is misfolded to form a pathogenic protease-resistant form, PrPSc, which accumulates in neurons, astroglia and microglia in the CNS. Here using dual-staining immunohistochemistry, we compared the cell specificity of PrPSc accumulation at early preclinical times post-infection using three mouse scrapie strains that differ in brain regional pathology. PrPSc from each strain had a different pattern of cell specificity. Strain 22L was mainly associated with astroglia, whereas strain ME7 was mainly associated with neurons and neuropil. In thalamus and cortex, strain RML was similar to 22L, but in substantia nigra, RML was similar to ME7. Expression of 90 genes involved in neuroinflammation was studied quantitatively using mRNA from thalamus at preclinical times. Surprisingly, despite the cellular differences in PrPSc accumulation, the pattern of upregulated genes was similar for all three strains, and the small differences observed correlated with variations in the early disease tempo. Gene upregulation correlated with activation of both astroglia and microglia detected in early disease prior to vacuolar pathology or clinical signs. Interestingly, the profile of upregulated genes in scrapie differed markedly from that seen in two acute viral CNS diseases (LaCrosse virus and BE polytropic Friend retrovirus) that had reactive gliosis at levels similar to our prion-infected mice. PMID:27046083

Production of cellobionate from cellulose using an engineered Neurospora crassa strain with laccase and redox mediator addition

USDA-ARS?s Scientific Manuscript database

We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5'ace-1'cre-1'ndvB) was shown to produce cellobionate directly from cellulose ...

Optimization of the IPP Precursor Supply for the Production of Lycopene, Decaprenoxanthin and Astaxanthin by Corynebacterium glutamicum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heider, Sabine A. E.; Wolf, Natalie; Hofemeier, Arne

The biotechnologically relevant bacterium Corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides. The precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (MEP) or non-mevalonate pathway. Terminal pathway engineering in recombinant C. glutamicum permitted the production of various non-native C50 and C40 carotenoids. Here, the role of engineering isoprenoid precursor supply for lycopene production by C. glutamicum was characterized. Overexpression of dxs encodingmore » the enzyme that catalyzes the first committed step of the MEP-pathway by chromosomal promoter exchange in a prophage-cured, genome-reduced C. glutamicum strain improved lycopene formation. Similarly, an increased IPP supply was achieved by chromosomal integration of two artificial operons comprising MEP pathway genes under the control of a constitutive promoter. Combined overexpression of dxs and the other six MEP pathways genes in C. glutamicum strain LYC3-MEP was not synergistic with respect to improving lycopene accumulation. Based on C. glutamicum strain LYC3-MEP, astaxanthin could be produced in the milligrams per gram cell dry weight range when the endogenous genes crtE, crtB, and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were coexpressed with the genes for lycopene cyclase and β-carotene hydroxylase from Pantoea ananatis and carotene C(4) oxygenase from Brevundimonas aurantiaca.« less

In-Plane Heterostructures Enable Internal Stress Assisted Strain Engineering in 2D Materials.

PubMed

Liu, Feng; Wang, Tzu-Chiang; Tang, Qiheng

2018-04-01

Conventional methods to induce strain in 2D materials can hardly catch up with the sharp increase in requirements to design specific strain forms, such as the pseudomagnetic field proposed in graphene, funnel effect of excitons in MoS 2 , and also the inverse funnel effect reported in black phosphorus. Therefore, a long-standing challenge in 2D materials strain engineering is to find a feasible scheme that can be used to design given strain forms. In this article, combining the ability of experimentally synthetizing in-plane heterostructures and elegant Eshelby inclusion theory, the possibility of designing strain fields in 2D materials to manipulate physical properties, which is called internal stress assisted strain engineering, is theoretically demonstrated. Particularly, through changing the inclusion's size, the stress or strain gradient can be controlled precisely, which is never achieved. By taking advantage of it, the pseudomagnetic field as well as the funnel effect can be accurately designed, which opens an avenue to practical applications for strain engineering in 2D materials. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced plasmid DNA production by enzyme-controlled glucose release and an engineered Escherichia coli.

PubMed

Ramírez, Elisa A; Velázquez, Daniela; Lara, Alvaro R

2016-04-01

To evaluate the combination of a culture medium employing glucoamylase-mediated glucose reléase from a gluco-polysaccharide and an E. coli strain engineered in its glucose transport system for improving plasmid DNA (pDNA) production. The production of pDNA was tested using E. coli DH5α grown in shake-flasks and the recently developed VH33 Δ(recA deoR)-engineered strain, which utilizes glucose more efficiently than wild type strains. Three glucoamylase concentrations for releasing glucose from the polysaccharide carbon source were used: 1, 2 and 3 U l(-1). Both strains reached similar cell densities ranging from 5 to 8.8 g l(-1) under the different conditions. The highest pDNA yields on biomass (YpDNA/X) for both strains were obtained when 3 U enzyme l(-1)were used. Under these conditions, 35 ± 3 mgof pDNA l(-1) were produced by DH5α after 24 h of culture. Under the same conditions, the engineered strain produced 66 ± 1 mgpDNAl(-1) after 20 h. pDNA supercoiled fractionswere close to 80 % for both strains. The pDNA concentration achieved by the engineered E. coli was 89 % higher than that of DH5α. The combination of the engineered strain and enzyme-controlled glucose release is an attractive alternative for pDNA production in shake-flasks.

Genetically engineered Pseudomonas putida X3 strain and its potential ability to bioremediate soil microcosms contaminated with methyl parathion and cadmium.

PubMed

Zhang, Rong; Xu, Xingjian; Chen, Wenli; Huang, Qiaoyun

2016-02-01

A multifunctional Pseudomonas putida X3 strain was successfully engineered by introducing methyl parathion (MP)-degrading gene and enhanced green fluorescent protein (EGFP) gene in P. putida X4 (CCTCC: 209319). In liquid cultures, the engineered X3 strain utilized MP as sole carbon source for growth and degraded 100 mg L(-1) of MP within 24 h; however, this strain did not further metabolize p-nitrophenol (PNP), an intermediate metabolite of MP. No discrepancy in minimum inhibitory concentrations (MICs) to cadmium (Cd), copper (Cu), zinc (Zn), and cobalt (Co) was observed between the engineered X3 strain and its host strain. The inoculated X3 strain accelerated MP degradation in different polluted soil microcosms with 100 mg MP kg(-1) dry soil and/or 5 mg Cd kg(-1) dry soil; MP was completely eliminated within 40 h. However, the presence of Cd in the early stage of remediation slightly delayed MP degradation. The application of X3 strain in Cd-contaminated soil strongly affected the distribution of Cd fractions and immobilized Cd by reducing bioavailable Cd concentrations with lower soluble/exchangeable Cd and organic-bound Cd. The inoculated X3 strain also colonized and proliferated in various contaminated microcosms. Our results suggested that the engineered X3 strain is a potential bioremediation agent showing competitive advantage in complex contaminated environments.

The effect of energy substrates on PHB accumulation of Acidiphilium cryptum DX1-1.

PubMed

Xu, Ai-ling; Xia, Jin-lan; Song, Zhi-wen; Jiang, Peng; Xia, Yan; Wan, Min-xi; Zhang, Rui-yong; Yang, Yi; Liu, Ke-ke

2013-09-01

The effect of glucose and elemental sulfur on the growth and PHB accumulation of Acidiphilium cryptum DX1-1 was investigated. Meanwhile, the differential expressions of 19 genes related with PHB accumulation, sulfur metabolism and carbon fixed in heterotrophy, phytotrophy and mixotrophy were studied by RT-qPCR. The results showed that strain DX1-1 could accumulate PHB with sulfur as the energy substance and atmospheric CO2 as carbon resource. Glucose could improve the growth of strain DX1-1 cultured in medium with sulfur as the energy substance, and almost all the key enzyme-encoding genes related with PHB, sulfur metabolism and carbon fixed were basically up-regulated. PHB polymerase (Arcy_3030), ribulose-bisphosphate carboxylase (Acry_0825), ribulose-phosphate-epimerase (Acry_0022), and cysteine synthase A (Acry_2560) played important role in PHB accumulation, the modified expression of which could influence the PHB yield. With CO2 as carbon resource, the main initial substance of PHB accumulation for strain DX1-1 was acetyl-CoA, instead of acetate with the glucose as the carbon resource. Because of accumulating PHB by fixed atmospheric CO2 while independent of light, A. cryptum DX1-1 may have specifically potential in production of PHB.

Progress of a Cross-Correlation Based Optical Strain Measurement Technique for Detecting Radial Growth on a Rotating Disk

NASA Technical Reports Server (NTRS)

Clem, Michelle M.; Abdul-Aziz, Ali; Woike, Mark R.; Fralick, Gustave C.

2015-01-01

The modern turbine engine operates in a harsh environment at high speeds and is repeatedly exposed to combined high mechanical and thermal loads. The cumulative effects of these external forces lead to high stresses and strains on the engine components, such as the rotating turbine disks, which may eventually lead to a catastrophic failure if left undetected. The operating environment makes it difficult to use conventional strain gauges, therefore, non-contact strain measurement techniques is of interest to NASA and the turbine engine community. This presentation describes one such approach; the use of cross correlation analysis to measure strain experienced by the engine turbine disk with the goal of assessing potential faults and damage.

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening

PubMed Central

Li, Guoliang; Yuan, Hui; Zhang, Hongchao; Li, Yanjun; Xie, Xixian; Chen, Ning

2017-01-01

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5′-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain. PMID:28472077

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening.

PubMed

Fan, Xiaoguang; Wu, Heyun; Li, Guoliang; Yuan, Hui; Zhang, Hongchao; Li, Yanjun; Xie, Xixian; Chen, Ning

2017-01-01

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5'-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain.

Construction and fed-batch cultivation of Candida famata with enhanced riboflavin production.

PubMed

Dmytruk, Kostyantyn; Lyzak, Oleksy; Yatsyshyn, Valentyna; Kluz, Maciej; Sibirny, Vladimir; Puchalski, Czeslaw; Sibirny, Andriy

2014-02-20

Riboflavin (vitamin B2) is an essential nutrition component serving as a precursor of coenzymes FMN and FAD that are involved mostly in reactions of oxidative metabolism. Riboflavin is produced in commercial scale and is used in feed and food industries, and in medicine. The yeast Candida famata (Candida flareri) belongs to the group of so called "flavinogenic yeasts" which overproduce riboflavin under iron limitation. Three genes SEF1, RIB1 and RIB7 coding for a putative transcription factor, GTP cyclohydrolase II and riboflavin synthase, respectively were simultaneously overexpressed in the background of a non-reverting riboflavin producing mutant AF-4, obtained earlier in our laboratory using methods of classical selection (Dmytruk et al. (2011), Metabolic Engineering 13, 82-88). Cultivation conditions of the constructed strain were optimized for shake-flasks and bioreactor cultivations. The constructed strain accumulated up to 16.4g/L of riboflavin in optimized medium in a 7L laboratory bioreactor during fed-batch fermentation. Copyright © 2013 Elsevier B.V. All rights reserved.

Bacterial Interactions with CdSe Quantum Dots

NASA Astrophysics Data System (ADS)

Holden, P.; Nadeau, J. L.; Kumar, A.; Clarke, S.; Priester, J. H.; Stucky, G. D.

2007-12-01

Cadmium selenide quantum dots (QDs) are semiconductor nanoparticles that are manufactured for biomedical imaging, photovoltaics, and other applications. While metallic nanoparticles can be made biotically by bacteria and fungi, and thus occur in nature, the fate of either natural or engineered QDs and relationships to nanoparticle size, conjugate and biotic conditions are mostly unknown. Working with several different bacterial strains and QDs of different sizes and conjugate chemistries, including QDs synthesized by a Fusarium fungal strain, we show that QDs can enter cells through specfic receptor-mediated processes, that QDs are broken down by bacteria during cell association, and that toxicity to cells is much like that imposed by Cd(II) ions. The mechanisms of entry and toxicity are not fully understood, but preliminary evidence suggests that electron transfer between cells and QDs occurs. Also, cell membranes are compromised, indicating oxidative stress is occurring. Results with planktonic and biofilm bacteria are similar, but differently, biofilms tend to accumulate Cd(II) associated with QD treatments.

Biological nitrogen fixation and biomass accumulation within poplar clones as a result of inoculations with diazotrophic endophyte consortia.

PubMed

Knoth, Jenny L; Kim, Soo-Hyung; Ettl, Gregory J; Doty, Sharon L

2014-01-01

Sustainable production of biomass for bioenergy relies on low-input crop production. Inoculation of bioenergy crops with plant growth-promoting endophytes has the potential to reduce fertilizer inputs through the enhancement of biological nitrogen fixation (BNF). Endophytes isolated from native poplar growing in nutrient-poor conditions were selected for a series of glasshouse and field trials designed to test the overall hypothesis that naturally occurring diazotrophic endophytes impart growth promotion of the host plants. Endophyte inoculations contributed to increased biomass over uninoculated control plants. This growth promotion was more pronounced with multi-strain consortia than with single-strain inocula. Biological nitrogen fixation was estimated through (15)N isotope dilution to be 65% nitrogen derived from air (Ndfa). Phenotypic plasticity in biomass allocation and branch production observed as a result of endophyte inoculations may be useful in bioenergy crop breeding and engineering programs. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Simultaneous accumulation of proline and trehalose in industrial baker's yeast enhances fermentation ability in frozen dough.

PubMed

Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

2012-05-01

Freeze tolerance is a necessary characteristic for industrial baker's yeast because frozen-dough baking is one of the key technologies for supplying oven-fresh bakery products to consumers. Both proline and trehalose are known to function as cryoprotectants in yeast cells. In order to enhance the freeze tolerance of yeast cells, we constructed a self-cloning diploid baker's yeast strain with simultaneous accumulation of proline, by expressing the PRO1-I150T allele, encoding the proline-feedback inhibition-less sensitive γ-glutamyl kinase, and trehalose, by disrupting the NTH1 gene, encoding neutral trehalase. The resultant strain retained higher tolerance to oxidative and freezing stresses than did the single proline- or trehalose-accumulating strain. Interestingly, our results suggest that proline and trehalose protect yeast cells from short-term and long-term freezing, respectively. Simultaneous accumulation of proline and trehalose in industrial baker's yeast also enhanced the fermentation ability in the frozen dough compared with the single accumulation of proline or trehalose. These results indicate that baker's yeast that accumulates both proline and trehalose is applicable for frozen-dough baking. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fungal accumulation of metals from building materials during brown rot wood decay.

PubMed

Hastrup, Anne Christine Steenkjær; Jensen, Bo; Jellison, Jody

2014-08-01

This study analyzes the accumulation and translocation of metal ions in wood during the degradation performed by one strain of each of the three brown rot fungi; Serpula lacrymans, Meruliporia incrassata and Coniophora puteana. These fungi species are inhabitants of the built environment where the prevention and understanding of fungal decay is of high priority. This study focuses on the influence of various building materials in relation to fungal growth and metal uptake. Changes in the concentration of iron, manganese, calcium and copper ions in the decayed wood were analyzed by induced coupled plasma spectroscopy and related to wood weight loss and oxalic acid accumulation. Metal transport into the fungal inoculated wood was found to be dependent on the individual strain/species. The S. lacrymans strain caused a significant increase in total iron whereas the concentration of copper ions in the wood appeared decreased after 10 weeks of decay. Wood inoculated with the M. incrassata isolate showed the contrary tendency with high copper accumulation and low iron increase despite similar weight losses for the two strains. However, significantly lower oxalic acid accumulation was recorded in M. incrassata degraded wood. The addition of a building material resulted in increased weight loss in wood degraded by C. puteana in the soil-block test; however, this could not be directly linked specifically to the accumulation of any of the four metals recorded. The accumulation of oxalic acid seemed to influence the iron uptake. The study assessing the influence of the presence of soil and glass in the soil-block test revealed that soil contributed the majority of the metals for uptake by the fungi and contributed to increased weight loss. The varying uptake observed among the three brown rot fungi strains toward the four metals analyzed may be related to the specific non-enzymatic and enzymatic properties including bio-chelators employed by each of the species during wood decay.

A mathematical model to describe the nonlinear elastic properties of the gastrocnemius tendon of chickens.

PubMed

Foutz, T L

1991-03-01

A phenomenological model was developed to describe the nonlinear elastic behavior of the avian gastrocnemius tendon. Quasistatic uniaxial tensile tests were used to apply a deformation and resulting load on the tendon at a deformation rate of 5 mm/min. Plots of deformation versus load indicated a nonlinear loading response. By calculating engineering stress and engineering strain, the experimental data were normalized for tendon shape. The elastic response was determined from stress-strain curves and was found to vary with engineering strain. The response to the applied engineering strain could best be described by a mathematical model that combined a linear function and a nonlinear function. Three parameters in the model were developed to represent the nonlinear elastic behavior of the tendon, thereby allowing analysis of elasticity without prior knowledge of engineering strain. This procedure reduced the amount of data needed for the statistical analysis of nonlinear elasticity.

Increased resveratrol production in wines using engineered wine strains Saccharomyces cerevisiae EC1118 and relaxed antibiotic or auxotrophic selection.

PubMed

Sun, Ping; Liang, Jing-Long; Kang, Lin-Zhi; Huang, Xiao-Yan; Huang, Jia-Jun; Ye, Zhi-Wei; Guo, Li-Qiong; Lin, Jun-Fang

2015-01-01

Resveratrol is a polyphenolic compound with diverse beneficial effects on human health. Red wine is the major dietary source of resveratrol but the amount that people can obtain from wines is limited. To increase the resveratrol production in wines, two expression vectors carrying 4-coumarate: coenzyme A ligase gene (4CL) from Arabidopsis thaliana and resveratrol synthase gene (RS) from Vitis vinifera were transformed into industrial wine strain Saccharomyces cerevisiae EC1118. When cultured with 1 mM p-coumaric acid, the engineered strains grown with and without the addition of antibiotics produced 8.249 and 3.317 mg/L of trans-resveratrol in the culture broth, respectively. Resveratrol content of the wine fermented with engineered strains was twice higher than that of the control, indicating that our engineered strains could increase the production of resveratrol during wine fermentation. © 2015 American Institute of Chemical Engineers.

[Effects of endophytic fungi from Dendrobium officinale on host growth and components metabolism of tissue culture seedlings].

PubMed

Zhu, Bo; Liu, Jing-Jing; Si, Jin-Ping; Qin, Lu-Ping; Han, Ting; Zhao, Li; Wu, Ling-Shang

2016-05-01

The paper aims to study the effects of endophytic fungi from D. officinale cultivated on living trees on growth and components metabolism of tissue culture seedlings. Morphological characteristics and agronomic characters of tissue culture seedlings infected and uninfected by endophytic fungus were observed and measured. Polysaccharides and alcohol-soluble extracts contents were determined by phenol-sulfuric acid method and hot-dipmethod, respectively. Monosacchride composition of polysaccharides and alcohol-soluble extracts components were analyzed by pre-column derivatives HPLC and HPLC method, respectively. It showed that effects of turning to purple of stem nodes could be changed by endophytic fungus. Besides, the endophytic fungus could affect the contents and constitutions of polysaccharides and alcohol-soluble extracts. The strains tested, expect DO34, could promote growth and polysaccharides content of tissue culture seedlings. The strains tested, expect DO12, could promote the accumulation of mannose. Furthermore, DO18, DO19 and DO120 could increase alcohol-soluble extracts. On the basis, four superior strains were selected for mechanism research between endophytic fungus and their hosts and microbiology engineering. Copyright© by the Chinese Pharmaceutical Association.

Cyclic stress effect on stress corrosion cracking of duplex stainless steel in chloride and caustic solutions

NASA Astrophysics Data System (ADS)

Yang, Di

Duplex stainless steel (DSS) is a dual-phase material with approximately equal volume amount of austenite and ferrite. It has both great mechanical properties (good ductility and high tensile/fatigue strength) and excellent corrosion resistance due to the mixture of the two phases. Cyclic loadings with high stress level and low frequency are experienced by many structures. However, the existing study on corrosion fatigue (CF) study of various metallic materials has mainly concentrated on relatively high frequency range. No systematic study has been done to understand the ultra-low frequency (˜10-5 Hz) cyclic loading effect on stress corrosion cracking (SCC) of DSSs. In this study, the ultra-low frequency cyclic loading effect on SCC of DSS 2205 was studied in acidified sodium chloride and caustic white liquor (WL) solutions. The research work focused on the environmental effect on SCC of DSS 2205, the cyclic stress effect on strain accumulation behavior of DSS 2205, and the combined environmental and cyclic stress effect on the stress corrosion crack initiation of DSS 2205 in the above environments. Potentiodynamic polarization tests were performed to investigate the electrochemical behavior of DSS 2205 in acidic NaCl solution. Series of slow strain rate tests (SSRTs) at different applied potential values were conducted to reveal the optimum applied potential value for SCC to happen. Room temperature static and cyclic creep tests were performed in air to illustrate the strain accumulation effect of cyclic stresses. Test results showed that cyclic loading could enhance strain accumulation in DSS 2205 compared to static loading. Moreover, the strain accumulation behavior of DSS 2205 was found to be controlled by the two phases of DSS 2205 with different crystal structures. The B.C.C. ferrite phase enhanced strain accumulation due to extensive cross-slips of the dislocations, whereas the F.C.C. austenite phase resisted strain accumulation due to cyclic strain hardening. Cyclic SSRTs were performed under the conditions that SCC occurs in sodium chloride and WL solutions. Test results show that cyclic stress facilitated crack initiations in DSS 2205. Stress corrosion cracks initiated from the intermetallic precipitates in acidic chloride environment, and the cracks initiated from austenite phase in WL environment. Cold-working has been found to retard the crack initiations induced by cyclic stresses.

The pmr gene, encoding a Ca2+-ATPase, is required for calcium and manganese homeostasis and normal development of hyphae and conidia in Neurospora crassa.

PubMed

Bowman, Barry J; Abreu, Stephen; Johl, Jessica K; Bowman, Emma Jean

2012-11-01

The pmr gene is predicted to encode a Ca(2+)-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr(RIP), in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl(2) (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca(2+)-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca(2+)-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr(RIP) Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.

Effect of Polyhydroxybutyrate (PHB) storage on L-arginine production in recombinant Corynebacterium crenatum using coenzyme regulation.

PubMed

Xu, Meijuan; Qin, Jingru; Rao, Zhiming; You, Hengyi; Zhang, Xian; Yang, Taowei; Wang, Xiaoyuan; Xu, Zhenghong

2016-01-19

Corynebacterium crenatum SYPA 5 is the industrial strain for L-arginine production. Poly-β-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and L-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the L-arginine biosynthesis in C. crenatum SYPA 5. C. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of L-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L L-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key L-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9-3.0 times compared with the parent strain. Accumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time L-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular L-arginine and intracellular PHB simultaneously. All of these have a potential biotechnological application as a strategy for high-yield L-arginine.

Characterization of a Selenium-Tolerant Rhizosphere Strain from a Novel Se-Hyperaccumulating Plant Cardamine hupingshanesis

PubMed Central

Yuan, Linxi; Luo, Lei; Yin, Xuebin

2014-01-01

A novel selenium- (Se-) hyperaccumulating plant, Cardamine hupingshanesis, accumulating Se as a form of SeCys2, was discovered in Enshi, Hubei, China, which could not be explained by present selenocysteine methyltransferase (SMT) theory. However, it is interesting to investigate if rhizosphere bacteria play some roles during SeCys2 accumulation. Here, one Se-tolerant rhizosphere strain, Microbacterium oxydans, was isolated from C. hupingshanesis. Phylogenetic analysis and 16S rRNA gene sequences determined the strain as a kind of Gram positive bacillus and belonged to the family Brevibacterium frigoritolerans. Furthermore, Se tolerance test indicated the strain could grow in extreme high Se level of 15.0 mg Se L−1. When exposed to 1.5 mg Se L−1, SeCys2 was the predominant Se species in the bacteria, consistent with the Se species in C. hupingshanesis. This coincidence might reveal that this strain played some positive effect in SeCys2 accumulation of C. hupingshanesis. Moreover, when exposed to 1.5 mg Se L−1 or 15.0 mg Se L−1, As absorption diminished in the logarithmic phase. In contrast, As absorption increased when exposed to 7.5 mg Se L−1, indicating As metabolism processes could be affected by Se on this strain. The present study provided a sight on the role of rhizosphere bacteria during Se accumulation for Se-hyperaccumulating plant. PMID:25478582

Carbon Monoxide, Hydrogen, and Formate Metabolism during Methanogenesis from Acetate by Thermophilic Cultures of Methanosarcina and Methanothrix Strains.

PubMed

Zinder, S H; Anguish, T

1992-10-01

CO and H(2) have been implicated in methanogenesis from acetate, but it is unclear whether they are directly involved in methanogenesis or electron transfer in acetotrophic methanogens. We compared metabolism of H(2), CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1 and Methanothrix sp. strain CALS-1. M. thermophila accumulated H(2) to partial pressures of 40 to 70 Pa (1 Pa = 0.987 x 10 atm), as has been previously reported for this and other Methanosarcina cultures. In contrast, Methanothrix sp. strain CALS-1 accumulated H(2) to maximum partial pressures near 1 Pa. Growing cultures of Methanothrix sp. strain CALS-1 initially accumulated CO, which reached partial pressures near 0.6 Pa (some CO came from the rubber stopper) during the middle of methanogenesis; this was followed by a decrease in CO partial pressures to less than 0.01 Pa by the end of methanogenesis. Accumulation or consumption of CO by cultures of M. thermophila growing on acetate was not detected. Late-exponential-phase cultures of Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased by flushing with N(2)-CO(2), accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it reached this partial pressure. Cyanide (1 mM) blocked CO consumption but not production. High partial pressures of H(2) (40 kPa) inhibited methanogenesis from acetate by M. thermophila but not by Methanothrix sp. strain CALS-1, and 2 kPa of CO was not inhibitory to M. thermophila but was inhibitory to Methanothrix sp. strain CALS-1. Levels of CO dehydrogenase, hydrogenase, and formate dehydrogenase in Methanothrix sp. strain CALS-1 were 9.1, 0.045, and 5.8 mumol of viologen reduced min mg of protein. These results suggest that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H(2) in M. thermophila and are consistent with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain CALS-1; however, they could also be explained by passive equilibration of CO with a metabolic intermediate.

Carbon Monoxide, Hydrogen, and Formate Metabolism during Methanogenesis from Acetate by Thermophilic Cultures of Methanosarcina and Methanothrix Strains

PubMed Central

Zinder, S. H.; Anguish, T.

1992-01-01

CO and H2 have been implicated in methanogenesis from acetate, but it is unclear whether they are directly involved in methanogenesis or electron transfer in acetotrophic methanogens. We compared metabolism of H2, CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1 and Methanothrix sp. strain CALS-1. M. thermophila accumulated H2 to partial pressures of 40 to 70 Pa (1 Pa = 0.987 × 10-5 atm), as has been previously reported for this and other Methanosarcina cultures. In contrast, Methanothrix sp. strain CALS-1 accumulated H2 to maximum partial pressures near 1 Pa. Growing cultures of Methanothrix sp. strain CALS-1 initially accumulated CO, which reached partial pressures near 0.6 Pa (some CO came from the rubber stopper) during the middle of methanogenesis; this was followed by a decrease in CO partial pressures to less than 0.01 Pa by the end of methanogenesis. Accumulation or consumption of CO by cultures of M. thermophila growing on acetate was not detected. Late-exponential-phase cultures of Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased by flushing with N2-CO2, accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it reached this partial pressure. Cyanide (1 mM) blocked CO consumption but not production. High partial pressures of H2 (40 kPa) inhibited methanogenesis from acetate by M. thermophila but not by Methanothrix sp. strain CALS-1, and 2 kPa of CO was not inhibitory to M. thermophila but was inhibitory to Methanothrix sp. strain CALS-1. Levels of CO dehydrogenase, hydrogenase, and formate dehydrogenase in Methanothrix sp. strain CALS-1 were 9.1, 0.045, and 5.8 μmol of viologen reduced min-1 mg of protein-1. These results suggest that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H2 in M. thermophila and are consistent with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain CALS-1; however, they could also be explained by passive equilibration of CO with a metabolic intermediate. PMID:16348788

Attenuated Escherichia coli strains expressing the colonization factor antigen I (CFA/I) and a detoxified heat-labile enterotoxin (LThK63) enhance clearance of ETEC from the lungs of mice and protect mice from intestinal ETEC colonization and LT-induced fluid accumulation.

PubMed

Byrd, Wyatt; Boedeker, Edgar C

2013-03-15

Although enterotoxigenic Escherichia coli (ETEC) infections are important causes of infantile and traveler's diarrhea there is no licensed vaccine available for those at-risk. Our goal is to develop a safe, live attenuated ETEC vaccine. We used an attenuated E. coli strain (O157:H7, Δ-intimin, Stx1-neg, Stx2-neg) as a vector (ZCR533) to prepare two vaccine strains, one strain expressing colonization factor antigen I (ZCR533-CFA/I) and one strain expressing CFA/I and a detoxified heat-labile enterotoxin (ZCR533-CFA/I+LThK63) to deliver ETEC antigens to mucosal sites in BALB/c mice. Following intranasal and intragastric immunization with the vaccine strains, serum IgG and IgA antibodies were measured to the CFA/I antigen, however, only serum IgG antibodies were detected to the heat-labile enterotoxin. Intranasal administration of the vaccine strains induced respiratory and intestinal antibody responses to the CFA/I and LT antigens, while intragastric administration induced only intestinal antibody responses with no respiratory antibodies detected to the CFA/I and LT antigens. Mice immunized intranasally with the vaccine strains showed enhanced clearance of wild-type (wt) ETEC bacteria from the lungs. Mice immunized intranasally and intragastrically with the vaccine strains were protected from intestinal colonization following oral challenge with ETEC wt bacteria. Mice immunized intragastrically with the ZCR533-CFA/I+LThK63 vaccine strain had less fluid accumulate in their intestine following challenge with ETEC wt bacteria or with purified LT as compared to the sham mice indicating that the immunized mice were protected from LT-induced intestinal fluid accumulation. Thus, mice intragastrically immunized with the ZCR533-CFA/I+LThK63 vaccine strain were able to effectively neutralize the activity of the LT enterotoxin. However, no difference in intestinal fluid accumulation was detected in the mice immunized intranasally with the vaccine strain as compared to the sham mice as the immunized mice induced insufficient intestinal anti-LT antibody to neutralize the activity of the enterotoxin. These results show that our ETEC vaccine induced serum and mucosal antibody responses to CFA/I and LT after mucosal administration which then acted to protect the immunized mice against lung and intestinal colonization, as well as, intestinal fluid accumulation. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of key triacylglycerol biosynthesis processes in rhodococci

DOE PAGES

Amara, Sawsan; Seghezzi, Nicolas; Otani, Hiroshi; ...

2016-04-29

In this study, oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcriptsmore » were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δ atf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.« less

Characterization of key triacylglycerol biosynthesis processes in rhodococci

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amara, Sawsan; Seghezzi, Nicolas; Otani, Hiroshi

In this study, oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcriptsmore » were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δ atf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.« less

Strain gage system evaluation program

NASA Technical Reports Server (NTRS)

Dolleris, G. W.; Mazur, H. J.; Kokoszka, E., Jr.

1978-01-01

A program was conducted to determine the reliability of various strain gage systems when applied to rotating compressor blades in an aircraft gas turbine engine. A survey of current technology strain gage systems was conducted to provide a basis for selecting candidate systems for evaluation. Testing and evaluation was conducted in an F 100 engine. Sixty strain gage systems of seven different designs were installed on the first and third stages of an F 100 engine fan. Nineteen strain gage failures occurred during 62 hours of engine operation, for a survival rate of 68 percent. Of the failures, 16 occurred at blade-to-disk leadwire jumps (84 percent), two at a leadwire splice (11 percent), and one at a gage splice (5 percent). Effects of erosion, temperature, G-loading, and stress levels are discussed. Results of a post-test analysis of the individual components of each strain gage system are presented.

Probabilistic analysis of structures involving random stress-strain behavior

NASA Technical Reports Server (NTRS)

Millwater, H. R.; Thacker, B. H.; Harren, S. V.

1991-01-01

The present methodology for analysis of structures with random stress strain behavior characterizes the uniaxial stress-strain curve in terms of (1) elastic modulus, (2) engineering stress at initial yield, (3) initial plastic-hardening slope, (4) engineering stress at point of ultimate load, and (5) engineering strain at point of ultimate load. The methodology is incorporated into the Numerical Evaluation of Stochastic Structures Under Stress code for probabilistic structural analysis. The illustrative problem of a thick cylinder under internal pressure, where both the internal pressure and the stress-strain curve are random, is addressed by means of the code. The response value is the cumulative distribution function of the equivalent plastic strain at the inner radius.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holland, Steven C.; Artier, Juliana; Miller, Neil T.

Genetic engineering of photosynthetic organisms typically redirects native metabolism towards desirable products, which thereby represent new metabolic sinks. There is limited information on how these modifications impact the evolved mechanisms of photosynthetic energy metabolism and cellular growth. Two engineered strains of Synechocystis sp. PCC 6803 with altered carbon sink capacity were assayed for their photosynthetic and CO 2 concentrating mechanism properties in conditions of high and low inorganic carbon (Ci) availability. In the ΔglgC mutant, glycogen cannot be synthesized and a carbon sink pathway has been effectively removed. The JU547 strain has been engineered by integration of the Pseudomonas syringaemore » ethylene forming enzyme and provides a new sink. When cultured under high carbon conditions, ΔglgC displayed diminished photochemical efficiency, a more reduced NADPH pool, delayed initiation of the Calvin-Benson-Bassham cycle, and impairment of linear and cyclic electron flows. It also exhibited a large decrease in photochemical quenching indicative of the accumulation of Q A-, normally associated with a reduced PQ pool, but appears instead to be the result of an undefined dissipative mechanism to spill excess energy. In the case of carbon sink integration, JU547 displayed slightly more oxidized PQ and NADPH pools and increased rates of cyclic electron flow and an enhanced demand for inorganic carbon as suggested by increase in the expression of the bicarbonate transporter, SbtA. Overall, the results highlight the importance of the native regulatory network of autotrophic metabolism in governing photosynthetic performance and provide cogent examples of both predicable and difficult to predict phenotypic consequences upon installation of new pathways in autotrophs.« less

Enhanced ethanol fermentation by engineered Saccharomyces cerevisiae strains with high spermidine contents.

PubMed

Kim, Sun-Ki; Jo, Jung-Hyun; Jin, Yong-Su; Seo, Jin-Ho

2017-05-01

Construction of robust and efficient yeast strains is a prerequisite for commercializing a biofuel production process. We have demonstrated that high intracellular spermidine (SPD) contents in Saccharomyces cerevisiae can lead to improved tolerance against various fermentation inhibitors, including furan derivatives and acetic acid. In this study, we examined the potential applicability of the S. cerevisiae strains with high SPD contents under two cases of ethanol fermentation: glucose fermentation in repeated-batch fermentations and xylose fermentation in the presence of fermentation inhibitors. During the sixteen times of repeated-batch fermentations using glucose as a sole carbon source, the S. cerevisiae strains with high SPD contents maintained higher cell viability and ethanol productivities than a control strain with lower SPD contents. Specifically, at the sixteenth fermentation, the ethanol productivity of a S. cerevisiae strain with twofold higher SPD content was 31% higher than that of the control strain. When the SPD content was elevated in an engineered S. cerevisiae capable of fermenting xylose, the resulting S. cerevisiae strain exhibited much 40-50% higher ethanol productivities than the control strain during the fermentations of synthetic hydrolysate containing high concentrations of fermentation inhibitors. These results suggest that the strain engineering strategy to increase SPD content is broadly applicable for engineering yeast strains for robust and efficient production of ethanol.

Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications.

PubMed Central

Kim, J; Alizadeh, P; Harding, T; Hefner-Gravink, A; Klionsky, D J

1996-01-01

The accumulation of trehalose is a critical determinant of stress resistance in the yeast Saccharomyces cerevisiae. We have constructed a yeast strain in which the activity of the trehalose-hydrolyzing enzyme, acid trehalase (ATH), has been abolished. Loss of ATH activity was accomplished by disrupting the ATH1 gene, which is essential for ATH activity. The delta ath1 strain accumulated greater levels of cellular trehalose and grew to a higher cell density than the isogenic wild-type strain. In addition, the elevated levels of trehalose in the delta ath1 strain correlated with increased tolerance to dehydration, freezing, and toxic levels of ethanol. The improved resistance to stress conditions exhibited by the delta ath1 strain may make this strain useful in commercial applications, including baking and brewing. PMID:8633854

Degradation of carbazole, dibenzothiophene, and dibenzofuran at low temperature by Pseudomonas sp. strain C3211.

PubMed

Jensen, Anne-Mette; Finster, Kai Waldemar; Karlson, Ulrich

2003-04-01

Pseudomonas sp. strain C3211 was isolated from a temperate climate soil contaminated with creosote. This strain was able to degrade carbazole, dibenzothiophene and dibenzofuran at 10 degrees C with acetone as a co-substrate. When dibenzothiophene was degraded by strain C3211, an orange compound, which absorbed at 472 nm, accumulated in the medium. Degradation of dibenzofuran was followed by accumulation of a yellowish compound, absorbing at 462 nm. The temperature optimum of strain C3211 for degradation of dibenzothiophene and dibenzofuran was at 20 to 21 degrees C, while the maximum temperature for degradation was at 27 degrees C. Both compounds were degraded at 4 degrees C. Degradation at 10 degrees C was faster than degradation at 25 degrees C. This indicates that strain C3211 is adapted to life at low temperatures.

Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides: An Explanation for the High Oleaginicity of Strain WJ11.

PubMed

Tang, Xin; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

2015-01-01

The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA); however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (CDW). We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w) lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD+:isocitrate dehydrogenase and NADP+:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL) to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.

Sensitivity of Texas strains of Ceratocystis fagacearum to triazole fungicides

Treesearch

A. Dan Wilson; L.B. Forse

1997-01-01

Ten geographically diverse Texas strains of the oak wilt fungus Ceratocystis fagacearum were tested in vitro for their sensitivity to five triazole fungicides based on accumulated linear growth, linear growth rates, and dry weight accumulation in response to fungicide concentrations of 0.1 to 600 parts per billion (ppb). None of the triazoles inhibited growth at 0.1...

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

DOE PAGES

Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; ...

2018-02-20

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. Furthermore, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to constructmore » xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.« less

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lian, Jiazhang; Bao, Zehua; Hu, Sumeng

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. Furthermore, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to constructmore » xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.« less

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains.

PubMed

Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; Zhao, Huimin

2018-06-01

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories. © 2018 Wiley Periodicals, Inc.

Effect of material inhomogeneity on the cyclic plastic deformation behavior at the microstructural level: micromechanics-based modeling of dual-phase steel

NASA Astrophysics Data System (ADS)

Paul, Surajit Kumar

2013-07-01

The microstructure of dual-phase (DP) steels typically consists of a soft ferrite matrix with dispersed islands of hard martensite phase. Due to the composite effect of ferrite and martensite, DP steels exhibit a unique combination of strain hardening, strength and ductility. A microstructure-based micromechanical modeling approach is adopted in this work to capture the tensile and cyclic plastic deformation behavior of DP steel. During tensile straining, strain incompatibility between the softer ferrite matrix and the harder martensite phase arises due to a difference in the flow characteristics of these two phases. Microstructural-level inhomogeneity serves as the initial imperfection, triggering strain incompatibility, strain partitioning and finally shear band localization during tensile straining. The local deformation in the ferrite phase is constrained by adjacent martensite islands, which locally results in stress triaxiality development in the ferrite phase. As the martensite distribution varies within the microstructure, the stress triaxiality also varies in a band within the microstructure. Inhomogeneous stress and strain distribution within the softer ferrite phase arises even during small tensile straining because of material inhomogeneity. The magnitude of cyclic plastic deformation within the softer ferrite phase also varies according to the stress distribution in the first-quarter cycle tensile loading. Accumulation of tensile/compressive plastic strain with number of cycles is noted in different locations within the ferrite phase during both symmetric stress and strain controlled cycling. The basic mode of cyclic plastic deformation in an inhomogeneous material is cyclic strain accumulation, i.e. ratcheting. Microstructural inhomogeneity results in cyclic strain accumulation in the aggregate DP material even in symmetric stress cycling.

Osmoregulation in Agrobacterium tumefaciens: accumulation of a novel disaccharide is controlled by osmotic strength and glycine betaine.

PubMed Central

Smith, L T; Smith, G M; Madkour, M A

1990-01-01

We have investigated the mechanism of osmotic stress adaptation (osmoregulation) in Agrobacterium tumefaciens biotype I (salt-tolerant) and biotype II (salt-sensitive) strains. Using natural-abundance 13C nuclear magnetic resonance spectroscopy, we identified all organic solutes that accumulated to significant levels in osmotically stressed cultures. When stressed, biotype I strains (C58, NT1, and A348) accumulated glutamate and a novel disaccharide, beta-fructofuranosyl-alpha-mannopyranoside, commonly known as mannosucrose. In the salt-sensitive biotype II strain K84, glutamate was observed but mannosucrose was not. We speculate that mannosucrose confers the extra osmotic tolerance observed in the biotype I strains. In addition to identifying the osmoregulated solutes that this species synthesizes, we investigated the ability of A. tumefaciens to utilize the powerful osmotic stress protectant glycine betaine when it is supplied in the medium. Results from growth experiments, nuclear magnetic resonance spectroscopy, and a 14C labeling experiment demonstrated that in the absence of osmotic stress, glycine betaine was metabolized, while in stressed cultures, glycine betaine accumulated intracellularly and conferred enhanced osmotic stress tolerance. Furthermore, when glycine betaine was taken up in stressed cells, its accumulation caused the intracellular concentration of mannosucrose to drop significantly. The possible role of osmoregulation of A. tumefaciens in the transformation of plants is discussed. PMID:2254260

Cyclic deformation leads to defect healing and strengthening of small-volume metal crystals

DOE PAGES

Wang, Zhang-Jie; Li, Qing-Jie; Cui, Yi-Nan; ...

2015-10-19

When microscopic and macroscopic specimens of metals are subjected to cyclic loading, the creation, interaction, and accumulation of defects lead to damage, cracking, and failure. We demonstrate that when aluminum single crystals of submicrometer dimensions are subjected to low-amplitude cyclic deformation at room temperature, the density of preexisting dislocation lines and loops can be dramatically reduced with virtually no change of the overall sample geometry and essentially no permanent plastic strain. Furthermore, this “cyclic healing” of the metal crystal leads to significant strengthening through dramatic reductions in dislocation density, in distinct contrast to conventional cyclic strain hardening mechanisms arising frommore » increases in dislocation density and interactions among defects in microcrystalline and macrocrystalline metals and alloys. Our real-time, in situ transmission electron microscopy observations of tensile tests reveal that pinned dislocation lines undergo shakedown during cyclic straining, with the extent of dislocation unpinning dependent on the amplitude, sequence, and number of strain cycles. Those unpinned mobile dislocations moving close enough to the free surface of the thin specimens as a result of such repeated straining are then further attracted to the surface by image forces that facilitate their egress from the crystal. Our results point to a versatile pathway for controlled mechanical annealing and defect engineering in submicrometer-sized metal crystals, thereby obviating the need for thermal annealing or significant plastic deformation that could cause change in shape and/or dimensions of the specimen.« less

Cyclic deformation leads to defect healing and strengthening of small-volume metal crystals

PubMed Central

Wang, Zhang-Jie; Li, Qing-Jie; Cui, Yi-Nan; Liu, Zhan-Li; Ma, Evan; Li, Ju; Sun, Jun; Zhuang, Zhuo; Dao, Ming; Shan, Zhi-Wei; Suresh, Subra

2015-01-01

When microscopic and macroscopic specimens of metals are subjected to cyclic loading, the creation, interaction, and accumulation of defects lead to damage, cracking, and failure. Here we demonstrate that when aluminum single crystals of submicrometer dimensions are subjected to low-amplitude cyclic deformation at room temperature, the density of preexisting dislocation lines and loops can be dramatically reduced with virtually no change of the overall sample geometry and essentially no permanent plastic strain. This “cyclic healing” of the metal crystal leads to significant strengthening through dramatic reductions in dislocation density, in distinct contrast to conventional cyclic strain hardening mechanisms arising from increases in dislocation density and interactions among defects in microcrystalline and macrocrystalline metals and alloys. Our real-time, in situ transmission electron microscopy observations of tensile tests reveal that pinned dislocation lines undergo shakedown during cyclic straining, with the extent of dislocation unpinning dependent on the amplitude, sequence, and number of strain cycles. Those unpinned mobile dislocations moving close enough to the free surface of the thin specimens as a result of such repeated straining are then further attracted to the surface by image forces that facilitate their egress from the crystal. These results point to a versatile pathway for controlled mechanical annealing and defect engineering in submicrometer-sized metal crystals, thereby obviating the need for thermal annealing or significant plastic deformation that could cause change in shape and/or dimensions of the specimen. PMID:26483463

Systems metabolic engineering design: Fatty acid production as an emerging case study

PubMed Central

Tee, Ting Wei; Chowdhury, Anupam; Maranas, Costas D; Shanks, Jacqueline V

2014-01-01

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C6 to C16 are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel-like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high-yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high-yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain-length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain-lengths and functionalities. PMID:24481660

Systems metabolic engineering design: fatty acid production as an emerging case study.

PubMed

Tee, Ting Wei; Chowdhury, Anupam; Maranas, Costas D; Shanks, Jacqueline V

2014-05-01

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C6 to C16 are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel-like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high-yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high-yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain-length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain-lengths and functionalities. © 2014 Wiley Periodicals, Inc.

A viscoplastic study of crack-tip deformation and crack growth in a nickel-based superalloy at elevated temperature

NASA Astrophysics Data System (ADS)

Zhao, L. G.; Tong, J.

Viscoplastic crack-tip deformation behaviour in a nickel-based superalloy at elevated temperature has been studied for both stationary and growing cracks in a compact tension (CT) specimen using the finite element method. The material behaviour was described by a unified viscoplastic constitutive model with non-linear kinematic and isotropic hardening rules, and implemented in the finite element software ABAQUS via a user-defined material subroutine (UMAT). Finite element analyses for stationary cracks showed distinctive strain ratchetting behaviour near the crack tip at selected load ratios, leading to progressive accumulation of tensile strain normal to the crack-growth plane. Results also showed that low frequencies and superimposed hold periods at peak loads significantly enhanced strain accumulation at crack tip. Finite element simulation of crack growth was carried out under a constant Δ K-controlled loading condition, again ratchetting was observed ahead of the crack tip, similar to that for stationary cracks. A crack-growth criterion based on strain accumulation is proposed where a crack is assumed to grow when the accumulated strain ahead of the crack tip reaches a critical value over a characteristic distance. The criterion has been utilized in the prediction of crack-growth rates in a CT specimen at selected loading ranges, frequencies and dwell periods, and the predictions were compared with the experimental results.

Influence of the Strain History on TWIP Steel Deformation Mechanisms in the Deep-Drawing Process

NASA Astrophysics Data System (ADS)

Lapovok, R.; Timokhina, I.; Mester, A.-K.; Weiss, M.; Shekhter, A.

2018-03-01

A study of preferable deformation modes on strain path and strain level in a TWIP steel sheet was performed. Different strain paths were obtained by stretch forming of specimens with various shapes and tensile tests. TEM analysis was performed on samples cut from various locations in the deformed specimens, which had different strain paths and strain levels and the preferable deformation modes were identified. Stresses caused by various strain paths were considered and an analytical analysis performed to identify the preferable deformation modes for the case of single crystal. For a single crystal, in assumption of the absence of lattice rotation, the strain path and the level of accumulated equivalent strain define the preferable deformation mode. For a polycrystalline material, such analytical analysis is not possible due to the large number of grains and, therefore, numerical simulation was employed. For the polycrystalline material, the role of strain path diminishes due to the presence of a large number of grains with random orientations and the effect of accumulated strain becomes dominant. However, at small strains the strain path still defines the level of twinning activity. TEM analysis experimentally confirmed that various deformation modes lead to different deformation strengthening mechanisms.

Influence of the Strain History on TWIP Steel Deformation Mechanisms in the Deep-Drawing Process

NASA Astrophysics Data System (ADS)

Lapovok, R.; Timokhina, I.; Mester, A.-K.; Weiss, M.; Shekhter, A.

2018-06-01

A study of preferable deformation modes on strain path and strain level in a TWIP steel sheet was performed. Different strain paths were obtained by stretch forming of specimens with various shapes and tensile tests. TEM analysis was performed on samples cut from various locations in the deformed specimens, which had different strain paths and strain levels and the preferable deformation modes were identified. Stresses caused by various strain paths were considered and an analytical analysis performed to identify the preferable deformation modes for the case of single crystal. For a single crystal, in assumption of the absence of lattice rotation, the strain path and the level of accumulated equivalent strain define the preferable deformation mode. For a polycrystalline material, such analytical analysis is not possible due to the large number of grains and, therefore, numerical simulation was employed. For the polycrystalline material, the role of strain path diminishes due to the presence of a large number of grains with random orientations and the effect of accumulated strain becomes dominant. However, at small strains the strain path still defines the level of twinning activity. TEM analysis experimentally confirmed that various deformation modes lead to different deformation strengthening mechanisms.

Ratcheting induced cyclic softening behaviour of 42CrMo4 steel

NASA Astrophysics Data System (ADS)

Kreethi, R.; Mondal, A. K.; Dutta, K.

2015-02-01

Ratcheting is an important field of fatigue deformation which happens under stress controlled cyclic loading of materials. The aim of this investigation is to study the uniaxial ratcheting behavior of 42CrMo4 steel in annealed condition, under various applied stresses. In view of this, stress controlled fatigue tests were carried out at room temperature up to 200 cycles using a servo-hydraulic universal testing machine. The results indicate that accumulation of ratcheting strain increases monotonically with increasing maximum applied stress however; the rate of strain accumulation attains a saturation plateau after few cycles. The investigated steel shows cyclic softening behaviour under the applied stress conditions. The nature of strain accumulation and cyclic softening has been discussed in terms of dislocation distribution and plastic damage incurred in the material.

Life prediction technologies for aeronautical propulsion systems

NASA Technical Reports Server (NTRS)

Mcgaw, Michael A.

1987-01-01

Fatigue and fracture problems continue to occur in aeronautical gas turbine engines. Components whose useful life is limited by these failure modes include turbine hot-section blades, vanes and disks. Safety considerations dictate that catastrophic failures be avoided, while economic considerations dictate that noncatastrophic failures occur as infrequently as possible. The design decision is therefore in making the tradeoff between engine performance and durability. The NASA Lewis Research Center has contributed to the aeropropulsion industry in the areas of life prediction technology for 30 years, developing creep and fatigue life prediction methodologies for hot-section materials. Emphasis is placed on the development of methods capable of handling both thermal and mechanical fatigue under severe environments. Recent accomplishments include the development of more accurate creep-fatigue life prediction methods such as the total strain version of Lewis' Strainrange Partitioning (SRP) and the HOST-developed Cyclic Damage Accumulation (CDA) model. Other examples include the Double Damage Curve Approach (DDCA), which provides greatly improved accuracy for cumulative fatigue design rules.

Isolation and identification of poly beta hydroxybutyric acid accumulating bacteria of Staphylococcal sp. and characterization of biodegradable polyester.

PubMed

Roy, Bappaditya; Banerjee, Rajat; Chatterjee, Sumana

2009-04-01

Staphylococcus sp. strain BP/SU1, capable of degrading the biopolymer and utilize it as a source of carbon and energy, was isolated from activated sludge using METABOLIX (MBX D411G). It was found that this strain was capable of accumulating poly(3-hydroxybutyric acid) P(3-HB), as granule poly (3-hydroxybutyric acid), p(3-HB), inclusion bodies when grown under suitable nutrient conditions. These strains could sustain cell growth up to a dry mass of 9.24 g/l with a doubling time of 8 to 10 hr and could accumulate P(3-HB) as granular inclusion bodies to a cell dry weight of more than 12%. P(3-HB) accumulated by this organism was isolated and characterized through NMR, FT-IR spectroscopy, UV Spectroscopy, Mass spectroscopy and Differential Scanning Calorimetry. P(3-HB) granules so isolated showed physical and chemical properties that should be possessed by a superior quality thermoplastic biopolymer.

Engineering Isoprene Synthase Expression and Activity in Cyanobacteria.

PubMed

Chaves, Julie E; Rueda-Romero, Paloma; Kirst, Henning; Melis, Anastasios

2017-12-15

Efforts to heterologously produce quantities of isoprene hydrocarbons (C 5 H 8 ) renewably from CO 2 and H 2 O through the photosynthesis of cyanobacteria face barriers, including low levels of recombinant enzyme accumulation compounded by their slow innate catalytic activity. The present work sought to alleviate the "expression level" barrier upon placing the isoprene synthase (IspS) enzyme in different fusion configurations with the cpcB protein, the highly expressed β-subunit of phycocyanin. Different cpcB*IspS fusion constructs were made, distinguished by the absence or presence of linker amino acids between the two proteins. Composition of linker amino acids was variable with lengths of 7, 10, 16, and 65 amino acids designed to test for optimal activity of the IspS through spatial positioning between the cpcB and IspS. Results showed that fusion constructs with the highly expressed cpcB gene, as the leader sequence, improved transgene expression in the range of 61 to 275-fold over what was measured with the unfused IspS control. However, the specific activity of the IspS enzyme was attenuated in all fusion transformants, possibly because of allosteric effects exerted by the leader cpcB fusion protein. This inhibition varied depending on the nature of the linker amino acids between the cpcB and IspS proteins. In terms of isoprene production, the results further showed a trade-off between specific activity and transgenic enzyme accumulation. For example, the cpcB*L7*IspS strain showed only about 10% the isoprene synthase specific-activity of the unfused cpcB-IspS control, but it accumulated 254-fold more IspS enzyme. The latter more than countered the slower specific activity and made the cpcB*L7*IspS transformant the best isoprene producing strain in this work. Isoprene to biomass yield ratios improved from 0.2 mg g -1 in the unfused cpcB-IspS control to 5.4 mg g -1 in the cpcB*L7*IspS strain, a 27-fold improvement.

Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens.

PubMed

Zhu, Tao; Cheng, Xueqing; Liu, Yuntian; Deng, Zixin; You, Delin

2013-09-01

Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants of Streptomyces aureofaciens and the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0g/l CTC predominated along with 1.2g/l TC, and discovered that chlorination by ctcP (an FADH2-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the ΔctcP strain accumulated about 18.9g/l "clean" TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with low kcat of 0.51±0.01min(-1), while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression of ctcP in S. aureofaciens and improved CTC production to a final titer of 25.9g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Toward Homosuccinate Fermentation: Metabolic Engineering of Corynebacterium glutamicum for Anaerobic Production of Succinate from Glucose and Formate

PubMed Central

Litsanov, Boris; Brocker, Melanie

2012-01-01

Previous studies have demonstrated the capability of Corynebacterium glutamicum for anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (Δcat Δpqo Δpta-ackA ΔldhA), was constructed. Chromosomal integration of the pyruvate carboxylase gene pycP458S into BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, the fdh gene from Mycobacterium vaccae, coding for an NAD+-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase gene gapA. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-gap accumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose). PMID:22389371

[Selection of acetate-tolerant mutants from Escherichia coli DH5alpha and the metabolic properties of mutant DA19].

PubMed

Zhu, Caiqing; Ye, Qin

2003-08-01

Esherichia coli DH5alpha is one of the widely used host strains in genetic engineering. However, foreign gene expression level in this strain is seriously inhibited due to its great sensitivity to the accumulated metabolite, acetate. This study aimed at improving the tolerance of this strain against acetate. Cells of E. coli DH5alpha were irradiated with 60Co, and subsequently continuous culture of the irradiated cells was conducted with gradual increase in the dilution rate and the selective pressure, acetate concentration in the medium. The mutants were picked up on MA plates which contained 5g/L sodium acetate. 5 strains with great improvement in acetate tolerance were obtained, among which DA19 was the best. In cultivation of DA19 in complex media YPS and YPS2G, the cell density, maximum specific growth rate and acetate produced were respectively 1.17 and 1.05, 1.08 and 1.27, and 0.06 and 0.59 times of those of DH5alpha. In a chemically defined medium, the cell density of DA19 was 3.4-fold of that of DH5alpha. The cell density of DA19 in a medium containing 10g/L sodium acetate was comparable to that of DH5alpha in the same medium without the addition of acetate.

Nitrogen and phosphorus removal coupled with carbohydrate production by five microalgae cultures cultivated in biogas slurry.

PubMed

Tan, Fen; Wang, Zhi; Zhouyang, Siyu; Li, Heng; Xie, Youping; Wang, Yuanpeng; Zheng, Yanmei; Li, Qingbiao

2016-12-01

In this study, five microalgae strains were cultured for their ability to survive in biogas slurry, remove nitrogen resources and accumulate carbohydrates. It was proved that five microalgae strains adapted in biogas slurry well without ammonia inhibition. Among them, Chlorella vulgaris ESP-6 showed the best performance on carbohydrate accumulation, giving the highest carbohydrate content of 61.5% in biogas slurry and the highest ammonia removal efficiency and rate of 96.3% and 91.7mg/L/d respectively in biogas slurry with phosphorus and magnesium added. Additionally, the absence of phosphorus and magnesium that can be adverse for biomass accumulation resulted in earlier timing of carbohydrate accumulation and magnesium was firstly recognized and proved as the influence factor for carbohydrate accumulation. Microalgae that cultured in biogas slurry accumulated more carbohydrate in cell, making biogas slurry more suitable medium for the improvement of carbohydrate content, thus can be regarded as a new strategy to accumulate carbohydrate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Macro- to microscale strain transfer in fibrous tissues is heterogeneous and tissue-specific.

PubMed

Han, Woojin M; Heo, Su-Jin; Driscoll, Tristan P; Smith, Lachlan J; Mauck, Robert L; Elliott, Dawn M

2013-08-06

Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35-70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15-25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Induced Protoporphyrin IX Accumulation by the δ-Aminolevulinic Acid in Bacteria and its Potential Use in the Photodynamic Therapy

NASA Astrophysics Data System (ADS)

Brígido-Aparicio, Cyntiha; Ramón-Gallegos, Eva; Arenas-Huertero, Francisco Jesús; Uribe-Hernández, Raúl

2008-08-01

The increasing incident of resistant strains to antibiotic has encouraged the search of new antibacterial treatments, such as the photodynamic therapy. In recent years, photodynamic therapy has demonstrated being a good technology for the treatment of recurrent bacteria infection. PDT presents a hopeful approach to eliminate Gram positive and negative bacteria in immunological compromised patients. This therapy uses a laser in combination with a photosensibilizer in presence of intracellular molecular oxygen. The process generates an effect of phototoxicity in bacterial cells. The aim of this work was to determine the in vitro conditions to accumulate PpIX in effective concentrations in Staphylococcus aureus ATCC25923 and Streptococcus pyogenes, which are responsible of human cutaneous diseases. A cellular suspension of both strains was prepared in TSB to obtain growth in Log-phase, then, the suspensions were adjusted to a final concentration of 2.61×108 cells/mL. The strains were exposed to increasing concentrations from 0 to 160μg/mL of δ-ALA in order to determinate the concentration that induces the biggest accumulation of PpIX. PpIX was measured using the Piomelli method modified for bacteria. The concentration selected was 40 mg/mL of ALA. It was found that in basal concentration of δ-ALA (0 μg/mL) both strains accumulated similar amount of PpIX. In concentrations of 5 mg/mL of δ-ALA it was observed a significant (p<0.001) increment in PpIX concentration. Finally it was realized a kinetic to determinate the optimal accumulation over the time at 0, 5, 10, 15 and 30 min, and 1, 2, 4, 8, 16 and 32 h. It was found that the ideal time for PDT application, in both strains, was 24 h because in smaller times there was not statistically significant difference. The S. aureus ATCC25923 accumulated significantly the biggest concentration of PpIX with regard to S. pyogenes. In conclusion, it was found that the optimal conditions to apply PDT will be to expose both strains to 40 mg/mL of ALA and to irradiate at 24 h after the exposition.

Metabolism of waste engine oil by Pseudomonas species.

PubMed

Salam, Lateef B

2016-06-01

Two bacterial strains phylogenetically identified as Pseudomonas aeruginosa strains RM1 and SK1 displayed extensive degradation ability on waste engine oil (SAE 40W) in batch cultures. Spectrophotometric analysis revealed the presence of various heavy metals such as lead, chromium and nickel in the waste engine oil. The rate of degradation of waste engine oil by the isolates, for the first 12 days and the last 9 days were 66.3, 31.6 mg l -1  day -1   and 69.6, 40.0 mg l -1  day -1 for strains RM1 and SK1, respectively. Gas chromatographic (GC) analyses of residual waste engine oil, revealed that 66.58, 89.06 % and 63.40, 90.75 % of the initial concentration of the waste engine oil were degraded by strains RM1 and SK1 within 12 and 21 days. GC fingerprints of the waste engine oil after 12 days of incubation of strains RM1 and SK1 showed total disappearance of C 15 , C 23 , C 24 , C 25 and C 26 hydrocarbon fractions as well as drastic reductions of C 13 , C 14 , C 16 and PAHs fractions such as C 19 -anthracene and C 22 -pyrene. At the end of 21 days incubation, total disappearance of C 17 -pristane, C 22 -pyrene, one of the C 19 -anthracene and significant reduction of C 18 -phytane (97.2 %, strain RM1; 95.1 %, strain SK1) fractions were observed. In addition, <10 % of Day 0 values of medium fraction ranges C 13 , and C 16 were discernible after 21 days. This study has established the potentials of P. aeruginosa strains RM1 and SK1 in the degradation of aliphatic, aromatic and branched alkane components of waste engine oils.

Strain Gage Signal Interpretation.

DTIC Science & Technology

1986-02-01

blades and vanes in many engines have been collected, played back and examined. The engine types encompass GE’s stable of turbine engines from the small...aeromechanical engineer . 1.3 SUMMARY OF RESULTS Strain gage signals from vibrating rotor blades and vanes were collected, examined, classified, and generalized...turboprops, to turbojets and to the large high bypass turbofan engines . Test conditions include all the phases that are investigated

Functional Requirements for DjlA- and RraA-Mediated Enhancement of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR.

PubMed

Gialama, Dimitra; Delivoria, Dafni Chrysanthi; Michou, Myrsini; Giannakopoulou, Artemis; Skretas, Georgios

2017-06-16

In previous work, we have generated the engineered Escherichia coli strains SuptoxD and SuptoxR, which upon co-expression of the effector genes djlA or rraA, respectively, are capable of suppressing the cytotoxicity caused by membrane protein (MP) overexpression and of producing dramatically enhanced yields for a variety of recombinant MPs of both prokaryotic and eukaryotic origin. Here, we investigated the functional requirements for DnaJ-like protein A (DjlA)- and regulator of ribonuclease activity A (RraA)-mediated enhancement of recombinant MP production in these strains and show that: (i) DjlA and RraA act independently, that is, the beneficial effects of each protein on recombinant MP production occur through a mechanism that does not involve the other, and in a non-additive manner; (ii) full-length and membrane-bound DjlA is required for exerting its beneficial effects on recombinant MP production in E. coli SuptoxD; (iii) the MP production-promoting properties of DjlA in SuptoxD involve the action of the molecular chaperone DnaK but do not rely on the activation of the regulation of capsular synthesis response, a well-established consequence of djlA overexpression; (iv) the observed RraA-mediated effects in E. coli SuptoxR involve the ribonucleolytic activity of RNase E, but not that of its paralogous ribonuclease RNase G; and (v) DjlA and RraA are unique among similar E. coli proteins in their ability to promote bacterial recombinant MP production. These observations provide important clues about the molecular requirements for suppressed toxicity and enhanced MP accumulation in SuptoxD/SuptoxR and will guide future studies aiming to decipher the exact mechanism of DjlA- and RraA-mediated enhancement of recombinant MP production in these strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of calcium-depositing bacteria Agrobacterium tumefaciens and its influence on corrosion of different engineering metals used in cooling water system.

PubMed

Narenkumar, Jayaraman; Sathishkumar, Kuppusamy; Selvi, Adikesavan; Gobinath, Rajagopalan; Murugan, Kadarkarai; Rajasekar, Aruliah

2017-12-01

The present investigation deals with the role of calcium-depositing bacterial community on corrosion of various engineering metals, namely, brass alloy (BS), copper (Cu), stainless steel (SS) and mild steel (MS). Based on the corrosion behavior, Agrobacterium tumefaciens EN13, an aerobic bacterium is identified as calcium-depositing bacteria on engineering metals. The results of the study are supported with biochemical characterization, 16S rRNA gene sequencing, calcium quantification, weight loss, electrochemical (impedance and polarization) and surface analysis (XRD and FTIR) studies. The calcium quantification study showed carbonate precipitation in abiotic system/biotic system as 50 and 700 ppm, respectively. FTIR results too confirmed the accumulation of calcium deposits from the environment on the metal surface by EN13. Electrochemical studies too supported the corrosion mechanism by showing a significant increase in the charge transfer resistance ( R ct ) of abiotic system (44, 33.6, 45, 29.6 Ω cm 2 ) than compared to biotic system (41, 10.1 29 and 25 Ω cm 2 ). Hence, the outcome of the present study confirmed the enhanced bioaccumulation behavior of calcium by the strain, EN13.

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function.

PubMed

Saiki, Asako; Ishida, Yasuaki; Segawa, Shuichi; Hirota, Ryuichi; Nakamura, Takeshi; Kuroda, Akio

2016-05-01

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500 nmol/mg protein-approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.

Collagen incorporation within electrospun conduits reduces lipid oxidation and impacts conduit mechanics.

PubMed

Birthare, Karamveer; Shojaee, Mozhgan; Jones, Carlos Gross; Brenner, James R; Bashur, Chris A

2016-04-21

Modulating the host response, including the accumulation of oxidized lipid species, is important for improving tissue engineered vascular graft (TEVG) viability. Accumulation of oxidized lipids promotes smooth muscle cell (SMC) hyper-proliferation and inhibits endothelial cell migration, which can lead to several of the current challenges for small-diameter TEVGs. Generating biomaterials that reduce lipid oxidation is important for graft survival and this assessment can provide a reliable correlation to clinical situations. In this study, we determined the collagen to poly(ε-caprolactone) (PCL) ratio required to limit the production of pro-inflammatory species, while maintaining the required mechanical strength for the graft. Electrospun conduits were prepared from 0%, 10%, and 25% blends of collagen/PCL (w/w) and implanted in the rat peritoneal cavity for four weeks. The results showed that adding collagen to the PCL conduits reduced the accumulation of oxidized lipid species within the implanted conduits. In addition, the ratio of collagen had a significant impact on the recruited cell phenotype and construct mechanics. All conduits exhibited greater than 44% yield strain and sufficient tensile strength post-implantation. In conclusion, these results demonstrate that incorporating collagen into synthetic electrospun scaffolds, both 10% and 25% blend conditions, appears to limit the pro-inflammatory characteristics after in vivo implantation.

Damage Assessment of Heat Resistant Steels through Electron BackScatter Diffraction Strain Analysis under Creep and Creep-Fatigue Conditions

NASA Astrophysics Data System (ADS)

Fujiyama, Kazunari; Kimachi, Hirohisa; Tsuboi, Toshiki; Hagiwara, Hiroyuki; Ogino, Shotaro; Mizutani, Yoshiki

EBSD(Electron BackScatter Diffraction) analyses were conducted for studying the quantitative microstructural metrics of creep and creep-fatigue damage for austenitic SUS304HTB boiler tube steel and ferritic Mod.9Cr piping steel. KAM(Kernel Average Misorientation) maps and GOS(Grain Orientation Spread) maps were obtained for these samples and the area averaged values KAMave and GOSave were obtained. While the increasing trends of these misorientation metrics were observed for SUS304HTB steel, the decreasing trends were observed for damaged Mod.9Cr steel with extensive recovery of subgrain structure. To establish more universal parameter representing the accumulation of damage to compensate these opposite trends, the EBSD strain parameters were introduced for converting the misorientation changes into the quantities representing accumulated permanent strains during creep and creep-fatigue damage process. As KAM values were dependent on the pixel size (inversely proportional to the observation magnification) and the permanent strain could be expressed as the shear strain which was the product of dislocation density, Burgers vector and dislocation movement distance, two KAM strain parameters MεKAMnet and MεδKAMave were introduced as the sum of product of the noise subtracted KAMnet and the absolute change from initial value δKAMave with dislocation movement distance divided by pixel size. MεδKAMave parameter showed better relationship both with creep strain in creep tests and accumulated creep strain range in creep-fatigue tests. This parameter can be used as the strain-based damage evaluation and detector of final failure.

Spring 2014 Internship Diffuser Data Analysis

NASA Technical Reports Server (NTRS)

Laigaie, Robert T.; Ryan, Harry M.

2014-01-01

J-2X engine testing on the A-2 test stand at the NASA John C. Stennis Space Center (SSC) has recently concluded. As part of that test campaign, the engine was operated at lower power levels in support of expanding the use of J-2X to other missions. However, the A-2 diffuser was not designed for engine testing at the proposed low power levels. To evaluate the risk of damage to the diffuser, computer simulations were created of the rocket engine exhaust plume inside the 50ft long, water-cooled, altitude-simulating diffuser. The simulations predicted that low power level testing would cause the plume to oscillate in the lower sections of the diffuser. This can possibly cause excessive vibrations, stress, and heat transfer from the plume to the diffuser walls. To understand and assess the performance of the diffuser during low power level engine testing, nine accelerometers and four strain gages were installed around the outer surface of the diffuser. The added instrumentation also allowed for the verification of the rocket exhaust plume computational model. Prior to engine hot-fire testing, a diffuser water-flow test was conducted to verify the proper operation of the newly installed instrumentation. Subsequently, two J-2X engine hot-fire tests were completed. Hot-Fire Test 1 was 11.5 seconds in duration, and accelerometer and strain data verified that the rocket engine plume oscillated in the lower sections of the diffuser. The accelerometers showed very different results dependent upon location. The diffuser consists of four sections, with Section 1 being closest to the engine nozzle and Section 4 being farthest from the engine nozzle. Section 1 accelerometers showed increased amplitudes at startup and shutdown, but low amplitudes while the diffuser was started. Section 3 accelerometers showed the opposite results with near zero G amplitudes prior to and after diffuser start and peak amplitudes to +/- 100G while the diffuser was started. Hot-Fire Test 1 strain gages showed different data dependent on section. Section 1 strains were small, and were in the range of 50 to 150 microstrain, which would result in stresses from 1.45 to 4.35 ksi. The yield stress of the material, A-285 Grade C Steel, is 29.7 ksi. Section 4 strain gages showed much higher values with strains peaking at 1600 microstrain. This strain corresponds to a stress of 46.41 ksi, which is in excess of the yield stress, but below the ultimate stress of 55 to 75 ksi. The decreased accelerations and strain in Section 1, and the increased accelerations and strain in Sections 3 and 4 verified the computer simulation prediction of increased plume oscillations in the lower sections of the diffuser. Hot-Fire Test 2 ran for a duration of 125 seconds. The engine operated at a slightly higher power level than Hot-Fire Test 1 for the initial 35 seconds of the test. After 35 seconds the power level was lowered to Hot-Fire Test 1 levels. The acceleration and strain data for Hot-Fire Test 2 was similar during the initial part of the test. However, just prior to the engine being lowered to the Hot-Fire Test 1 power level, the strain gage data in Section 4 showed a large decrease to strains near zero microstrain from their peak at 1500 microstrain. Future work includes further strain and acceleration data analysis and evaluation.

Engineering Rhodosporidium toruloides with a membrane transporter facilitates production and separation of carotenoids and lipids in a bi-phasic culture.

PubMed

Lee, Jaslyn J L; Chen, Liwei; Cao, Bin; Chen, Wei Ning

2016-01-01

The oleaginous yeast Rhodosporidium toruloides has great biotechnological potential. It accumulates a high amount of lipids which can be used for biofuels and also produces carotenoids which are valuable in the food and pharmaceutical industry. However, the location of these two hydrophobic products in the cell membrane prohibits its efficient harvesting and separation. Here, the transporter Pdr10 was engineered into R. toruloides and cultured in two-phase media containing oil. This enabled the production and in situ export of carotenoids into the oil and concurrent separation from intracellular lipids in the cells. When Pdr10 strain was cultured in the two-phase media, carotenoids and fatty acids yield increased from 1.9 to 2.9 μg/mg and 0.07 to 0.09 mg/mg, respectively. A total of 1.8 μg/mg carotenoids was exported by Pdr10 strain, as compared to 0.3 μg/mg in the wild type. In the Pdr10 strain, the composition of carotenoids and fatty acid it produced also changed. Torulene became the major carotene produced instead of torularhodin. Also, the unsaturated fatty acid C18:2 became the dominant fatty acid produced instead of the saturated C16:0, which was similar to the grape seed oil used in the two-phase media. This indicated that oil was being consumed by the cells, which was supported by the increased intracellular glycerol levels detected by gas chromatography-mass spectrometry (GC-MS). Our approach represents an easy and greener extraction method which could serve to increase the yield and facilitate separation of carotenoids and fatty acids.

Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate.

PubMed

Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

2015-01-01

Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 .

Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate

PubMed Central

Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

2015-01-01

Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. Results: Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. Conclusion: The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 . PMID:26306151

Quantitative analysis and predictive engineering of self-rolling of nanomembranes under anisotropic mismatch strain

NASA Astrophysics Data System (ADS)

Chen, Cheng; Song, Pengfei; Meng, Fanchao; Li, Xiao; Liu, Xinyu; Song, Jun

2017-12-01

The present work presents a quantitative modeling framework for investigating the self-rolling of nanomembranes under different lattice mismatch strain anisotropy. The effect of transverse mismatch strain on the roll-up direction and curvature has been systematically studied employing both analytical modeling and numerical simulations. The bidirectional nature of the self-rolling of nanomembranes and the critical role of transverse strain in affecting the rolling behaviors have been demonstrated. Two fabrication strategies, i.e., third-layer deposition and corner geometry engineering, have been proposed to predictively manipulate the bidirectional rolling competition of strained nanomembranes, so as to achieve controlled, unidirectional roll-up. In particular for the strategy of corner engineering, microfabrication experiments have been performed to showcase its practical application and effectiveness. Our study offers new mechanistic knowledge towards understanding and predictive engineering of self-rolling of nanomembranes with improved roll-up yield.

Investigation of Linum flavum (L.) Hairy Root Cultures for the Production of Anticancer Aryltetralin Lignans.

PubMed

Renouard, Sullivan; Corbin, Cyrielle; Drouet, Samantha; Medvedec, Barbara; Doussot, Joël; Colas, Cyril; Maunit, Benoit; Bhambra, Avninder S; Gontier, Eric; Jullian, Nathalie; Mesnard, François; Boitel, Michèle; Abbasi, Bilal Haider; Arroo, Randolph R J; Lainé, Eric; Hano, Christophe

2018-03-26

Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.

Characterizing Strain Variation in Engineered E. coli Using a Multi-Omics-Based Workflow

DOE PAGES

Brunk, Elizabeth; George, Kevin W.; Alonso-Gutierrez, Jorge; ...

2016-05-19

Understanding the complex interactions that occur between heterologous and native biochemical pathways represents a major challenge in metabolic engineering and synthetic biology. We present a workflow that integrates metabolomics, proteomics, and genome-scale models of Escherichia coli metabolism to study the effects of introducing a heterologous pathway into a microbial host. This workflow incorporates complementary approaches from computational systems biology, metabolic engineering, and synthetic biology; provides molecular insight into how the host organism microenvironment changes due to pathway engineering; and demonstrates how biological mechanisms underlying strain variation can be exploited as an engineering strategy to increase product yield. As a proofmore » of concept, we present the analysis of eight engineered strains producing three biofuels: isopentenol, limonene, and bisabolene. Application of this workflow identified the roles of candidate genes, pathways, and biochemical reactions in observed experimental phenomena and facilitated the construction of a mutant strain with improved productivity. The contributed workflow is available as an open-source tool in the form of iPython notebooks.« less

Strain-Specific Induction of Endometrial Periglandular Fibrosis in Mice Exposed During Adulthood to the Endocrine Disrupting Chemical Bisphenol A

PubMed Central

Kendziorski, Jessica A.; Belcher, Scott M.

2015-01-01

The aim of this study was to compare effects of bisphenol A (BPA) on collagen accumulation in uteri of two mouse strains. Adult C57Bl/6N and CD-1 mice were exposed to dietary BPA (0.004–40 mg/kg/day) or 17α-ethinyl estradiol (0.00002–0.001 mg/kg/day) as effect control. An equine endometrosis-like phenotype with increased gland nesting and periglandular collagen accumulation was characteristic of unexposed C57Bl/6N, but not CD-1, endometrium. BPA non-monotonically increased gland nest density and periglandular collagen accumulation in both strains. Increased collagen I and III expression, decreased matrix metalloproteinase 2 (MMP2) and MMP14 expression, and increased immune response were associated with the endometrosis phenotype in the C57Bl/6N strain and the 30 ppm BPA CD-1 group. The association between the pro-collagen shift in increased collagen expression and decreased MMP2 expression and activity implies that strain differences and BPA exposure salter regulation of endometrial remodeling and contributes to increased fibrosis, a component of several human uterine diseases. PMID:26307436

Enhancing activity of antibiotics against Staphylococcus aureus: Zanthoxylum capense constituents and derivatives.

PubMed

Cabral, Vanessa; Luo, Xuan; Junqueira, Elisabete; Costa, Sofia S; Mulhovo, Silva; Duarte, Aida; Couto, Isabel; Viveiros, Miguel; Ferreira, Maria-José U

2015-04-15

Six compounds (1-6), isolated from the methanol extract of the roots of the African medicinal plant Zanthoxylum capense Thunb. (Rutaceae), and seven ester derivatives (7-13) were evaluated for their antibacterial activities and modulatory effects on the MIC of antibiotics (erythromycin, oxacillin, and tetracycline) and ethidium bromide (EtBr) against a Staphylococcus aureus reference strain (ATCC 6538). Using the same model, compounds 1-13 were also assessed for their potential as efflux pump inhibitors by a fluorometric assay that measures the accumulation of the broad range efflux pump substrate EtBr. Compounds 8 and 11 were further evaluated for their antibacterial, modulatory and EtBr accumulation effects against four additional S. aureus strains, which included two clinical methicillin-resistant S. aureus (MRSA) strains. Compounds (1-13) have not shown antibacterial activity at the concentration ranges tested. When evaluated against S. aureus ATCC 6538, oxychelerythrine (1) a benzophenanthridine alkaloid, showed the highest modulatory activity enhancing the susceptibility of this strain to all the tested antibiotics from two to four-fold. Ailanthoidiol diacetate (8) and ailanthoidiol di-2-ethylbutanoate (11) were also good modulators when combined with EtBr, increasing the bacteria susceptibility by four and two-fold, respectively. In the EtBr accumulation assay, using ATCC 6538 strain, the phenylpropanoid (+)-ailanthoidiol (6) and most of its ester derivatives (8-11) exhibited higher activity than the positive control verapamil. The highest effects were found for compounds 8 and 11 that also increased the accumulation of EtBr, using S. aureus ATCC 25923 as model. Furthermore, both compounds (8, 11) were able to enhance the ciprofloxacin activity against the MRSA clinical strains tested, causing a reduction of the antibiotic MIC values from two to four-fold. The EtBr accumulation assay revealed that this modulation activity was not due to an inhibition of efflux pumps mechanism. These results suggested that Z. capense constituents may be valuable as leads for restoring antibiotic activity against MRSA strains. Copyright © 2015 Elsevier GmbH. All rights reserved.

Accumulation of Polyphosphate in Lactobacillus spp. and Its Involvement in Stress Resistance

PubMed Central

Alcántara, Cristina; Blasco, Amalia; Zúñiga, Manuel

2014-01-01

Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus, a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus, 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase (ppk) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk-disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms. PMID:24375133

Anisotropic-Strain-Induced Band Gap Engineering in Nanowire-Based Quantum Dots.

PubMed

Francaviglia, Luca; Giunto, Andrea; Kim, Wonjong; Romero-Gomez, Pablo; Vukajlovic-Plestina, Jelena; Friedl, Martin; Potts, Heidi; Güniat, Lucas; Tütüncüoglu, Gözde; Fontcuberta I Morral, Anna

2018-04-11

Tuning light emission in bulk and quantum structures by strain constitutes a complementary method to engineer functional properties of semiconductors. Here, we demonstrate the tuning of light emission of GaAs nanowires and their quantum dots up to 115 meV by applying strain through an oxide envelope. We prove that the strain is highly anisotropic and clearly results in a component along the NW longitudinal axis, showing good agreement with the equations of uniaxial stress. We further demonstrate that the strain strongly depends on the oxide thickness, the oxide intrinsic strain, and the oxide microstructure. We also show that ensemble measurements are fully consistent with characterizations at the single-NW level, further elucidating the general character of the findings. This work provides the basic elements for strain-induced band gap engineering and opens new avenues in applications where a band-edge shift is necessary.

Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides.

PubMed

Zhao, Lina; Cánovas-Márquez, José T; Tang, Xin; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

2016-02-01

Fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, NADPH, and the precursor of fatty acids, acetyl-CoA, which is generated in the cytosol being produced by ATP: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. This transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (Fig. 1). The role of malate transporter in lipid accumulation in oleaginous fungi is not fully understood, however. Therefore, the expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70 % (from 13 to 22 % dry cell weight, CDW), whereas the lipid content in mt knockout mutant decreased about 27 % (from 13 to 9.5 % CDW) compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain and increased in the mt knockout strain compared with the wild-type strain. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.

Hydrocarbon productivities in different Botryococcus strains: comparative methods in product quantification.

PubMed

Eroglu, Ela; Okada, Shigeru; Melis, Anastasios

2011-08-01

Six different strains of the green microalgae Botryococcus belonging to the A-race or B-race, accumulating alkadiene or botryococcene hydrocarbons, respectively, were compared for biomass and hydrocarbon productivities. Biomass productivity was assessed gravimetrically upon strain growth in the laboratory under defined conditions. Hydrocarbon productivities were measured by three different and independent experimental approaches, including density equilibrium of the intact cells and micro-colonies, spectrophotometric analysis of hydrocarbon extracts, and gravimetric quantitation of eluted hydrocarbons. All three hydrocarbon-quantitation methods yielded similar results for each of the strains examined. The B-race microalgae Botryococcus braunii var. Showa and Kawaguchi-1 constitutively accumulated botryococcene hydrocarbons equivalent to 30% and 20%, respectively, of their overall biomass. The A-race microalgae Botryococcus braunii, varieties Yamanaka, UTEX 2441 and UTEX LB572 constitutively accumulated alkadiene hydrocarbons ranging from 14% to 13% and 10% of their overall biomass, respectively. Botryococcus sudeticus (UTEX 2629), a morphologically different green microalga, had the lowest hydrocarbon accumulation, equal to about 3% of its overall biomass. Results validate the density equilibrium and spectrophotometric analysis methods in the quantitation of botryococcene-type hydrocarbons. These analytical advances will serve in the screening and selection of B. braunii and of other microalgae in efforts to identify those having a high hydrocarbon content for use in commercial applications.

Accumulation of astaxanthin by a new Haematococcus pluvialis strain BM1 from the white sea coastal rocks (Russia).

PubMed

Chekanov, Konstantin; Lobakova, Elena; Selyakh, Irina; Semenova, Larisa; Sidorov, Roman; Solovchenko, Alexei

2014-08-15

We report on a novel arctic strain BM1 of a carotenogenic chlorophyte from a coastal habitat with harsh environmental conditions (wide variations in solar irradiance, temperature, salinity and nutrient availability) identified as Haematococcus pluvialis Flotow. Increased (25‰) salinity exerted no adverse effect on the growth of the green BM1 cells. Under stressful conditions (high light, nitrogen and phosphorus deprivation), green vegetative cells of H. pluvialis BM1 grown in BG11 medium formed non-motile palmelloid cells and, eventually, hematocysts capable of a massive accumulation of the keto-carotenoid astaxanthin with a high nutraceutical and therapeutic potential. Routinely, astaxanthin was accumulated at the level of 4% of the cell dry weight (DW), reaching, under prolonged stress, 5.5% DW. Astaxanthin was predominantly accumulated in the form of mono- and diesters of fatty acids from C16 and C18 families. The palmelloids and hematocysts were characterized by the formation of red-colored cytoplasmic lipid droplets, increasingly large in size and number. The lipid droplets tended to merge and occupied almost the entire volume of the cell at the advanced stages of stress-induced carotenogenesis. The potential application of the new strain for the production of astaxanthin is discussed in comparison with the H. pluvialis strains currently employed in microalgal biotechnology.

Cyclic Axial-Torsional Deformation Behavior of a Cobalt-Base Superalloy

NASA Technical Reports Server (NTRS)

Bonacuse, Peter J.; Kalluri, Sreeramesh

1995-01-01

The cyclic, high-temperature deformation behavior of a wrought cobalt-base super-alloy, Haynes 188, is investigated under combined axial and torsional loads. This is accomplished through the examination of hysteresis loops generated from a biaxial fatigue test program. A high-temperature axial, torsional, and combined axial-torsional fatigue database has been generated on Haynes 188 at 760 C. Cyclic loading tests have been conducted on uniform gage section tubular specimens in a servohydraulic axial-torsional test rig. Test control and data acquisition were accomplished with a minicomputer. The fatigue behavior of Haynes 188 at 760 C under axial, torsional, and combined axial-torsional loads and the monotonic and cyclic deformation behaviors under axial and torsional loads have been previously reported. In this paper, the cyclic hardening characteristics and typical hysteresis loops in the axial stress versus axial strain, shear stress ,versus engineering shear strain, axial strain versus engineering shear strain. and axial stress versus shear stress spaces are presented for cyclic in-phase and out-of-phase axial-torsional tests. For in-phase tests, three different values of the proportionality constant lambda (the ratio of engineering shear strain amplitude to axial strain amplitude, are examined, viz. 0.86, 1.73, and 3.46. In the out-of-phase tests, three different values of the phase angle, phi (between the axial and engineering shear strain waveforms), are studied, viz., 30, 60, and 90 degrees with lambda equals 1.73. The cyclic hardening behaviors of all the tests conducted on Haynes 188 at 760 C are evaluated using the von Mises equivalent stress-strain and the maximum shear stress-maximum engineering shear strain (Tresca) curves. Comparisons are also made between the hardening behaviors of cyclic axial, torsional, and combined in-phase (lambda = 1.73 and phi = 0) and out-of-phase (lambda = 1.73 and phi = 90') axial-torsional fatigue tests. These comparisons are accomplished through simple Ramberg-Osgood type stress-strain functions for cyclic, axial stress-strain and shear stress-engineering shear strain curves.

Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion

USDA-ARS?s Scientific Manuscript database

Saccharomyces physiology and fermentation related properties vary broadly among industrial strains. In this study, six industrial strains of varied genetic background were engineered to ferment xylose. Aerobic growth rates on xylose were 0.040 h**-1 to 0.167 h**-1. Fermentation of xylose, glucose/xy...

ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

DOE PAGES

Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.; ...

2016-03-03

With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsicmore » physiological constraints—in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. In conclusion, by combining the push/pull/block strategies, we significantly improved mevalonate production. We anticipate that this strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae convert feedstocks to acetyl-CoA derived fuels and chemicals.« less

ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.

With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsicmore » physiological constraints—in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. In conclusion, by combining the push/pull/block strategies, we significantly improved mevalonate production. We anticipate that this strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae convert feedstocks to acetyl-CoA derived fuels and chemicals.« less

Engineering calcium oxalate crystal formation in Arabidopsis

USDA-ARS?s Scientific Manuscript database

Many plants accumulate crystals of calcium oxalate. Just how these crystals form remains unknown. To gain insight into the mechanisms regulating calcium oxalate crystal formation, a crystal engineering approach was initiated utilizing the non-crystal accumulating plant, Arabidopsis. The success of t...

Screening high oleaginous Chlorella strains from different climate zones.

PubMed

Xu, Jin; Hu, Hanhua

2013-09-01

In outdoor cultivation, screening strains adapted to a wide temperature range or suitable strains for different environmental temperatures is of great importance. In this study, triacylglycerol (TAG) content of 23 oil-producing Chlorella strains from different climate zones were analyzed by thin layer chromatography. Four selected Chlorella strains (NJ-18, NJ-7, NMX35N and NMX139N) with rather high TAG content had higher total lipid content compared with Chlorella vulgaris SAG 211-11b. In particular, NJ-18 displayed the highest TAG productivity among the four high oil-producing Chlorella strains. Accumulation of TAGs in strain NMX35N changed a little from 30 to 40°C, showing a desirable characteristic of accumulating TAGs at high temperatures. Our results demonstrated that NJ-18 and NMX35N had suitable fatty acid profiles and good adaption to low and high temperatures respectively. Therefore, cultivation of the two Chlorella strains together might be a good option for economic biodiesel production during the whole seasons of the year. Copyright © 2013 Elsevier Ltd. All rights reserved.

Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol.

PubMed

Liu, Yiqi; Tu, Xiaohu; Xu, Qin; Bai, Chenxiao; Kong, Chuixing; Liu, Qi; Yu, Jiahui; Peng, Qiangqiang; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2018-01-01

As a promising one-carbon renewable substrate for industrial biotechnology, methanol has attracted much attention. However, engineering of microorganisms for industrial production of pharmaceuticals using a methanol substrate is still in infancy. In this study, the methylotrophic yeast Pichia pastoris was used to produce anti-hypercholesterolemia pharmaceuticals, lovastatin and its precursor monacolin J, from methanol. The biosynthetic pathways for monacolin J and lovastatin were first assembled and optimized in single strains using single copies of the relevant biosynthetic genes, and yields of 60.0mg/L monacolin J and 14.4mg/L lovastatin were obtained using methanol following pH controlled monoculture. To overcome limitations imposed by accumulation of intermediates and metabolic stress in monoculture, approaches using pathway splitting and co-culture were developed. Two pathway splitting strategies for monacolin J, and four for lovastatin were tested at different metabolic nodes. Biosynthesis of monacolin J and lovastatin was improved by 55% and 71%, respectively, when the upstream and downstream modules were separately accommodated in two different fluorescent strains, split at the metabolic node of dihydromonacolin L. However, pathway distribution at monacolin J blocked lovastatin biosynthesis in all designs, mainly due to its limited ability of crossing cellular membranes. Bioreactor fermentations were tested for the optimal co-culture strategies, and yields of 593.9mg/L monacolin J and 250.8mg/L lovastatin were achieved. This study provides an alternative method for production of monacolin J and lovastatin and reveals the potential of a methylotrophic yeast to produce complicated pharmaceuticals from methanol. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Compatibility of a wild type and its genetically modified Sinorhizobium strain with two mycorrhizal fungi on Medicago species as affected by drought stress.

PubMed

Vázquez, M M.; Azcón, R; Barea, J M.

2001-07-01

The effect of double inoculation with two strains of Sinorhizobium meliloti [the wild type (WT) strain GR4 and its genetically modified (GM) derivative GR4(pCK3)], and two species of arbuscular mycorrhizal (AM) fungi (Glomus deserticola and Glomus intraradices) was examined in a microcosm system on three species of Medicago (M. nolana, M. rigidula, M. rotata). Two water regimes (80 and 100% water holding capacity, WHC) were assayed. The efficiency of each AM fungus increasing plant growth, nutrient content, nodulation and water-stress tolerance was related to the Sinorhizobium strains and Medicago species. This indicates selective and specific compatibilities between microsymbionts and the common host plant. Differential effects of the mycorrhizal isolates were not associated with their colonizing ability. Nodulation and mycorrhizal dependency (MD) changed in each plant genotype in accordance with the Sinorhizobium strain and AM fungi involved. Generally, Medicago sp. MD decreased under water-stress conditions even when these conditions did not affect AM colonization (%). Proline accumulation in non-mycorrhizal plant leaves was increased by water stress, except in M. rotata plants. Differences in proline accumulation in AM-colonized plants suggest that both the AM fungus and the Sinorhizobium strain were able to induce different degrees of osmotic adjustment. Mycorrhizal plants nodulated by the WT strain accumulated more proline in M. rigidula and M. rotata under water stress than non-mycorrhizal plants. Conversely, mycorrhizal plants nodulated by the GM strain accumulated less proline in response to both AM colonization and drought. These results indicated changes in the synthesis of this nitrogenous osmoregulator product associated with microbial inoculation and drought tolerance. Mycorrhizal plants nodulated by the GM Sinorhizobium strain seem to suffer less from the detrimental effect of water stress, since under water limitation relative plant growth, percentage of AM colonization, root dry weight and the highest R/S ratio remained the same. The fact that GM nodulated plants are better adapted to drought stress could be of practical interest and the management of GM microorganism inoculation may be crucial for biotechnological approaches to improving crop yield in dry environments.

An Assessment of Engineered Calcium Oxalate Crystal Formation on Plant Growth and Development as a Step toward Evaluating Its Use to Enhance Plant Defense.

PubMed

Nakata, Paul A

2015-01-01

The establishment of new approaches to control chewing insects has been sought not only for direct use in reducing crop loss but also in managing resistance to the pesticides already in use. Engineered formation of calcium oxalate crystals is a potential strategy that could be developed to fulfill both these needs. As a step toward this development, this study investigates the effects of transforming a non-calcium oxalate crystal accumulating plant, Arabidopsis thaliana, into a crystal accumulating plant. Calcium oxalate crystal accumulating A. thaliana lines were generated by ectopic expression of a single bacterial gene encoding an oxalic acid biosynthetic enzyme. Biochemical and cellular studies suggested that the engineered A. thaliana lines formed crystals of calcium oxalate in a manner similar to naturally occurring crystal accumulating plants. The amount of calcium oxalate accumulated in leaves also reached levels similar to those measured in the leaves of Medicago truncatula in which the crystals are known to play a defensive role. Visual inspection of the different engineered lines, however, suggested a phenotypic consequence on plant growth and development with higher calcium oxalate concentrations. The restoration of a near wild-type plant phenotype through an enzymatic reduction of tissue oxalate supported this observation. Overall, this study is a first to provide initial insight into the potential consequences of engineering calcium oxalate crystal formation in non-crystal accumulating plants.

rRNA and Poly-β-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast and Famine Cycles

PubMed Central

Frigon, Dominic; Muyzer, Gerard; van Loosdrecht, Mark; Raskin, Lutgarde

2006-01-01

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-β-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage. PMID:16597926

rRNA and poly-beta-hydroxybutyrate dynamics in bioreactors subjected to feast and famine cycles.

PubMed

Frigon, Dominic; Muyzer, Gerard; van Loosdrecht, Mark; Raskin, Lutgarde

2006-04-01

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-beta-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.

Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis.

PubMed

Križanović, Stela; Butorac, Ana; Mrvčić, Jasna; Krpan, Maja; Cindrić, Mario; Bačun-Družina, Višnja; Stanzer, Damir

2015-06-01

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate.

PubMed

Guzik, Maciej W; Kenny, Shane T; Duane, Gearoid F; Casey, Eoin; Woods, Trevor; Babu, Ramesh P; Nikodinovic-Runic, Jasmina; Murray, Michael; O'Connor, Kevin E

2014-05-01

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1.

PubMed

Martinez-Gomez, N Cecilia; Good, Nathan M; Lidstrom, Mary E

2015-06-15

During an environmental perturbation, the survival of a cell and its response to the perturbation depend on both the robustness and functionality of the metabolic network. The regulatory mechanisms that allow the facultative methylotrophic bacterium Methylobacterium extorquens AM1 to effect the metabolic transition from succinate to methanol growth are not well understood. Methenyl-dephosphotetrahydromethanopterin (methenyl-dH4MPT), an early intermediate during methanol metabolism, transiently accumulated 7- to 11-fold after addition of methanol to a succinate-limited culture. This accumulation partially inhibited the activity of the methylene-H4MPT dehydrogenase, MtdA, restricting carbon flux to the assimilation cycles. A strain overexpressing the gene (mch) encoding the enzyme that consumes methenyl-dH4MPT did not accumulate methenyl-dH4MPT and had a growth rate that was 2.7-fold lower than that of the wild type. This growth defect demonstrates the physiological relevance of this enzymatic regulatory mechanism during the acclimation period. Changes in metabolites and enzymatic activities were analyzed in the strain overexpressing mch. Under these conditions, the activity of the enzyme coupling formaldehyde with dH4MPT (Fae) remained constant, with concomitant formaldehyde accumulation. Release of methenyl-dH4MPT regulation did not affect the induction of the serine cycle enzyme activities immediately after methanol addition, but after 1 h, the activity of these enzymes decreased, likely due to the toxicity of formaldehyde accumulation. Our results support the hypothesis that in a changing environment, the transient accumulation of methenyl-dH4MPT and inhibition of MtdA activity are strategies that permit flexibility and acclimation of the metabolic network while preventing the accumulation of the toxic compound formaldehyde. The identification and characterization of regulatory mechanisms for methylotrophy are in the early stages. We report a nontranscriptional regulatory mechanism that was found to operate as an immediate response for acclimation during changes in substrate availability. Methenyl-dH4MPT, an early intermediate during methanol oxidation, reversibly inhibits the methylene-H4MPT dehydrogenase, MtdA, when Methylobacterium extorquens is challenged to switch from succinate to methanol growth. Bypassing this regulatory mechanism causes formaldehyde to accumulate. Fae, the enzyme catalyzing the conversion of formaldehyde to methylene-dH4MPT, was also identified as another potential regulatory target using this strategy. The results herein further our understanding of the complex regulatory network in methylotrophy and will allow us to improve metabolic engineering strategies of methylotrophs for the production of value-added products. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Integration of systems biology with bioprocess engineering: L: -threonine production by systems metabolic engineering of Escherichia coli.

PubMed

Lee, Sang Yup; Park, Jin Hwan

2010-01-01

Random mutation and selection or targeted metabolic engineering without consideration of its impact on the entire metabolic and regulatory networks can unintentionally cause genetic alterations in the region, which is not directly related to the target metabolite. This is one of the reasons why strategies for developing industrial strains are now shifted towards targeted metabolic engineering based on systems biology, which is termed systems metabolic engineering. Using systems metabolic engineering strategies, all the metabolic engineering works are conducted in systems biology framework, whereby entire metabolic and regulatory networks are thoroughly considered in an integrated manner. The targets for purposeful engineering are selected after all possible effects on the entire metabolic and regulatory networks are thoroughly considered. Finally, the strain, which is capable of producing the target metabolite to a high level close to the theoretical maximum value, can be constructed. Here we review strategies and applications of systems biology successfully implemented on bioprocess engineering, with particular focus on developing L: -threonine production strains of Escherichia coli.

Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

PubMed Central

Pais, Thiago M.; Foulquié-Moreno, María R.; Hubmann, Georg; Duitama, Jorge; Swinnen, Steve; Goovaerts, Annelies; Yang, Yudi; Dumortier, Françoise; Thevelein, Johan M.

2013-01-01

The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance. PMID:23754966

An Outbreak of Diarrhea in Mandera, Kenya, Due to Escherichia coli Serogroup O-Nontypable Strain That Had a Coding Gene for Enteroaggregative E. coli Heat-Stable Enterotoxin 1

PubMed Central

Ochi, Sadayuki; Shah, Mohammad; Odoyo, Erick; Bundi, Martin; Miringu, Gabriel; Guyo, Sora; Wandera, Ernest; Kathiiko, Cyrus; Kariuki, Samuel; Karama, Mohamed; Tsuji, Takao; Ichinose, Yoshio

2017-01-01

In an outbreak of gastroenteritis in December 2009, in Mandera, Kenya, Escherichia coli O-nontypable (ONT) strain was isolated from stool specimens of patients (18/24, 75%). The E. coli ONT organisms could not be assigned to any of the recognized diarrheagenic groups of E. coli. However, they possessed the enteroaggregative E. coli heat-stable enterotoxin-1 gene. The cell-free culture filtrates of the E. coli ONT strain isolated from the outbreak cases induced considerable amount of fluid accumulation in suckling mouse intestine, indicating production of an enterotoxic factor(s). These results identify E. coli that did not have any diarrheagenic characteristics except astA as the etiological agent of the diarrheal outbreak in Mandera. It is however considered necessary to characterize the fluid accumulation factor(s) to determine whether any novel toxins were responsible for the fluid accumulation. Moreover, it is important to study dissemination of strains producing the enterotoxic factor(s) to assess their public health significance distribution in the environment. PMID:27994101

Correlation of Electrical Resistance to CMC Stress-Strain and Fracture Behavior Under High Heat-Flux Thermal and Stress Gradients

NASA Technical Reports Server (NTRS)

Appleby, Matthew; Morscher, Gregory; Zhu, Dongming

2015-01-01

Because SiCSiC ceramic matrix composites (CMCs) are under consideration for use as turbine engine hot-section components in extreme environments, it becomes necessary to investigate their performance and damage morphologies under complex loading and environmental conditions. Monitoring of electrical resistance (ER) has been shown as an effective tool for detecting damage accumulation of woven melt-infiltrated SiCSiC CMCs. However, ER change under complicated thermo-mechanical loading is not well understood. In this study a systematic approach is taken to determine the capabilities of ER as a relevant non-destructive evaluation technique for high heat-flux testing, including thermal gradients and localized stress concentrations. Room temperature and high temperature, laser-based tensile tests were conducted in which stress-dependent damage locations were determined using modal acoustic emission (AE) monitoring and compared to full-field strain mapping using digital image correlation (DIC). This information is then compared with the results of in-situ ER monitoring, post-test ER inspection and fractography in order to correlate ER response to convoluted loading conditions and damage evolution.

Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

PubMed Central

Dave, Khyati K.; Punekar, Narayan S.

2015-01-01

Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production. PMID:26683313

Thermal barrier coating life prediction model development, phase 1

NASA Technical Reports Server (NTRS)

Demasi, Jeanine T.; Ortiz, Milton

1989-01-01

The objective of this program was to establish a methodology to predict thermal barrier coating (TBC) life on gas turbine engine components. The approach involved experimental life measurement coupled with analytical modeling of relevant degradation modes. Evaluation of experimental and flight service components indicate the predominant failure mode to be thermomechanical spallation of the ceramic coating layer resulting from propagation of a dominant near interface crack. Examination of fractionally exposed specimens indicated that dominant crack formation results from progressive structural damage in the form of subcritical microcrack link-up. Tests conducted to isolate important life drivers have shown MCrAlY oxidation to significantly affect the rate of damage accumulation. Mechanical property testing has shown the plasma deposited ceramic to exhibit a non-linear stress-strain response, creep and fatigue. The fatigue based life prediction model developed accounts for the unusual ceramic behavior and also incorporates an experimentally determined oxide rate model. The model predicts the growth of this oxide scale to influence the intensity of the mechanic driving force, resulting from cyclic strains and stresses caused by thermally induced and externally imposed mechanical loads.

Fatigue life prediction modeling for turbine hot section materials

NASA Technical Reports Server (NTRS)

Halford, G. R.; Meyer, T. G.; Nelson, R. S.; Nissley, D. M.; Swanson, G. A.

1989-01-01

A major objective of the fatigue and fracture efforts under the NASA Hot Section Technology (HOST) program was to significantly improve the analytic life prediction tools used by the aeronautical gas turbine engine industry. This was achieved in the areas of high-temperature thermal and mechanical fatigue of bare and coated high-temperature superalloys. The cyclic crack initiation and propagation resistance of nominally isotropic polycrystalline and highly anisotropic single crystal alloys were addressed. Life prediction modeling efforts were devoted to creep-fatigue interaction, oxidation, coatings interactions, multiaxiality of stress-strain states, mean stress effects, cumulative damage, and thermomechanical fatigue. The fatigue crack initiation life models developed to date include the Cyclic Damage Accumulation (CDA) and the Total Strain Version of Strainrange Partitioning (TS-SRP) for nominally isotropic materials, and the Tensile Hysteretic Energy Model for anisotropic superalloys. A fatigue model is being developed based upon the concepts of Path-Independent Integrals (PII) for describing cyclic crack growth under complex nonlinear response at the crack tip due to thermomechanical loading conditions. A micromechanistic oxidation crack extension model was derived. The models are described and discussed.

Microalgae cultivation in a tubular bioreactor and utilization of their cells

NASA Astrophysics Data System (ADS)

Koyu, Hon-Nami; Shunji, Kunito

1998-03-01

In this study on the possiblities of microalgae technology as an option for CO2 mitigation, many microalgae were isolated from seawater. Some species of the isolates, Chlamydomonas sp. strain YA-SH-1, which accumulates starch in cells under light and ferment ethanol in dark and anaerobic condition, was grown outdoors by using 50-L tubular bioreactors in batch cultivation and harvested. Using these cells, the performance of ethanol production was examined quantitatively in a 0.5-L scale fermentor. Another species, Tetraselmis sp. strain Tt-1, was cultivated in a semi-batch manner by a similar type of tubular bioreactor indoors and examined for its utilization. Tests showed these cells could be used as partial substitute for wood and kenaf pulp for processing into paper. With the idea of making microalgae produce cellulose by genetic engineering in their minds, the authors studied the structure of bacterial cellulose synthase genes and the low temperature-induced, reversible flocculation in a thermophilic blue green alga (Cyanobacterium), Synechocystis vulcanus in order to examine the feasibility of using these genes as gene source and the cynanobacterium as host.

Fatigue life prediction modeling for turbine hot section materials

NASA Technical Reports Server (NTRS)

Halford, G. R.; Meyer, T. G.; Nelson, R. S.; Nissley, D. M.; Swanson, G. A.

1988-01-01

A major objective of the fatigue and fracture efforts under the Hot Section Technology (HOST) program was to significantly improve the analytic life prediction tools used by the aeronautical gas turbine engine industry. This was achieved in the areas of high-temperature thermal and mechanical fatigue of bare and coated high-temperature superalloys. The cyclic crack initiation and propagation resistance of nominally isotropic polycrystalline and highly anisotropic single crystal alloys were addressed. Life prediction modeling efforts were devoted to creep-fatigue interaction, oxidation, coatings interactions, multiaxiality of stress-strain states, mean stress effects, cumulative damage, and thermomechanical fatigue. The fatigue crack initiation life models developed to date include the Cyclic Damage Accumulation (CDA) and the Total Strain Version of Strainrange Partitioning (TS-SRP) for nominally isotropic materials, and the Tensile Hysteretic Energy Model for anisotropic superalloys. A fatigue model is being developed based upon the concepts of Path-Independent Integrals (PII) for describing cyclic crack growth under complex nonlinear response at the crack tip due to thermomechanical loading conditions. A micromechanistic oxidation crack extension model was derived. The models are described and discussed.

Fumaric acid production in Saccharomyces cerevisiae by simultaneous use of oxidative and reductive routes.

PubMed

Xu, Guoqiang; Chen, Xiulai; Liu, Liming; Jiang, Linghuo

2013-11-01

In this study, the simultaneous use of reductive and oxidative routes to produce fumaric acid was explored. The strain FMME003 (Saccharomyces cerevisiae CEN.PK2-1CΔTHI2) exhibited capability to accumulate pyruvate and was used for fumaric acid production. The fum1 mutant FMME004 could produce fumaric acid via oxidative route, but the introduction of reductive route derived from Rhizopus oryzae NRRL 1526 led to lower fumaric acid production. Analysis of the key factors associated with fumaric acid production revealed that pyruvate carboxylase had a low degree of control over the carbon flow to malic acid. The fumaric acid titer was improved dramatically when the heterologous gene RoPYC was overexpressed and 32 μg/L of biotin was added. Furthermore, under the optimal carbon/nitrogen ratio, the engineered strain FMME004-6 could produce up to 5.64 ± 0.16 g/L of fumaric acid. These results demonstrated that the proposed fermentative method is efficient for fumaric acid production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Experimental Investigation of the Influence of Joint Geometric Configurations on the Mechanical Properties of Intermittent Jointed Rock Models Under Cyclic Uniaxial Compression

NASA Astrophysics Data System (ADS)

Liu, Yi; Dai, Feng; Fan, Pengxian; Xu, Nuwen; Dong, Lu

2017-06-01

Intermittent joints in rock mass are quite sensitive to cyclic loading conditions. Understanding the fatigue mechanical properties of jointed rocks is beneficial for rational design and stability analysis of rock engineering projects. This study experimentally investigated the influences of joint geometry (i.e., dip angle, persistency, density and spacing) on the fatigue mechanism of synthetic jointed rock models. Our results revealed that the stress-strain curve of jointed rock under cyclic loadings is dominated by its curve under monotonic uniaxial loadings; the terminal strain in fatigue curve is equal to the post-peak strain corresponding to the maximum cyclic stress in the monotonic stress-strain curve. The four joint geometrical parameters studied significantly affect the fatigue properties of jointed rocks, including the irreversible strains, the fatigue deformation modulus, the energy evolution, the damage variable and the crack coalescence patterns. The higher the values of the geometrical parameters, the lower the elastic energy stores in this jointed rock, the higher the fatigue damage accumulates in the first few cycles, and the lower the fatigue life. The elastic energy has certain storage limitation, at which the fatigue failure occurs. Two basic micro-cracks, i.e., tensile wing crack and shear crack, are observed in cyclic loading and unloading tests, which are controlled principally by joint dip angle and persistency. In general, shear cracks only occur in the jointed rock with higher dip angle or higher persistency, and the jointed rock is characterized by lower fatigue strength, larger damage variable and lower fatigue life.

Benefits of a Recombination-Proficient Escherichia coli System for Adaptive Laboratory Evolution.

PubMed

Peabody, George; Winkler, James; Fountain, Weston; Castro, David A; Leiva-Aravena, Enzo; Kao, Katy C

2016-11-15

Adaptive laboratory evolution typically involves the propagation of organisms asexually to select for mutants with the desired phenotypes. However, asexual evolution is prone to competition among beneficial mutations (clonal interference) and the accumulation of hitchhiking and neutral mutations. The benefits of horizontal gene transfer toward overcoming these known disadvantages of asexual evolution were characterized in a strain of Escherichia coli engineered for superior sexual recombination (genderless). Specifically, we experimentally validated the capacity of the genderless strain to reduce the mutational load and recombine beneficial mutations. We also confirmed that inclusion of multiple origins of transfer influences both the frequency of genetic exchange throughout the chromosome and the linkage of donor DNA. We built a simple kinetic model to estimate recombination frequency as a function of transfer size and relative genotype enrichment in batch transfers; the model output correlated well with the experimental data. Our results provide strong support for the advantages of utilizing the genderless strain over its asexual counterpart during adaptive laboratory evolution for generating beneficial mutants with reduced mutational load. Over 80 years ago Fisher and Muller began a debate on the origins of sexual recombination. Although many aspects of sexual recombination have been examined at length, experimental evidence behind the behaviors of recombination in many systems and the means to harness it remain elusive. In this study, we sought to experimentally validate some advantages of recombination in typically asexual Escherichia coli and determine if a sexual strain of E. coli can become an effective tool for strain development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Benefits of a Recombination-Proficient Escherichia coli System for Adaptive Laboratory Evolution

PubMed Central

Peabody, George; Winkler, James; Fountain, Weston; Castro, David A.; Leiva-Aravena, Enzo

2016-01-01

ABSTRACT Adaptive laboratory evolution typically involves the propagation of organisms asexually to select for mutants with the desired phenotypes. However, asexual evolution is prone to competition among beneficial mutations (clonal interference) and the accumulation of hitchhiking and neutral mutations. The benefits of horizontal gene transfer toward overcoming these known disadvantages of asexual evolution were characterized in a strain of Escherichia coli engineered for superior sexual recombination (genderless). Specifically, we experimentally validated the capacity of the genderless strain to reduce the mutational load and recombine beneficial mutations. We also confirmed that inclusion of multiple origins of transfer influences both the frequency of genetic exchange throughout the chromosome and the linkage of donor DNA. We built a simple kinetic model to estimate recombination frequency as a function of transfer size and relative genotype enrichment in batch transfers; the model output correlated well with the experimental data. Our results provide strong support for the advantages of utilizing the genderless strain over its asexual counterpart during adaptive laboratory evolution for generating beneficial mutants with reduced mutational load. IMPORTANCE Over 80 years ago Fisher and Muller began a debate on the origins of sexual recombination. Although many aspects of sexual recombination have been examined at length, experimental evidence behind the behaviors of recombination in many systems and the means to harness it remain elusive. In this study, we sought to experimentally validate some advantages of recombination in typically asexual Escherichia coli and determine if a sexual strain of E. coli can become an effective tool for strain development. PMID:27613685

Driving carbon flux through exogenous butyryl-CoA: Acetate CoA-transferase to produce butyric acid at high titer in Thermobifida fusca.

PubMed

Deng, Yu; Mao, Yin; Zhang, Xiaojuan

2015-12-20

Butyric acid, a 4-carbon short chain fatty acid, is widely used in chemical, food, and pharmaceutical industries. The low activity of butyryl-CoA: acetate CoA-transferase in Thermobifida fusca muS, a thermophilic actinobacterium whose optimal temperature was 55°C, was found to hinder the accumulation of high yield of butyric acid. In order to solve this problem, an exogenous butyryl-CoA: acetate CoA-transferase gene (actA) from Thermoanaerobacterium thermosaccharolyticum DSM571 was integrated into the chromosome of T. fusca muS by replacing celR gene, forming T. fusca muS-1. We demonstrated that on 5g/L cellulose, the yield of butyric acid by the engineered muS-1 strain was increased by 42.9 % compared to the muS strain. On 100g/L of cellulose, the muS-1 strain could consume 90.5% of total cellulose in 144h, with 33.2g/L butyric acid produced. Furthermore, on the mix substrates including the major components of biomass: cellulose, xylose, mannose and galactose, 70.4g/L butyric acid was produced in 168h by fed-batch fermentation. To validate the ability of fermenting biomass, the muS-1 strain was grown on the milled corn stover ranging from 200 to 250μm. The muS-1 strain had the highest butyrate titer 17.1g/L on 90g/L corn stover. Copyright © 2015 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hershkovitz, N.; Oren, A.; Cohen, Y.

The drought-resistant cyanobacteria Phormidium autumnale, strain LPP{sub 4}, and a Chroococcidiopsis sp. accumulated trehalose, sucrose, and both trehalose and sucrose, respectively, in response to matric water stress. Accumulated sugar concentrations reached values of up to 6.2 {mu}g of trehalose per {mu}g of chlorophyll in P. autumnale, 6.9 {mu}g of sucrose per {mu}g of chlorophyll in LPP{sub 4}, and 4.1 {mu}g of sucrose and 3.2 {mu}g of trehalose per {mu}g of chlorophyll in the Chroococcidiopsis sp. The same sugars were accumulated by these cyanobacteria in similar concentrations under osmotic water stress. Cyanobacteria that did not show drought resistance (Plectonema boryanum andmore » Synechococcus strain PCC 7942) did not accumulate significant amounts of sugars when matric water stress was applied.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Tianyong; Olson, Daniel G.; Tian, Liang

Clostridium thermocellum and Thermoanaerobacterium saccharolyticumare thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticumproduce ethanol with a yield of 90% of the theoretical maximum, engineered strains ofC. thermocellumproduce ethanol at lower yields (~50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in theiradhEgenes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, theadhEgenes from six strains ofC. thermocellumandT. saccharolyticumwere cloned and expressed inEscherichia coli, the enzymesmore » produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains ofT. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain ofC. thermocellumhas acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced intoC. thermocellumandT. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. This work describes the characterization of the AdhE enzyme from different strains ofC. thermocellumandT. saccharolyticum.C. thermocellumandT. saccharolyticumare thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production byC. thermocellumandT. saccharolyticum.« less

Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast.

PubMed

Kamisaka, Yasushi; Kimura, Kazuyoshi; Uemura, Hiroshi; Yamaoka, Masakazu

2013-08-01

Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np), which was previously found to be an active form in the ∆snf2 mutant. Overexpression of Dga1∆Np in the ∆snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1∆Np in lipid accumulation. We found that the overexpression of Dga1∆Np in the ∆dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1∆Np to stimulate lipid accumulation, the ∆dga1-1 mutant, in which the 3'-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ∆dga1-2 mutant, in which the 3'-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1∆Np in the ∆dga1-1 mutant had a lower lipid content than the original ∆dga1 mutant, whereas overexpression in the ∆dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3' terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1∆Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1∆Np in the ∆dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1∆Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ∆dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is involved in the effect of dga1 disruption on Dga1∆Np-dependent lipid accumulation. These results provide a new strategy to engineer S. cerevisiae for optimal lipid production.

Ratcheting in a nonlinear viscoelastic adhesive

NASA Astrophysics Data System (ADS)

Lemme, David; Smith, Lloyd

2017-11-01

Uniaxial time-dependent creep and cycled stress behavior of a standard and toughened film adhesive were studied experimentally. Both adhesives exhibited progressive accumulation of strain from an applied cycled stress. Creep tests were fit to a viscoelastic power law model at three different applied stresses which showed nonlinear response in both adhesives. A third order nonlinear power law model with a permanent strain component was used to describe the creep behavior of both adhesives and to predict creep recovery and the accumulation of strain due to cycled stress. Permanent strain was observed at high stress but only up to 3% of the maximum strain. Creep recovery was under predicted by the nonlinear model, while cycled stress showed less than 3% difference for the first cycle but then over predicted the response above 1000 cycles by 4-14% at high stress. The results demonstrate the complex response observed with structural adhesives, and the need for further analytical advancements to describe their behavior.

Engineering Bacillus licheniformis as a thermophilic platform for the production of l-lactic acid from lignocellulose-derived sugars.

PubMed

Li, Chao; Gai, Zhongchao; Wang, Kai; Jin, Liping

2017-01-01

Bacillus licheniformis MW3 as a GRAS and thermophilic strain is a promising microorganism for chemical and biofuel production. However, its capacity to co-utilize glucose and xylose, the major sugars found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, a "dual-channel" process was implemented to engineer strain MW3 for simultaneous utilization of glucose and xylose, using l-lactic acid as a target product. A non-phosphotransferase system (PTS) glucose uptake route was activated via deletion of the glucose transporter gene ptsG and introduction of the galactose permease gene galP . After replacing the promoter of glucokinase gene glck with the strong promoter P als , the engineered strain recovered glucose consumption and utilized glucose and xylose simultaneously. Meanwhile, to improve the consumption rate of xylose in this strain, several measures were undertaken, such as relieving the regulation of the xylose repressor XylR, reducing the catabolite-responsive element, and optimizing the rate-limiting step. Knockout of ethanol and acetic acid pathway genes further increased lactic acid yield by 6.2%. The resultant strain, RH15, was capable of producing 121.9 g/L l-lactic acid at high yield (95.3%) after 40 h of fermentation from a mixture of glucose and xylose. When a lignocellulosic hydrolysate was used as the substrate, 99.3 g/L l-lactic acid was produced within 40 h, with a specific productivity of 2.48 g/[L h] and a yield of 94.6%. Our engineered strain B. licheniformis RH15 could thermophilically produced l-lactic acid from lignocellulosic hydrolysate with relatively high concentration and productivity at levels that were competitive with most reported cases of l-lactic acid-producers. Thus, the engineered strain might be used as a platform for the production of other chemicals. In addition to engineering the B. licheniformis strain, the "dual-channel" process might serve as an alternative method for engineering a variety of other strains.

Field performance of a genetically engineered strain of pink bollworm.

PubMed

Simmons, Gregory S; McKemey, Andrew R; Morrison, Neil I; O'Connell, Sinead; Tabashnik, Bruce E; Claus, John; Fu, Guoliang; Tang, Guolei; Sledge, Mickey; Walker, Adam S; Phillips, Caroline E; Miller, Ernie D; Rose, Robert I; Staten, Robert T; Donnelly, Christl A; Alphey, Luke

2011-01-01

Pest insects harm crops, livestock and human health, either directly or by acting as vectors of disease. The Sterile Insect Technique (SIT)--mass-release of sterile insects to mate with, and thereby control, their wild counterparts--has been used successfully for decades to control several pest species, including pink bollworm, a lepidopteran pest of cotton. Although it has been suggested that genetic engineering of pest insects provides potential improvements, there is uncertainty regarding its impact on their field performance. Discrimination between released and wild moths caught in monitoring traps is essential for estimating wild population levels. To address concerns about the reliability of current marking methods, we developed a genetically engineered strain of pink bollworm with a heritable fluorescent marker, to improve discrimination of sterile from wild moths. Here, we report the results of field trials showing that this engineered strain performed well under field conditions. Our data show that attributes critical to SIT in the field--ability to find a mate and to initiate copulation, as well as dispersal and persistence in the release area--were comparable between the genetically engineered strain and a standard strain. To our knowledge, these represent the first open-field experiments with a genetically engineered insect. The results described here provide encouragement for the genetic control of insect pests.

Production of 3-hydroxypropionic acid by balancing the pathway enzymes using synthetic cassette architecture.

PubMed

Sankaranarayanan, Mugesh; Somasundar, Ashok; Seol, Eunhee; Chauhan, Ashish Singh; Kwon, Seongjin; Jung, Gyoo Yeol; Park, Sunghoon

2017-10-10

Biological 3-hydroxypropionic acid (3-HP) production from glycerol is a two-step reaction catalyzed by glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH). Recombinant strains developed for 3-HP production often suffer from the accumulation of a toxic intermediate, 3-hydroxypropionaldehyde (3-HPA). In order to avoid 3-HPA accumulation, balancing of the two enzymatic activities, in the present study, was attempted by employment of synthetic-regulatory cassettes comprising varying-strength promoters and bicistronic ribosome-binding sites (RBSs). When tested in recombinant Escherichia coli, the cassettes could precisely and differentially control the gene expression in transcription, protein expression and enzymatic activity. Five recombinant strains showing different expressions for GDHt were developed and studied for 3-HPA accumulation and 3-HP production. It was found that 3-HPA accumulation could be completely abolished when expressing ALDH at a level approximately 8-fold higher than that of GDHt. One of the strains, SP4, produced 625mM (56.4g/L) of 3-HP in a fed-batch bioreactor, though late-period production was limited by acetate accumulation. Overall, this study demonstrated the importance of pathway balancing in 3-HP production as well as the utility of the synthetic cassette architecture for precise control of bacterial gene expression. Copyright © 2017 Elsevier B.V. All rights reserved.

Finite Strain Behavior of Polyurea for a Wide Range of Strain Rates

DTIC Science & Technology

2010-02-01

dimensional dynamic compressive behavior of EPDM rubber ," Journal of Engineering Materials and Technology, Transaction of the ASME, 125:294-301. [97] Song, B...and Chen, W. (2004) "Dynamic compressive behavior of EPDM rubber un- der nearly uniaxial strain conditions," Journal of Engineering Materials and... rubber elastic springs to describe the steep initial stiffness of virgin butadiene rubber under tensile and compressive loading at intermediate strain

Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering.

PubMed

Chen, Yan; Xiao, Wenhai; Wang, Ying; Liu, Hong; Li, Xia; Yuan, Yingjin

2016-06-21

Microbial production of lycopene, a commercially and medically important compound, has received increasing concern in recent years. Saccharomyces cerevisiae is regarded as a safer host for lycopene production than Escherichia coli. However, to date, the lycopene yield (mg/g DCW) in S. cerevisiae was lower than that in E. coli and did not facilitate downstream extraction process, which might be attributed to the incompatibility between host cell and heterologous pathway. Therefore, to achieve lycopene overproduction in S. cerevisiae, both host cell and heterologous pathway should be delicately engineered. In this study, lycopene biosynthesis pathway was constructed by integration of CrtE, CrtB and CrtI in S. cerevisiae CEN.PK2. When YPL062W, a distant genetic locus, was deleted, little acetate was accumulated and approximately 100 % increase in cytosolic acetyl-CoA pool was achieved relative to that in parental strain. Through screening CrtE, CrtB and CrtI from diverse species, an optimal carotenogenic enzyme combination was obtained, and CrtI from Blakeslea trispora (BtCrtI) was found to have excellent performance on lycopene production as well as lycopene proportion in carotenoid. Then, the expression level of BtCrtI was fine-tuned and the effect of cell mating types was also evaluated. Finally, potential distant genetic targets (YJL064W, ROX1, and DOS2) were deleted and a stress-responsive transcription factor INO2 was also up-regulated. Through the above modifications between host cell and carotenogenic pathway, lycopene yield was increased by approximately 22-fold (from 2.43 to 54.63 mg/g DCW). Eventually, in fed-batch fermentation, lycopene production reached 55.56 mg/g DCW, which is the highest reported yield in yeasts. Saccharomyces cerevisiae was engineered to produce lycopene in this study. Through combining host engineering (distant genetic loci and cell mating types) with pathway engineering (enzyme screening and gene fine-tuning), lycopene yield was stepwise improved by 22-fold as compared to the starting strain. The highest lycopene yield (55.56 mg/g DCW) in yeasts was achieved in 5-L bioreactors. This study provides a good reference of combinatorial engineering of host cell and heterologous pathway for microbial overproduction of pharmaceutical and chemical products.

Open Field Release of Genetically Engineered Sterile Male Aedes aegypti in Malaysia

PubMed Central

Raduan, Norzahira; Kwee Wee, Lim; Hong Ming, Wong; Guat Ney, Teoh; Rahidah A.A., Siti; Salman, Sawaluddin; Subramaniam, Selvi; Nordin, Oreenaiza; Hanum A.T., Norhaida; Angamuthu, Chandru; Marlina Mansor, Suria; Lees, Rosemary S.; Naish, Neil; Scaife, Sarah; Gray, Pam; Labbé, Geneviève; Beech, Camilla; Nimmo, Derric; Alphey, Luke; Vasan, Seshadri S.; Han Lim, Lee; Wasi A., Nazni; Murad, Shahnaz

2012-01-01

Background Dengue is the most important mosquito-borne viral disease. In the absence of specific drugs or vaccines, control focuses on suppressing the principal mosquito vector, Aedes aegypti, yet current methods have not proven adequate to control the disease. New methods are therefore urgently needed, for example genetics-based sterile-male-release methods. However, this requires that lab-reared, modified mosquitoes be able to survive and disperse adequately in the field. Methodology/Principal Findings Adult male mosquitoes were released into an uninhabited forested area of Pahang, Malaysia. Their survival and dispersal was assessed by use of a network of traps. Two strains were used, an engineered ‘genetically sterile’ (OX513A) and a wild-type laboratory strain, to give both absolute and relative data about the performance of the modified mosquitoes. The two strains had similar maximum dispersal distances (220 m), but mean distance travelled of the OX513A strain was lower (52 vs. 100 m). Life expectancy was similar (2.0 vs. 2.2 days). Recapture rates were high for both strains, possibly because of the uninhabited nature of the site. Conclusions/Significance After extensive contained studies and regulatory scrutiny, a field release of engineered mosquitoes was safely and successfully conducted in Malaysia. The engineered strain showed similar field longevity to an unmodified counterpart, though in this setting dispersal was reduced relative to the unmodified strain. These data are encouraging for the future testing and implementation of genetic control strategies and will help guide future field use of this and other engineered strains. PMID:22970102

Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum

DOE PAGES

Papanek, Beth A.; Biswas, Ranjita; Rydzak, Thomas; ...

2015-09-12

Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further improve its CBP capabilities, we study a mutant strain of C. thermocellum that was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulosemore » Avicel, effectively eliminating formate, acetate, and lactate production and reducing H 2 production by fivefold. On 5 g/L Avicel, strain AG553 reached an ethanol yield of 63.5% of the theoretical maximum compared with 19.9% by the wild type, and it showed similar yields on pretreated switchgrass and poplar. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Final ethanol titer peaked at 73.4 mM in mutant AG553 on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to CO 2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1 mM ethanol on 10 g/L Avicel. In conclusion, with the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, AG553 is the best ethanol-yielding CBP strain to date and will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass.« less

Microbial control of hydrogen sulfide production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montgomery, A.D.; Bhupathiraju, V.K.; Wofford, N.

1995-12-31

A sulfide-resistant strain of Thiobacillus denitrificans, strain F, prevented the accumulation of sulfide by Desulfovibrio desulfuricans when both organisms were grown in liquid medium. The wild-type strain of T. denitrificans did not prevent the accumulation of sulfide produced by D. desulfuricans. Strain F also prevented the accumulation of sulfide by a mixed population of sulfate-reducing bacteria enriched from an oil field brine. Fermentation balances showed that strain F stoichiometrically oxidized the sulfide produced by D. desulfuricans and the oil field brine enrichment to sulfate. The ability of a strain F to control sulfide production in an experimental system of coresmore » and formation water from the Redfield, Iowa, natural gas storage facility was also investigated. A stable, sulfide-producing biofilm was established in two separate core systems, one of which was inoculated with strain F while the other core system (control) was treated in an identical manner, but was not inoculated with strain F. When formation water with 10 mM acetate and 5 mM nitrate was injected into both core systems, the effluent sulfide concentrations in the control core system ranged from 200 to 460 {mu}M. In the test core system inoculated with strain F, the effluent sulfide concentrations were lower, ranging from 70 to 110 {mu}M. In order to determine whether strain F could control sulfide production under optimal conditions for sulfate-reducing bacteria, the electron donor was changed to lactate and inorganic nutrients (nitrogen and phosphate sources) were added to the formation water. When nutrient-supplemented formation water with 3.1 mM lactate and 10 mM nitrate was used, the effluent sulfide concentrations of the control core system initially increased to about 3,800 {mu}M, and then decreased to about 1,100 {mu}M after 5 weeks. However, in the test core system inoculated with strain F, the effluent sulfide concentrations were much lower, 160 to 330 {mu}M.« less

Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.

PubMed

Berlanga, Mercedes; Montero, M T; Fernández-Borrell, Jordi; Guerrero, Ricardo

2006-06-01

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications.

Thermal research of infrared sight thermoelectric cooler control circuit under temperature environment

NASA Astrophysics Data System (ADS)

Gao, Youtang; Ding, Huan; Xue, Xiao; Xu, Yuan; Chang, Benkang

2010-10-01

Testing device TST-05B, which is suitable for adaptability test of semiconductor devices, electronic products and other military equipment under the condition of the surrounding air temperature rapidly changing, is used here for temperature shock test.Thermal stability technology of thermoelectric cooler control circuit infrared sight under temperature shock is studied in this paper. Model parameters and geometry is configured for ADI devices (ADN8830), welding material and PCB which are used in system. Thermoelectric cooler control circuit packaged by CSP32 distribution are simulated and analyzed by thermal shock and waveform through engineering finite element analysis software ANSYYS. Because solders of the whole model have much stronger stress along X direction than that of other directions, initial stress constraints along X direction are primarily considered when the partial model of single solder is imposed by thermal load. When absolute thermal loads stresses of diagonal nodes with maximum strains are separated from the whole model, interpolation is processed according to thermal loads circulation. Plastic strains and thermal stresses of nodes in both sides of partial model are obtained. The analysis results indicates that with thermal load circulation, maximum forces of each circulation along X direction are increasingly enlarged and with the accumulation of plastic strains of danger point, at the same time structural deformation and the location of maximum equivalent plastic strain in the solder joints at the first and eighth, the composition will become invalid in the end.

Strain accumulation in southern California, 1973-1980.

USGS Publications Warehouse

Savage, J.C.; Prescott, W.H.; Lisowski, M.; King, N.E.

1981-01-01

Frequent surveys of seven trilateration networks in southern California over the interval 1973-1980 suggest that a regional increment in strain may have occurred in 1978-1979. Prior to 1978 and after late 1979 the strain accumulation has been predominantly a uniaxial north-south compression. This secular trend was interrupted sometime in 1978-1979 by an increment in both north-south and east-west extension in five of the seven networks. The onset of this change appears to have occurred first in the networks farthest south. The changes occurred without any unusual seismicity within the networks, but the overall seismicity in southern California was unusually low prior to and has been unusually high since the occurrence. The average principal strain rates for the seven networks in the 1973-1980 interval are 0.17 mu strain/yr north- south contraction and 0.08 mu strain/yr east-west extension. Although the observed increment in strain could be related to unidentified systematic error in the measuring system, a careful review of the measurements and comparisons with three other measuring systems reveal no appreciable cumulative systematic error. -Authors

Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation.

PubMed

Yi, Zhiqian; Xu, Maonian; Magnusdottir, Manuela; Zhang, Yuetuan; Brynjolfsson, Sigurdur; Fu, Weiqi

2015-09-29

Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress.

Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation

PubMed Central

Yi, Zhiqian; Xu, Maonian; Magnusdottir, Manuela; Zhang, Yuetuan; Brynjolfsson, Sigurdur; Fu, Weiqi

2015-01-01

Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress. PMID:26426027

Accumulation of Astaxanthin by a New Haematococcus pluvialis Strain BM1 from the White Sea Coastal Rocks (Russia)

PubMed Central

Chekanov, Konstantin; Lobakova, Elena; Selyakh, Irina; Semenova, Larisa; Sidorov, Roman; Solovchenko, Alexei

2014-01-01

We report on a novel arctic strain BM1 of a carotenogenic chlorophyte from a coastal habitat with harsh environmental conditions (wide variations in solar irradiance, temperature, salinity and nutrient availability) identified as Haematococcus pluvialis Flotow. Increased (25‰) salinity exerted no adverse effect on the growth of the green BM1 cells. Under stressful conditions (high light, nitrogen and phosphorus deprivation), green vegetative cells of H. pluvialis BM1 grown in BG11 medium formed non-motile palmelloid cells and, eventually, hematocysts capable of a massive accumulation of the keto-carotenoid astaxanthin with a high nutraceutical and therapeutic potential. Routinely, astaxanthin was accumulated at the level of 4% of the cell dry weight (DW), reaching, under prolonged stress, 5.5% DW. Astaxanthin was predominantly accumulated in the form of mono- and diesters of fatty acids from C16 and C18 families. The palmelloids and hematocysts were characterized by the formation of red-colored cytoplasmic lipid droplets, increasingly large in size and number. The lipid droplets tended to merge and occupied almost the entire volume of the cell at the advanced stages of stress-induced carotenogenesis. The potential application of the new strain for the production of astaxanthin is discussed in comparison with the H. pluvialis strains currently employed in microalgal biotechnology. PMID:25196836

Thinking outside the "bug": a unique assay to measure intracellular drug penetration in gram-negative bacteria.

PubMed

Zhou, Ying; Joubran, Camil; Miller-Vedam, Lakshmi; Isabella, Vincent; Nayar, Asha; Tentarelli, Sharon; Miller, Alita

2015-04-07

Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

PubMed

Lee, Kang-Hoon; Lim, Debora; Chiu, Sophia; Greenhalgh, David; Cho, Kiho

2016-04-01

Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans. Copyright © 2016 Elsevier Inc. All rights reserved.

40 CFR 1042.345 - Reporting.

Code of Federal Regulations, 2010 CFR

2010-07-01

... test engine, including the engine family's identification and the engine's model year, build date... you developed and applied the Green Engine Factor, if applicable. (5) Identify how you accumulated...

Creep Life of Ceramic Components Using a Finite-Element-Based Integrated Design Program (CARES/CREEP)

NASA Technical Reports Server (NTRS)

Powers, L. M.; Jadaan, O. M.; Gyekenyesi, J. P.

1998-01-01

The desirable properties of ceramics at high temperatures have generated interest in their use for structural application such as in advanced turbine engine systems. Design lives for such systems can exceed 10,000 hours. The long life requirement necessitates subjecting the components to relatively low stresses. The combination of high temperatures and low stresses typically places failure for monolithic ceramics in the creep regime. The objective of this paper is to present a design methodology for predicting the lifetimes of structural components subjected to creep rupture conditions. This methodology utilizes commercially available finite element packages and takes into account the time-varying creep strain distributions (stress relaxation). The creep life, of a component is discretized into short time steps, during which the stress and strain distributions are assumed constant. The damage is calculated for each time step based on a modified Monkman-Grant creep rupture criterion. Failure is assumed to occur when the normalized accumulated damage at any point in the component is greater than or equal to unity. The corresponding time will be the creep rupture life for that component. Examples are chosen to demonstrate the Ceramics Analysis and Reliability Evaluation of Structures/CREEP (CARES/CREEP) integrated design program, which is written for the ANSYS finite element package. Depending on the component size and loading conditions, it was found that in real structures one of two competing failure modes (creep or slow crack growth) will dominate. Applications to benchmark problems and engine components are included.

Life prediction technologies for aeronautical propulsion systems

NASA Technical Reports Server (NTRS)

Mcgaw, Michael A.

1990-01-01

Fatigue and fracture problems continue to occur in aeronautical gas turbine engines. Components whose useful life is limited by these failure modes include turbine hot-section blades, vanes, and disks. Safety considerations dictate that catastrophic failures be avoided, while economic considerations dictate that catastrophic failures be avoided, while economic considerations dictate that noncatastrophic failures occur as infrequently as possible. Therefore, the decision in design is making the tradeoff between engine performance and durability. LeRC has contributed to the aeropropulsion industry in the area of life prediction technology for over 30 years, developing creep and fatigue life prediction methodologies for hot-section materials. At the present time, emphasis is being placed on the development of methods capable of handling both thermal and mechanical fatigue under severe environments. Recent accomplishments include the development of more accurate creep-fatigue life prediction methods such as the total strain version of LeRC's strain-range partitioning (SRP) and the HOST-developed cyclic damage accumulation (CDA) model. Other examples include the development of a more accurate cumulative fatigue damage rule - the double damage curve approach (DDCA), which provides greatly improved accuracy in comparison with usual cumulative fatigue design rules. Other accomplishments in the area of high-temperature fatigue crack growth may also be mentioned. Finally, we are looking to the future and are beginning to do research on the advanced methods which will be required for development of advanced materials and propulsion systems over the next 10-20 years.

Production of L-lactic acid from metabolically engineered strain of Enterobacter aerogenes ATCC 29007.

PubMed

Thapa, Laxmi Prasad; Lee, Sang Jun; Park, Chulhwan; Kim, Seung Wook

2017-07-01

In this study, L-lactic acid production was investigated from metabolically engineered strain of E. aerogenes ATCC 29007. The engineered strain E. aerogenes SUMI01 (Δpta) was generated by the deletion of phosphate acetyltransferase (pta) gene from the chromosome of E. aerogenes ATCC 29007 and deletion was confirmed by colony PCR. Under the optimized fermentation conditions, at 37°C and pH 6 for 84h, the L-lactic acid produced by engineered strain E. aerogenes SUMI01 (Δpta) in flask fermentation using 100g/L mannitol as the carbon source was 40.05g/L as compared to that of the wild type counterpart 20.70g/L. At the end of the batch fermentation in bioreactor the production of L-lactic acid reached to 46.02g/L and yield was 0.41g/g by utilizing 112.32g/L mannitol. This is the first report regarding the production of L-lactic acid from Enterobacter species. We believe that this result may provide valuable guidelines for further engineering Enterobacter strain for the improvement of L-lactic acid production. Copyright © 2017 Elsevier Inc. All rights reserved.

Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae.

PubMed

Inoue, Hiroyuki; Hashimoto, Seitaro; Matsushika, Akinori; Watanabe, Seiya; Sawayama, Shigeki

2014-12-01

The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.

[Culture conditions of engineered strain of L-asparaginase and the recombinant plasmid stability].

PubMed

Wang, Y; Qian, S; Ye, J; Meng, G; Zhang, S

1999-12-01

The growth curves of engineered strain JM105(pASN) were different in LB and M-3 media. The expression level and activity of L-asparaginase were affected apparently by both biomass and induction time. Glucose repression of production of L-asparaginase was found. The stability of the recombinant plasmid pASN in different host strains and in LB and M-3 media was determined. After cultivation inLB broth and M-3 media at 30 degrees C for more than 50 generations without antibiotic selection, then induced at 42 degrees C for up to 5 h, the engineered strains were proved to be stable, except for DHA alpha (pASN).

Genetic diversity in natural populations of a soil bacterium across a landscape gradient

PubMed Central

McArthur, J. Vaun; Kovacic, David A.; Smith, Michael H.

1988-01-01

Genetic diversity in natural populations of the bacterium Pseudomonas cepacia was surveyed in 10 enzymes from 70 clones isolated along a landscape gradient. Estimates of genetic diversity, ranging from 0.54 to 0.70, were higher than any previously reported values of which we are aware and were positively correlated with habitat variability. Patterns of bacterial genetic diversity were correlated with habitat variability. Findings indicate that the source of strains used in genetic engineering will greatly affect the outcome of planned releases in variable environments. Selection of generalist strains may confer a large advantage to engineered populations, while selection of laboratory strains may result in quick elimination of the engineered strains. PMID:16594009

Elastic block and strain modeling of GPS data around the Haiyuan-Liupanshan fault, northeastern Tibetan Plateau

NASA Astrophysics Data System (ADS)

Li, Yanchuan; Shan, Xinjian; Qu, Chunyan; Zhang, Yingfeng; Song, Xiaogang; Jiang, Yu; Zhang, Guohong; Nocquet, Jean-Mathieu; Gong, Wenyu; Gan, Weijun; Wang, Chisheng

2017-12-01

Based on the dense GPS velocity field in the northeastern margin of the Tibetan Plateau from 1999 to 2016, we have produced the deformation and strain characteristics of the Haiyuan fault and the Liupanshan fault. Estimated long-term slip rate along the Haiyuan-Liupanshan fault zones show a gradual decrease from 6.4 ± 1.6 mm/yr at the Tuolaishan fault to 2.9 ± 1.2 mm/yr at the Southern Liupanshan fault. Left-lateral thrusting movement was inverted for the Xiangshan-Tianjingshan fault (XS-TJS), which has an average slip rate of 2.1 ± 3.4 mm/yr during the study period. We also calculated the heterogeneous distribution of interseismic coupling along the fault zones. Our result also shows the locking depth of the Tianzhu seismic gap is ∼22 km. The slip rate deficit, the seismic moment accumulation rate, and the Coulomb stress accumulation rate are high on the fault planes, whereas the second invariant of the strain rate is low at the surface. The Liupanshan fault is locked to a depth of ∼23 km, and the corresponding seismic moment accumulation rate on the fault plane is high, while the strain rate at the surface is low. The accumulated strain along the Tianzhu seismic gap and the Liupanshan fault could be balanced by earthquakes with magnitudes of Mw7.9 and Mw7.4, considering the absence of large earthquakes over the last 1000 years and 1400 years respectively. The Haiyuan segments had ruptured during 1920 Haiyuan earthquake, and the estimated locking depth for period 1999-2016 is 5-10 km. Its seismic moment accumulation rate at depth is low and the strain rate at the surface is high. Our result indicates that 70% of the strike-slip along the Haiyuan segments transforms into thrusting along the Liupanshan fault, while the remaining 30% is related to the orogeny of the Liupanshan. For slip between the Haiyuan fault and the XS-TJS, about 27-34% of the slip is partitioned on the XS-TJS.

Strain engineering of van der Waals heterostructures.

PubMed

Vermeulen, Paul A; Mulder, Jefta; Momand, Jamo; Kooi, Bart J

2018-01-18

Modifying the strain state of solids allows control over a plethora of functional properties. The weak interlayer bonding in van der Waals (vdWaals) materials such as graphene, hBN, MoS 2 , and Bi 2 Te 3 might seem to exclude strain engineering, since strain would immediately relax at the vdWaals interfaces. Here we present direct observations of the contrary by showing growth of vdWaals heterostructures with persistent in-plane strains up to 5% and we show that strain relaxation follows a not yet reported process distinctly different from strain relaxation in three-dimensionally bonded (3D) materials. For this, 2D bonded Bi 2 Te 3 -Sb 2 Te 3 and 2D/3D bonded Bi 2 Te 3 -GeTe multilayered films are grown using Pulsed Laser Deposition (PLD) and their structure is monitored in situ using Reflective High Energy Electron Diffraction (RHEED) and post situ analysis is performed using Transmission Electron Microscopy (TEM). Strain relaxation is modeled and found to solely depend on the layer being grown and its initial strain. This insight demonstrates that strain engineering of 2D bonded heterostructures obeys different rules than hold for epitaxial 3D materials and opens the door to precise tuning of the strain state of the individual layers to optimize functional performance of vdWaals heterostructures.

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials.

PubMed

Heyde, Keith C; Scott, Felicia Y; Paek, Sung-Ho; Zhang, Ruihua; Ruder, Warren C

2017-03-09

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain.

PubMed

Heher, Philipp; Maleiner, Babette; Prüller, Johanna; Teuschl, Andreas Herbert; Kollmitzer, Josef; Monforte, Xavier; Wolbank, Susanne; Redl, Heinz; Rünzler, Dominik; Fuchs, Christiane

2015-09-01

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca(2+) dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Engineering Aspergillus oryzae A-4 through the Chromosomal Insertion of Foreign Cellulase Expression Cassette to Improve Conversion of Cellulosic Biomass into Lipids

PubMed Central

Shen, Qi; Ma, Junwei; Fu, Jianrong; Zhao, Yuhua

2014-01-01

A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%–133% and 35.22%–59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass. PMID:25251435

Engineering Aspergillus oryzae A-4 through the chromosomal insertion of foreign cellulase expression cassette to improve conversion of cellulosic biomass into lipids.

PubMed

Lin, Hui; Wang, Qun; Shen, Qi; Ma, Junwei; Fu, Jianrong; Zhao, Yuhua

2014-01-01

A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%-133% and 35.22%-59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass.

Utilization of Sugarcane Bagasse by Halogeometricum borinquense Strain E3 for Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

PubMed Central

Salgaonkar, Bhakti B.; Bragança, Judith M.

2017-01-01

Sugarcane bagasse (SCB), one of the major lignocellulosic agro-industrial waste products, was used as a substrate for biosynthesis of polyhydroxyalkanoates (PHA) by halophilic archaea. Among the various wild-type halophilic archaeal strains screened, Halogeometricum borinquense strain E3 showed better growth and PHA accumulation as compared to Haloferaxvolcanii strain BBK2, Haloarcula japonica strain BS2, and Halococcus salifodinae strain BK6. Growth kinetics and bioprocess parameters revealed the maximum PHA accumulated by strain E3 to be 50.4 ± 0.1 and 45.7 ± 0.19 (%) with specific productivity (qp) of 3.0 and 2.7 (mg/g/h) using NaCl synthetic medium supplemented with 25% and 50% SCB hydrolysate, respectively. PHAs synthesized by strain E3 were recovered in chloroform using a Soxhlet apparatus. Characterization of the polymer using crotonic acid assay, X-ray diffraction (XRD), differential scanning calorimeter (DSC), Fourier transform infrared (FT-IR), and proton nuclear magnetic resonance (1H-NMR) spectroscopy analysis revealed the polymer obtained from SCB hydrolysate to be a co-polymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] comprising of 13.29 mol % 3HV units. PMID:28952529

Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

PubMed Central

Shima, Jun; Hino, Akihiro; Yamada-Iyo, Chie; Suzuki, Yasuo; Nakajima, Ryouichi; Watanabe, Hajime; Mori, Katsumi; Takano, Hiroyuki

1999-01-01

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δnth1), acid trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough. PMID:10388673

Protein design in systems metabolic engineering for industrial strain development.

PubMed

Chen, Zhen; Zeng, An-Ping

2013-05-01

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Generating favorable growth factor and protease release profiles to enable extracellular matrix accumulation within an in vitro tissue engineering environment.

PubMed

Zhang, Xiaoqing; Battiston, Kyle G; Labow, Rosalind S; Simmons, Craig A; Santerre, J Paul

2017-05-01

Tissue engineering (particularly for the case of load-bearing cardiovascular and connective tissues) requires the ability to promote the production and accumulation of extracellular matrix (ECM) components (e.g., collagen, glycosaminoglycan and elastin). Although different approaches have been attempted in order to enhance ECM accumulation in tissue engineered constructs, studies of underlying signalling mechanisms that influence ECM deposition and degradation during tissue remodelling and regeneration in multi-cellular culture systems have been limited. The current study investigated vascular smooth muscle cell (VSMC)-monocyte co-culture systems using different VSMC:monocyte ratios, within a degradable polyurethane scaffold, to assess their influence on ECM generation and degradation processes, and to elucidate relevant signalling molecules involved in this in vitro vascular tissue engineering system. It was found that a desired release profile of growth factors (e.g. insulin growth factor-1 (IGF-1)) and hydrolytic proteases (e.g. matrix-metalloproteinases 2, 9, 13 and 14 (MMP2, MMP9, MMP13 and MMP14)), could be achieved in co-culture systems, yielding an accumulation of ECM (specifically for 2:1 and 4:1 VSMC:monocyte culture systems). This study has significant implications for the tissue engineering field (including vascular tissue engineering), not only because it identified important cytokines and proteases that control ECM accumulation/degradation within synthetic tissue engineering scaffolds, but also because the established culture systems could be applied to improve the development of different types of tissue constructs. Sufficient extracellular matrix accumulation within cardiovascular and connective tissue engineered constructs is a prerequisite for their appropriate function in vivo. This study established co-culture systems with tissue specific cells (vascular smooth muscle cells (VSMCs)) and defined ratios of immune cells (monocytes) to investigate extracellular matrix (ECM) generation and degradation processes, revealing important mechanisms underlying ECM turnover during vascular tissue regeneration/remodelling. A specific growth factor (IGF-1), as well as hydrolytic proteases (e.g. MMP2, MMP9, MMP13 and MMP14), were identified as playing important roles in these processes. ECM accumulation was found to be dependent on achieving a desired release profile of these ECM-promoting and ECM-degrading factors within the multi-cellular microenvironment. The findings enhance our understanding of ECM deposition and degradation during in vitro tissue engineering and would be applicable to the repair or regeneration of a variety of tissues. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Identification and detoxification of glycolaldehyde, an unattended bioethanol fermentation inhibitor.

PubMed

Jayakody, Lahiru N; Ferdouse, Jannatul; Hayashi, Nobuyuki; Kitagaki, Hiroshi

2017-03-01

Although there have been approximately 60 chemical compounds identified as potent fermentation inhibitors in lignocellulose hydrolysate, our research group recently discovered glycolaldehyde as a key fermentation inhibitor during second generation biofuel production. Accordingly, we have developed a yeast S. cerevisiae strain exhibiting tolerance to glycolaldehyde. During this glycolaldehyde study, we established novel approaches for rational engineering of inhibitor-tolerant S. cerevisiae strains, including engineering redox cofactors and engineering the SUMOylation pathway. These new technical dimensions provide a novel platform for engineering S. cerevisiae strains to overcome one of the key barriers for industrialization of lignocellulosic ethanol production. As such, this review discusses novel biochemical insight of glycolaldehyde in the context of the biofuel industry.

Pseudomonas aeruginosa KUCD1, a possible candidate for cadmium bioremediation

PubMed Central

Sinha, Sangram; Mukherjee, Samir Kumar

2009-01-01

A cadmium (8 mM) resistant Pseudomonas aeruginosa strain KUCd1 exhibiting high Cd accumulation under in vitro aerobic condition has been reported. The isolate showed a significant ability to remove more than 75% and 89% of the soluble cadmium during the active growth phase from the growth medium and from Cd-amended industrial wastewater under growth supportive condition. Transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDXS) suggest the presence of Cd in the cells from mid stationary phase. The cell fractionation study revealed membrane and periplasm to be the major accumulating site in this strain. The chemical nature of the accumulated Cd was studied by X-ray powder diffraction analysis. PMID:24031411

Engineering cofactor flexibility enhanced 2,3-butanediol production in Escherichia coli.

PubMed

Liang, Keming; Shen, Claire R

2017-12-01

Enzymatic reduction of acetoin into 2,3-butanediol (2,3-BD) typically requires the reduced nicotinamide adenine dinucleotide (NADH) or its phosphate form (NADPH) as electron donor. Efficiency of 2,3-BD biosynthesis, therefore, is heavily influenced by the enzyme specificity and the cofactor availability which varies dynamically. This work describes the engineering of cofactor flexibility for 2,3-BD production by simultaneous overexpression of an NADH-dependent 2,3-BD dehydrogenase from Klebsiella pneumoniae (KpBudC) and an NADPH-specific 2,3-BD dehydrogenase from Clostridium beijerinckii (CbAdh). Co-expression of KpBudC and CbAdh not only enabled condition versatility for 2,3-BD synthesis via flexible utilization of cofactors, but also improved production stereo-specificity of 2,3-BD without accumulation of acetoin. With optimization of medium and fermentation condition, the co-expression strain produced 92 g/L of 2,3-BD in 56 h with 90% stereo-purity for (R,R)-isoform and 85% of maximum theoretical yield. Incorporating cofactor flexibility into the design principle should benefit production of bio-based chemical involving redox reactions.

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools.

PubMed

Siddiqui, Michael S; Thodey, Kate; Trenchard, Isis; Smolke, Christina D

2012-03-01

Secondary metabolites are an important source of high-value chemicals, many of which exhibit important pharmacological properties. These valuable natural products are often difficult to synthesize chemically and are commonly isolated through inefficient extractions from natural biological sources. As such, they are increasingly targeted for production by biosynthesis from engineered microorganisms. The budding yeast species Saccharomyces cerevisiae has proven to be a powerful microorganism for heterologous expression of biosynthetic pathways. S. cerevisiae's usefulness as a host organism is owed in large part to the wealth of knowledge accumulated over more than a century of intense scientific study. Yet many challenges are currently faced in engineering yeast strains for the biosynthesis of complex secondary metabolite production. However, synthetic biology is advancing the development of new tools for constructing, controlling, and optimizing complex metabolic pathways in yeast. Here, we review how the coupling between yeast biology and synthetic biology is advancing the use of S. cerevisiae as a microbial host for the construction of secondary metabolic pathways. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Biophysical Stimulation for Engineering Functional Skeletal Muscle.

PubMed

Somers, Sarah M; Spector, Alexander A; DiGirolamo, Douglas J; Grayson, Warren L

2017-08-01

Tissue engineering is a promising therapeutic strategy to regenerate skeletal muscle. However, ex vivo cultivation methods typically result in a low differentiation efficiency of stem cells as well as grafts that resemble the native tissues morphologically, but lack contractile function. The application of biomimetic tensile strain provides a potent stimulus for enhancing myogenic differentiation and engineering functional skeletal muscle grafts. We reviewed integrin-dependent mechanisms that potentially link mechanotransduction pathways to the upregulation of myogenic genes. Yet, gaps in our understanding make it challenging to use these pathways to theoretically determine optimal ex vivo strain regimens. A multitude of strain protocols have been applied to in vitro cultures for the cultivation of myogenic progenitors (adipose- and bone marrow-derived stem cells and satellite cells) and transformed murine myoblasts, C2C12s. Strain regimens are characterized by orientation, amplitude, and time-dependent factors (effective frequency, duration, and the rest period between successive strain cycles). Analysis of published data has identified possible minimum/maximum values for these parameters and suggests that uniaxial strains may be more potent than biaxial strains, possibly because they more closely mimic physiologic strain profiles. The application of these biophysical stimuli for engineering 3D skeletal muscle grafts is nontrivial and typically requires custom-designed bioreactors used in combination with biomaterial scaffolds. Consideration of the physical properties of these scaffolds is critical for effective transmission of the applied strains to encapsulated cells. Taken together, these studies demonstrate that biomimetic tensile strain generally results in improved myogenic outcomes in myogenic progenitors and differentiated myoblasts. However, for 3D systems, the optimization of the strain regimen may require the entire system including cells, biomaterials, and bioreactor, to be considered in tandem.

Neutral Lipid Biosynthesis in Engineered Escherichia coli: Jojoba Oil-Like Wax Esters and Fatty Acid Butyl Esters

PubMed Central

Kalscheuer, Rainer; Stöveken, Tim; Luftmann, Heinrich; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

2006-01-01

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms. PMID:16461689

Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

PubMed

Kalscheuer, Rainer; Stöveken, Tim; Luftmann, Heinrich; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

2006-02-01

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

Bioconversion of oil sludge into biomass of lipid metabolites for use as a source of biofuel

NASA Astrophysics Data System (ADS)

Shchemelinina, T. N.; Matistov, N. V.; Markarova, M. Yu; Anchugova, E. M.

2018-01-01

The possibilities for the generation of biofuel from the results of the accumulation of lipids in oil-contaminated environments were studied. This type of accumulation occurs in the biomass of yeast strains Rhodotorula sp. VKM Y-2993D; in bacteria like Pseudomonas libanensis B-3041D and in consortia of microalgal strains such as Acutodesmus obliquus Syko-A Ch-055-12, Chlorella sp. SYKO A Ch-011-10, Monoraphidium sp., and Anabaena sp. The most promising of these for processing petroleum hydrocarbons into biofuels was found to be the consortium of microalgal strains, the content of palmitic acid of which reached 49.0 %, thereby achieving a mid-range cetane number.

Is policy well-targeted to remedy financial strain among caregivers of severely injured U.S. service members?

PubMed

Van Houtven, Courtney Harold; Friedemann-Sánchez, Greta; Clothier, Barbara; Levison, Deborah; Taylor, Brent C; Jensen, Agnes C; Phelan, Sean M; Griffin, Joan M

U.S. military service members have sustained severe injuries since the start of the wars in Iraq and Afghanistan. This paper aims to determine the factors associated with financial strain of their caregivers and establish whether recent federal legislation targets caregivers experiencing financial strain. In our national survey, 62.3% of caregivers depleted assets and/or accumulated debt, and 41% of working caregivers left the labor force. If a severely injured veteran needed intensive help, the primary caregiver faced odds 4.63 times higher of leaving the labor force, and used $27,576 more in assets and/or accumulated debt compared to caregivers of veterans needing little or no assistance.

RNA-Seq Analysis Provides Insights for Understanding Photoautotrophic Polyhydroxyalkanoate Production in Recombinant Synechocystis Sp.

PubMed Central

Lau, Nyok-Sean; Foong, Choon Pin; Kurihara, Yukio; Sudesh, Kumar; Matsui, Minami

2014-01-01

The photosynthetic cyanobacterium, Synechocystis sp. strain 6803, is a potential platform for the production of various chemicals and biofuels. In this study, direct photosynthetic production of a biopolymer, polyhydroxyalkanoate (PHA), in genetically engineered Synechocystis sp. achieved as high as 14 wt%. This is the highest production reported in Synechocystis sp. under photoautotrophic cultivation conditions without the addition of a carbon source. The addition of acetate increased PHA accumulation to 41 wt%, and this value is comparable to the highest production obtained with cyanobacteria. Transcriptome analysis by RNA-seq coupled with real-time PCR was performed to understand the global changes in transcript levels of cells subjected to conditions suitable for photoautotrophic PHA biosynthesis. There was lower expression of most PHA synthesis-related genes in recombinant Synechocystis sp. with higher PHA accumulation suggesting that the concentration of these enzymes is not the limiting factor to achieving high PHA accumulation. In order to cope with the higher PHA production, cells may utilize enhanced photosynthesis to drive the product formation. Results from this study suggest that the total flux of carbon is the possible driving force for the biosynthesis of PHA and the polymerizing enzyme, PHA synthase, is not the only critical factor affecting PHA-synthesis. Knowledge of the regulation or control points of the biopolymer production pathways will facilitate the further use of cyanobacteria for biotechnological applications. PMID:24466058

Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

PubMed

Shui, Zong-Xia; Qin, Han; Wu, Bo; Ruan, Zhi-yong; Wang, Lu-shang; Tan, Fu-Rong; Wang, Jing-Li; Tang, Xiao-Yu; Dai, Li-Chun; Hu, Guo-Quan; He, Ming-Xiong

2015-07-01

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

Increased growth and root Cu accumulation of Sorghum sudanense by endophytic Enterobacter sp. K3-2: Implications for Sorghum sudanense biomass production and phytostabilization.

PubMed

Li, Ya; Wang, Qi; Wang, Lu; He, Lin-Yan; Sheng, Xia-Fang

2016-02-01

Endophytic bacterial strain K3-2 was isolated from the roots of Sorghum sudanense (an bioenergy plant) grown in a Cu mine wasteland soils and characterized. Strain K3-2 was identified as Enterobacter sp. based on 16S rRNA gene sequence analysis. Strain K3-2 exhibited Cu resistance and produced 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA), siderophores, and arginine decarboxylase. Pot experiments showed that strain K3-2 significantly increased the dry weight and root Cu accumulation of Sorghum sudanense grown in the Cu mine wasteland soils. Furthermore, increase in total Cu uptake (ranging from 49% to 95%) of the bacterial inoculated-Sorghum sudanense was observed compared to the control. Notably, most of Cu (83-86%) was accumulated in the roots of Sorghum sudanense. Furthermore, inoculation with strain K3-2 was found to significantly increase Cu bioconcentration factors and the proportions of IAA- and siderophore-producing bacteria in the root interiors and rhizosphere soils of Sorghum sudanense compared with the control. Significant decrease in the available Cu content was also observed in the rhizosphere soils of the bacterial-inoculated Sorghum sudanense. The results suggest that the endophytic bacterial strain K3-2 may be exploited for promoting Sorghum sudanense biomass production and Cu phytostabilization in the Cu mining wasteland soils. Copyright © 2015 Elsevier Inc. All rights reserved.

Poly(3-hydroxybutyrate) hyperproduction by a global nitrogen regulator NtrB mutant strain of Paracoccus denitrificans PD1222

PubMed Central

Olaya-Abril, Alfonso; Luque-Almagro, Víctor M; Manso, Isabel; Gates, Andrew J; Moreno-Vivián, Conrado; Richardson, David J

2017-01-01

Abstract Paracoccus denitrificans PD1222 accumulates short-length polyhydroxyalkanoates, poly(3-hydroxybutyrate), under nitrogen-deficient conditions. Polyhydroxybutyrate metabolism requires the 3-ketoacyl-CoA thiolase PhaA, the acetoacetyl-CoA dehydrogenase/reductase PhaB and the synthase PhaC for polymerization. Additionally, P. denitrificans PD1222 grows aerobically with nitrate as sole nitrogen source. Nitrate assimilation is controlled negatively by ammonium through the two-component NtrBC system. NtrB is a sensor kinase that autophosphorylates a histidine residue under low-nitrogen concentrations and, in turn, transfers a phosphoryl group to an aspartate residue of the response regulator NtrC protein, which acts as a transcriptional activator of the P. denitrificans PD1222 nasABGHC genes. The P. denitrificans PD1222 NtrB mutant was unable to use nitrate efficiently as nitrogen source when compared to the wild-type strain, and it also overproduced poly(3-hydroxybutyrate). Acetyl-CoA concentration in the P. denitrificans PD1222 NtrB mutant strain was higher than in the wild-type strain. The expression of the phaC gene was also increased in the NtrB mutant when compared to the wild-type strain. These results suggest that accumulation of poly(3-hydroxybutyrate) in the NtrB mutant strain of PD1222 responds to the high levels of acetyl-CoA that accumulate in the cytoplasm as consequence of its inability to efficiently use nitrate as nitrogen source. PMID:29228177

Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization.

PubMed

Gao, Qi; Cao, Xuan; Huang, Yu-Ying; Yang, Jing-Lin; Chen, Jun; Wei, Liu-Jing; Hua, Qiang

2018-05-18

Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y. lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated β-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y. lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.

Distinct Osmoadaptation Strategies in the Strict Halophilic and Halotolerant Bacteria Isolated from Lunsu Salt Water Body of North West Himalayas.

PubMed

Vaidya, Shivani; Dev, Kamal; Sourirajan, Anuradha

2018-07-01

Two strict halophilic bacterial strains, Halobacillus trueperi SS1, and Halobacillus trueperi SS3, and three halotolerant bacterial strains, Shewanella algae SS2, Halomonas venusta SS5, and Marinomonas sp. SS8 of Lunsu salt water body, Himachal Pradesh, India, were selected to study the mechanism of salt tolerance and the role of osmolytes therein. A combination of flame photometry, chromatographic and colorimetric assays was used to study the mechanism of salt tolerance in the selected strict halophilic and halotolerant bacterial strains. The strict halophiles and, one of the halotolerants, Marinomonas sp. SS8 were found to utilize both "salt-in strategy" and "accumulation of compatible solutes strategy" for osmoregulation in hypersaline conditions. On the contrary, the remaining two halotolerants used "accumulation of compatible solutes strategy" under saline stress and not the "salt-in strategy". The present study suggests towards distinct mechanisms of salt tolerance in the two classes, wherein strict halophiles accumulate compatible solutes as well as adopt salt-in strategy, while the halotolerant bacteria accumulate a range of compatible solutes, except Marinomonas sp. SS8, which utilizes both the strategies to combat salt stress.

Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus.

PubMed

Cario, Anaïs; Jebbar, Mohamed; Thiel, Axel; Kervarec, Nelly; Oger, Phil M

2016-07-05

The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG accumulation was strongly reduced under supra-optimal pressure conditions clearly demonstrating the structural adaptation of this proteome to high hydrostatic pressure. The lack of MG synthesis only slightly altered the growth characteristics of two different MG synthesis deletion mutants. No shift to other osmolytes was observed. Altogether our observations suggest that the salinity stress response in T. barophilus is not essential and may be under negative selective pressure, similarly to what has been observed for its thermal stress response.

Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus

PubMed Central

Cario, Anaïs; Jebbar, Mohamed; Thiel, Axel; Kervarec, Nelly; Oger, Phil M.

2016-01-01

The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG accumulation was strongly reduced under supra-optimal pressure conditions clearly demonstrating the structural adaptation of this proteome to high hydrostatic pressure. The lack of MG synthesis only slightly altered the growth characteristics of two different MG synthesis deletion mutants. No shift to other osmolytes was observed. Altogether our observations suggest that the salinity stress response in T. barophilus is not essential and may be under negative selective pressure, similarly to what has been observed for its thermal stress response. PMID:27378270

Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum.

PubMed

Schwentner, Andreas; Feith, André; Münch, Eugenia; Busche, Tobias; Rückert, Christian; Kalinowski, Jörn; Takors, Ralf; Blombach, Bastian

2018-03-06

Evolutionary approaches are often undirected and mutagen-based yielding numerous mutations, which need elaborate screenings to identify relevant targets. We here apply Metabolic engineering to Guide Evolution (MGE), an evolutionary approach evolving and identifying new targets to improve microbial producer strains. MGE is based on the idea to impair the cell's metabolism by metabolic engineering, thereby generating guided evolutionary pressure. It consists of three distinct phases: (i) metabolic engineering to create the evolutionary pressure on the applied strain followed by (ii) a cultivation phase with growth as straightforward screening indicator for the evolutionary event, and (iii) comparative whole genome sequencing (WGS), to identify mutations in the evolved strains, which are eventually re-engineered for verification. Applying MGE, we evolved the PEP and pyruvate carboxylase-deficient strain C. glutamicum Δppc Δpyc to grow on glucose as substrate with rates up to 0.31 ± 0.02 h -1 which corresponds to 80% of the growth rate of the wildtype strain. The intersection of the mutations identified by WGS revealed isocitrate dehydrogenase (ICD) as consistent target in three independently evolved mutants. Upon re-engineering in C. glutamicum Δppc Δpyc, the identified mutations led to diminished ICD activities and activated the glyoxylate shunt replenishing oxaloacetate required for growth. Intracellular relative quantitative metabolome analysis showed that the pools of citrate, isocitrate, cis-aconitate, and L-valine were significantly higher compared to the WT control. As an alternative to existing L-valine producer strains based on inactivated or attenuated pyruvate dehydrogenase complex, we finally engineered the PEP and pyruvate carboxylase-deficient C. glutamicum strains with identified ICD mutations for L-valine production by overexpression of the L-valine biosynthesis genes. Among them, C. glutamicum Δppc Δpyc ICD G407S (pJC4ilvBNCE) produced up to 8.9 ± 0.4 g L-valine L -1 , with a product yield of 0.22 ± 0.01 g L-valine per g glucose. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Genetic Engineering: A Promising Tool to Engender Physiological, Biochemical, and Molecular Stress Resilience in Green Microalgae

PubMed Central

Guihéneuf, Freddy; Khan, Asif; Tran, Lam-Son P.

2016-01-01

As we march into the 21st century, the prevailing scenario of depleting energy resources, global warming and ever increasing issues of human health and food security will quadruple. In this context, genetic and metabolic engineering of green microalgae complete the quest toward a continuum of environmentally clean fuel and food production. Evolutionarily related, but unlike land plants, microalgae need nominal land or water, and are best described as unicellular autotrophs using light energy to fix atmospheric carbon dioxide (CO2) into algal biomass, mitigating fossil CO2 pollution in the process. Remarkably, a feature innate to most microalgae is synthesis and accumulation of lipids (60–65% of dry weight), carbohydrates and secondary metabolites like pigments and vitamins, especially when grown under abiotic stress conditions. Particularly fruitful, such an application of abiotic stress factors such as nitrogen starvation, salinity, heat shock, etc., can be used in a biorefinery concept for production of multiple valuable products. The focus of this mini-review underlies metabolic reorientation practices and tolerance mechanisms as applied to green microalgae under specific stress stimuli for a sustainable pollution-free future. Moreover, we entail current progress on genetic engineering as a promising tool to grasp adaptive processes for improving strains with potential biotechnological interests. PMID:27066043

Characterization of Two Novel Propachlor Degradation Pathways in Two Species of Soil Bacteria

PubMed Central

Martin, Margarita; Mengs, Gerardo; Allende, Jose Luis; Fernandez, Javier; Alonso, Ramon; Ferrer, Estrella

1999-01-01

Propachlor (2-chloro-N-isopropylacetanilide) is an acetamide herbicide used in preemergence. In this study, we isolated and characterized a soil bacterium, Acinetobacter strain BEM2, that was able to utilize this herbicide as the sole and limiting carbon source. Identification of the intermediates of propachlor degradation by this strain and characterization of new metabolites in the degradation of propachlor by a previously reported strain of Pseudomonas (PEM1) support two different propachlor degradation pathways. Washed-cell suspensions of strain PEM1 with propachlor accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain PEM1 grew on propachlor with a generation time of 3.4 h and a Ks of 0.17 ± 0.04 mM. Acinetobacter strain BEM2 grew on propachlor with a generation time of 3.1 h and a Ks of 0.3 ± 0.07 mM. Incubations with strain BEM2 resulted in accumulation of N-isopropylacetanilide, N-isopropylaniline, isopropylamine, and catechol. Both degradative pathways were inducible, and the principal product of the carbon atoms in the propachlor ring was carbon dioxide. These results and biodegradation experiments with the identified metabolites indicate that metabolism of propachlor by Pseudomonas sp. strain PEM1 proceeds through a different pathway from metabolism by Acinetobacter sp. strain BEM2. PMID:9925619

Methods of producing protoporphyrin IX and bacterial mutants therefor

DOEpatents

Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

2016-03-01

The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

An Outbreak of Diarrhea in Mandera, Kenya, Due to Escherichia coli Serogroup O-Nontypable Strain That Had a Coding Gene for Enteroaggregative E. coli Heat-Stable Enterotoxin 1.

PubMed

Ochi, Sadayuki; Shah, Mohammad; Odoyo, Erick; Bundi, Martin; Miringu, Gabriel; Guyo, Sora; Wandera, Ernest; Kathiiko, Cyrus; Kariuki, Samuel; Karama, Mohamed; Tsuji, Takao; Ichinose, Yoshio

2017-02-08

In an outbreak of gastroenteritis in December 2009, in Mandera, Kenya, Escherichia coli O-nontypable (ONT) strain was isolated from stool specimens of patients (18/24, 75%). The E. coli ONT organisms could not be assigned to any of the recognized diarrheagenic groups of E. coli However, they possessed the enteroaggregative E. coli heat-stable enterotoxin-1 gene. The cell-free culture filtrates of the E. coli ONT strain isolated from the outbreak cases induced considerable amount of fluid accumulation in suckling mouse intestine, indicating production of an enterotoxic factor(s). These results identify E. coli that did not have any diarrheagenic characteristics except astA as the etiological agent of the diarrheal outbreak in Mandera. It is however considered necessary to characterize the fluid accumulation factor(s) to determine whether any novel toxins were responsible for the fluid accumulation. Moreover, it is important to study dissemination of strains producing the enterotoxic factor(s) to assess their public health significance distribution in the environment. © The American Society of Tropical Medicine and Hygiene.

Quiet Clean Short-haul Experimental Engine (QCSEE) under-the-wing engine composite fan blade design report

NASA Technical Reports Server (NTRS)

Ravenhall, R.; Salemme, C. T.

1977-01-01

A total of 38 quiet clean short haul experimental engine under the wing composite fan blades were manufactured for various component tests, process and tooling, checkout, and use in the QCSEE UTW engine. The component tests included frequency characterization, strain distribution, bench fatigue, platform static load, whirligig high cycle fatigue, whirligig low cycle fatigue, whirligig strain distribution, and whirligig over-speed. All tests were successfully completed. All blades planned for use in the engine were subjected to and passed a whirligig proof spin test.

Genetically based population divergence in overwintering energy mobilization in brook charr (Salvelinus fontinalis).

PubMed

Crespel, Amélie; Bernatchez, Louis; Garant, Dany; Audet, Céline

2013-03-01

Investigating the nature of physiological traits potentially related to fitness is important towards a better understanding of how species and/or populations may respond to selective pressures imposed by contrasting environments. In northern species in particular, the ability to mobilize energy reserves to compensate for the low external energy intake during winter is crucial. However, the phenotypic and genetic bases of energy reserve accumulation and mobilization have rarely been investigated, especially pertaining to variation in strategy adopted by different populations. In the present study, we documented variation in several energy reserve variables and estimated their quantitative genetic basis to test the null hypothesis of no difference in variation at those traits among three strains of brook charr (Salvelinus fontinalis) and their reciprocal hybrids. Our results indicate that the strategy of winter energy preparation and mobilization was specific to each strain, whereby (1) domestic fish accumulated a higher amount of energy reserves before winter and kept accumulating liver glycogen during winter despite lower feeding; (2) Laval fish used liver glycogen and lipids during winter and experienced a significant decrease in condition factor; (3) Rupert fish had relatively little energy reserves accumulated at the end of fall and preferentially mobilized visceral fat during winter. Significant heritability for traits related to the accumulation and use of energy reserves was found in the domestic and Laval but not in the Rupert strain. Genetic and phenotypic correlations also varied among strains, which suggested population-specific genetic architecture underlying the expression of these traits. Hybrids showed limited evidence of non-additive effects. Overall, this study provides the first evidence of a genetically based-and likely adaptive-population-specific strategy for energy mobilization related to overwinter survival.

THE SOURCE OF LIPID ACCUMULATION IN L CELLS

PubMed Central

Bensch, Klaus G.; King, Donald W.; Socolow, Edward L.

1961-01-01

Strain L cells accumulate lipid, concurrent with cessation of protein synthesis, in the stationary phase of growth from the extracellular medium and as a result of de novo synthesis. Cells which have been more severely damaged with an amino acid analogue also accumulate lipid from the extracellular medium, but synthesize very little lipid from labeled acetate. The possible roles which lipid accumulation may play in the cell are discussed. PMID:19866577

Whole Genome Re-Sequencing Identifies a Quantitative Trait Locus Repressing Carbon Reserve Accumulation during Optimal Growth in Chlamydomonas reinhardtii

PubMed Central

Goold, Hugh Douglas; Nguyen, Hoa Mai; Kong, Fantao; Beyly-Adriano, Audrey; Légeret, Bertrand; Billon, Emmanuelle; Cuiné, Stéphan; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua

2016-01-01

Microalgae have emerged as a promising source for biofuel production. Massive oil and starch accumulation in microalgae is possible, but occurs mostly when biomass growth is impaired. The molecular networks underlying the negative correlation between growth and reserve formation are not known. Thus isolation of strains capable of accumulating carbon reserves during optimal growth would be highly desirable. To this end, we screened an insertional mutant library of Chlamydomonas reinhardtii for alterations in oil content. A mutant accumulating five times more oil and twice more starch than wild-type during optimal growth was isolated and named constitutive oil accumulator 1 (coa1). Growth in photobioreactors under highly controlled conditions revealed that the increase in oil and starch content in coa1 was dependent on light intensity. Genetic analysis and DNA hybridization pointed to a single insertional event responsible for the phenotype. Whole genome re-sequencing identified in coa1 a >200 kb deletion on chromosome 14 containing 41 genes. This study demonstrates that, 1), the generation of algal strains accumulating higher reserve amount without compromising biomass accumulation is feasible; 2), light is an important parameter in phenotypic analysis; and 3), a chromosomal region (Quantitative Trait Locus) acts as suppressor of carbon reserve accumulation during optimal growth. PMID:27141848

Metabolic engineering of microbial competitive advantage for industrial fermentation processes.

PubMed

Shaw, A Joe; Lam, Felix H; Hamilton, Maureen; Consiglio, Andrew; MacEwen, Kyle; Brevnova, Elena E; Greenhagen, Emily; LaTouf, W Greg; South, Colin R; van Dijken, Hans; Stephanopoulos, Gregory

2016-08-05

Microbial contamination is an obstacle to widespread production of advanced biofuels and chemicals. Current practices such as process sterilization or antibiotic dosage carry excess costs or encourage the development of antibiotic resistance. We engineered Escherichia coli to assimilate melamine, a xenobiotic compound containing nitrogen. After adaptive laboratory evolution to improve pathway efficiency, the engineered strain rapidly outcompeted a control strain when melamine was supplied as the nitrogen source. We additionally engineered the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica to assimilate nitrogen from cyanamide and phosphorus from potassium phosphite, and they outcompeted contaminating strains in several low-cost feedstocks. Supplying essential growth nutrients through xenobiotic or ecologically rare chemicals provides microbial competitive advantage with minimal external risks, given that engineered biocatalysts only have improved fitness within the customized fermentation environment. Copyright © 2016, American Association for the Advancement of Science.

Strain response of thermal barrier coatings captured under extreme engine environments through synchrotron X-ray diffraction

NASA Astrophysics Data System (ADS)

Knipe, Kevin; Manero, Albert; Siddiqui, Sanna F.; Meid, Carla; Wischek, Janine; Okasinski, John; Almer, Jonathan; Karlsson, Anette M.; Bartsch, Marion; Raghavan, Seetha

2014-07-01

The mechanical behaviour of thermal barrier coatings in operation holds the key to understanding durability of jet engine turbine blades. Here we report the results from experiments that monitor strains in the layers of a coating subjected to thermal gradients and mechanical loads representing extreme engine environments. Hollow cylindrical specimens, with electron beam physical vapour deposited coatings, were tested with internal cooling and external heating under various controlled conditions. High-energy synchrotron X-ray measurements captured the in situ strain response through the depth of each layer, revealing the link between these conditions and the evolution of local strains. Results of this study demonstrate that variations in these conditions create corresponding trends in depth-resolved strains with the largest effects displayed at or near the interface with the bond coat. With larger temperature drops across the coating, significant strain gradients are seen, which can contribute to failure modes occurring within the layer adjacent to the interface.

Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast

PubMed Central

Oud, Bart; Maris, Antonius J A; Daran, Jean-Marc; Pronk, Jack T

2012-01-01

Successful reverse engineering of mutants that have been obtained by nontargeted strain improvement has long presented a major challenge in yeast biotechnology. This paper reviews the use of genome-wide approaches for analysis of Saccharomyces cerevisiae strains originating from evolutionary engineering or random mutagenesis. On the basis of an evaluation of the strengths and weaknesses of different methods, we conclude that for the initial identification of relevant genetic changes, whole genome sequencing is superior to other analytical techniques, such as transcriptome, metabolome, proteome, or array-based genome analysis. Key advantages of this technique over gene expression analysis include the independency of genome sequences on experimental context and the possibility to directly and precisely reproduce the identified changes in naive strains. The predictive value of genome-wide analysis of strains with industrially relevant characteristics can be further improved by classical genetics or simultaneous analysis of strains derived from parallel, independent strain improvement lineages. PMID:22152095

Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast.

PubMed

Oud, Bart; van Maris, Antonius J A; Daran, Jean-Marc; Pronk, Jack T

2012-03-01

Successful reverse engineering of mutants that have been obtained by nontargeted strain improvement has long presented a major challenge in yeast biotechnology. This paper reviews the use of genome-wide approaches for analysis of Saccharomyces cerevisiae strains originating from evolutionary engineering or random mutagenesis. On the basis of an evaluation of the strengths and weaknesses of different methods, we conclude that for the initial identification of relevant genetic changes, whole genome sequencing is superior to other analytical techniques, such as transcriptome, metabolome, proteome, or array-based genome analysis. Key advantages of this technique over gene expression analysis include the independency of genome sequences on experimental context and the possibility to directly and precisely reproduce the identified changes in naive strains. The predictive value of genome-wide analysis of strains with industrially relevant characteristics can be further improved by classical genetics or simultaneous analysis of strains derived from parallel, independent strain improvement lineages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Strain accumulation across the Coast Ranges at the latitude of San Francisco, 1994-2000

USGS Publications Warehouse

Savage, J.C.; Gan, Weijun; Prescott, W.H.; Svarc, J.L.

2004-01-01

A 66-monument geodetic array spanning the Coast Ranges near San Francisco has been surveyed more than eight times by GIPS between late 1993 and early 2001. The measured horizontal velocities of the monuments are well represented by uniform, right-lateral, simple shear parallel to N29??W. (The local strike of the San Andreas Fault is ???N34??W. The observed areal dilatation rate of 6.9 ?? 10.0 nstrain yr-1 (quoted uncertainty is one standard deviation and extension is reckoned positive) is not significantly different from zero, which implies that the observed strain accumulation could be released by strike-slip faulting alone. Our results are consistent with the slip rates assigned by the Working Group on California Earthquake Probabilities [2003] to the principal faults (San Gregorio, San Andreas, Hayward-Rodgers Creek, Calaveras-Concord-Green Valley, and Greenville Faults) cutting across the GPS array. The vector sum of those slip rates is 39.8 ?? 2.6 mm yr-1 N29.8??W ?? 2.8??, whereas the motion across the GPS array (breadth 120 km) inferred from the uniform strain rate approximation is 38.7 ?? 1.2 mm yr-1 N29.0?? ?? 0.9?? right-lateral shear and 0.4 ?? 0.9 mm yr-1 N61??E ?? 0.9?? extension. We interpret the near coincidence of these rates and the absence of significant accumulation of areal dilatation to imply that right-lateral slip on the principal faults can release the accumulating strain; major strain release on reverse faults subparallel to the San Andreas Fault within the Coast Ranges is not required. Copyright 2004 by the American Geophysical union.

Low-cycle fatigue analysis of a cooled copper combustion chamber

NASA Technical Reports Server (NTRS)

Miller, R. W.

1974-01-01

A three-dimensional finite element elastoplastic strain analysis was performed for the throat section of regeneratively cooled rocket engine combustion chamber. The analysis included thermal and pressure loads, and the effects of temperature dependent material properties, to determine the strain range corresponding to the engine operating cycle. The strain range was used in conjunction with OFHC copper isothermal fatigue test data to predict engine low-cycle fatigue life. The analysis was performed for chamber configuration and operating conditions corresponding to a hydrogen-oxygen chamber which was fatigue tested to failure at the NASA Lewis Research Center.

Full Flight Envelope Direct Thrust Measurement on a Supersonic Aircraft

NASA Technical Reports Server (NTRS)

Conners, Timothy R.; Sims, Robert L.

1998-01-01

Direct thrust measurement using strain gages offers advantages over analytically-based thrust calculation methods. For flight test applications, the direct measurement method typically uses a simpler sensor arrangement and minimal data processing compared to analytical techniques, which normally require costly engine modeling and multisensor arrangements throughout the engine. Conversely, direct thrust measurement has historically produced less than desirable accuracy because of difficulty in mounting and calibrating the strain gages and the inability to account for secondary forces that influence the thrust reading at the engine mounts. Consequently, the strain-gage technique has normally been used for simple engine arrangements and primarily in the subsonic speed range. This paper presents the results of a strain gage-based direct thrust-measurement technique developed by the NASA Dryden Flight Research Center and successfully applied to the full flight envelope of an F-15 aircraft powered by two F100-PW-229 turbofan engines. Measurements have been obtained at quasi-steady-state operating conditions at maximum non-augmented and maximum augmented power throughout the altitude range of the vehicle and to a maximum speed of Mach 2.0 and are compared against results from two analytically-based thrust calculation methods. The strain-gage installation and calibration processes are also described.

Rational, combinatorial, and genomic approaches for engineering L-tyrosine production in Escherichia coli.

PubMed

Santos, Christine Nicole S; Xiao, Wenhai; Stephanopoulos, Gregory

2012-08-21

Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.

Environmental risk assessment of a genetically-engineered microorganism: Erwinia carotovora

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orvos, D.R.

1989-01-01

Environmental use of genetically-engineered microorganisms (GEMs) has raised concerns over potential ecological impact. Development of microcosm systems useful in preliminary testing for risk assessment will provide useful information for predicting potential structural, functional, and genetic effects of GEM release. This study was executed to develop techniques that may be useful in risk assessment and microbial ecology, to ascertain which parameters are useful in determining risk and to predict risk from releasing an engineered strain of Erwinia carotovora. A terrestrial microcosm system for use in GEM risk assessment studies was developed for use in assessing alterations of microbial structure and functionmore » that may be caused by introducing the engineered strain of E. carotovora. This strain is being developed for use as a biological control agent for plant soft rot. Parameters that were monitored included survival and intraspecific competition of E. carotovora, structural effects upon both total bacterial populations and numbers of selected bacterial genera, effects upon activities of dehydrogenase and alkaline phosphatase, effects upon soil nutrients, and potential for gene transfer into or out of the engineered strain.« less

Rational, combinatorial, and genomic approaches for engineering L-tyrosine production in Escherichia coli

PubMed Central

Santos, Christine Nicole S.; Xiao, Wenhai; Stephanopoulos, Gregory

2012-01-01

Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations. PMID:22869698

Systematic engineering of TCA cycle for optimal production of a four-carbon platform chemical 4-hydroxybutyric acid in Escherichia coli.

PubMed

Choi, Sol; Kim, Hyun Uk; Kim, Tae Yong; Lee, Sang Yup

2016-11-01

To address climate change and environmental problems, it is becoming increasingly important to establish biorefineries for the production of chemicals from renewable non-food biomass. Here we report the development of Escherichia coli strains capable of overproducing a four-carbon platform chemical 4-hybroxybutyric acid (4-HB). Because 4-HB production is significantly affected by aeration level, genome-scale metabolic model-based engineering strategies were designed under aerobic and microaerobic conditions with emphasis on oxidative/reductive TCA branches and glyoxylate shunt. Several different metabolic engineering strategies were employed to develop strains suitable for fermentation both under aerobic and microaerobic conditions. It was found that microaerobic condition was more efficient than aerobic condition in achieving higher titer and productivity of 4-HB. The final engineered strain produced 103.4g/L of 4-HB by microaerobic fed-batch fermentation using glycerol. The aeration-dependent optimization strategy of TCA cycle will be useful for developing microbial strains producing other reduced derivative chemicals of TCA cycle intermediates. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic engineering of Saccharomyces cerevisiae ethanol strains PE-2 and CAT-1 for efficient lignocellulosic fermentation.

PubMed

Romaní, Aloia; Pereira, Filipa; Johansson, Björn; Domingues, Lucília

2015-03-01

In this work, Saccharomyces cerevisiae strains PE-2 and CAT-1, commonly used in the Brazilian fuel ethanol industry, were engineered for xylose fermentation, where the first fermented xylose faster than the latter, but also produced considerable amounts of xylitol. An engineered PE-2 strain (MEC1121) efficiently consumed xylose in presence of inhibitors both in synthetic and corn-cob hydrolysates. Interestingly, the S. cerevisiae MEC1121 consumed xylose and glucose simultaneously, while a CEN.PK based strain consumed glucose and xylose sequentially. Deletion of the aldose reductase GRE3 lowered xylitol production to undetectable levels and increased xylose consumption rate which led to higher final ethanol concentrations. Fermentation of corn-cob hydrolysate using this strain, MEC1133, resulted in an ethanol yield of 0.47 g/g of total sugars which is 92% of the theoretical yield. Copyright © 2014 Elsevier Ltd. All rights reserved.

Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains of Synechococcus sp. PCC 7002.

PubMed

Therien, Jesse B; Zadvornyy, Oleg A; Posewitz, Matthew C; Bryant, Donald A; Peters, John W

2014-01-01

The model alga Chlamydomonas reinhardtii requires acetate as a co-substrate for optimal production of lipids, and the addition of acetate to culture media has practical and economic implications for algal biofuel production. Here we demonstrate the growth of C. reinhardtii on acetate provided by mutant strains of the cyanobacterium Synechococcus sp. PCC 7002. Optimal growth conditions for co-cultivation of C. reinhardtii with wild-type and mutant strains of Synechococcus sp. 7002 were established. In co-culture, acetate produced by a glycogen synthase knockout mutant of Synechococcus sp. PCC 7002 was able to support the growth of a lipid-accumulating mutant strain of C. reinhardtii defective in starch production. Encapsulation of Synechococcus sp. PCC 7002 using an alginate matrix was successfully employed in co-cultures to limit growth and maintain the stability. The ability of immobilized strains of the cyanobacterium Synechococcus sp. PCC 7002 to produce acetate at a level adequate to support the growth of lipid-accumulating strains of C. reinhartdii offers a potentially practical, photosynthetic alternative to providing exogenous acetate into growth media.

Growth and lipid accumulation in three Chlorella strains from different regions in response to diurnal temperature fluctuations.

PubMed

Yang, Weinan; Zou, Shanmei; He, Meilin; Fei, Cong; Luo, Wei; Zheng, Shiyan; Chen, Bo; Wang, Changhai

2016-02-01

It was economically feasible to screen strains adaptive to wide temperature fluctuation for outdoor cultivation without temperature control. In this research, three Chlorella strains from arctic glacier, desert soil and temperate native lake were isolated and identified. The growth, biochemical composition, lipid content and fatty acid composition of each strain cultured under the mode of diurnal temperature fluctuations were compared. All the three Chlorella strains showed desirable abilities of accumulating lipid under diurnal temperature fluctuations and their fatty acid profiles were suitable for biodiesel production, although the growth and biochemical composition were seemed to be region-specific. The highest lipid content was at 51.83±2.49% DW, 42.80±2.97% DW and 36.13±2.27% DW under different temperature fluctuation of 11 °C, 25 °C, 7 °C, respectively. The results indicated that the three Chlorella strains could be promising biodiesel feedstock for outdoor cultivation by the cultural mode of diurnal temperature fluctuations. Copyright © 2015 Elsevier Ltd. All rights reserved.

Generation of lycopene-overproducing strains of the fungus Mucor circinelloides reveals important aspects of lycopene formation and accumulation.

PubMed

Zhang, Yingtong; Chen, Haiqin; Navarro, Eusebio; López-García, Sergio; Chen, Yong Q; Zhang, Hao; Chen, Wei; Garre, Victoriano

2017-03-01

To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation. Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains. A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

Influence of tryptophan and related compounds on ergot alkaloid formation in Claviceps purpurea (FR.) Tul.

PubMed

Erge, D; Schumann, B; Gröger, D

1984-01-01

L-Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine-accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high-yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mM stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5-Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

Significant strain accumulation between the deformation front and landward out-of-sequence thrusts in accretionary wedge of SW Taiwan revealed by cGPS and SAR interferometry

NASA Astrophysics Data System (ADS)

Tsai, M. C.

2017-12-01

High strain accumulation across the fold-and-thrust belt in Southwestern Taiwan are revealed by the Continuous GPS (cGPS) and SAR interferometry. This high strain is generally accommodated by the major active structures in fold-and-thrust belt of western Foothills in SW Taiwan connected to the accretionary wedge in the incipient are-continent collision zone. The active structures across the high strain accumulation include the deformation front around the Tainan Tableland, the Hochiali, Hsiaokangshan, Fangshan and Chishan faults. Among these active structures, the deformation pattern revealed from cGPS and SAR interferometry suggest that the Fangshan transfer fault may be a left-lateral fault zone with thrust component accommodating the westward differential motion of thrust sheets on both side of the fault. In addition, the Chishan fault connected to the splay fault bordering the lower-slope and upper-slope of the accretionary wedge which could be the major seismogenic fault and an out-of-sequence thrust fault in SW Taiwan. The big earthquakes resulted from the reactivation of out-of-sequence thrusts have been observed along the Nankai accretionary wedge, thus the assessment of the major seismogenic structures by strain accumulation between the frontal décollement and out-of-sequence thrusts is a crucial topic. According to the background seismicity, the low seismicity and mid-crust to mantle events are observed inland and the lower- and upper- slope domain offshore SW Taiwan, which rheologically implies the upper crust of the accretionary wedge is more or less aseimic. This result may suggest that the excess fluid pressure from the accretionary wedge not only has significantly weakened the prism materials as well as major fault zone, but also makes the accretionary wedge landward extension, which is why the low seismicity is observed in SW Taiwan area. Key words: Continuous GPS, SAR interferometry, strain rate, out-of-sequence thrust.

Direct observation of local magnetic properties in strain engineered lanthanum cobaltate thin films

NASA Astrophysics Data System (ADS)

Park, S.; Wu, Weida; Freeland, J. W.; Ma, J. X.; Shi, J.

2009-03-01

Strain engineered thin film devices with emergent properties have significant impacts on both technical application and material science. We studied strain-induced modification of magnetic properties (Co spin state) in epitaxially grown lanthanum cobaltate (LaCoO3) thin films with a variable temperature magnetic force microscopy (VT-MFM). The real space observation confirms long range magnetic ordering on a tensile-strained film and non-magnetic low-spin configuration on a low-strained film at low temperature. Detailed study of local magnetic properties of these films under various external magnetic fields will be discussed. Our results also demonstrate that VT-MFM is a very sensitive tool to detect the nanoscale strain induced magnetic defects.

Maximum-biomass prediction of homofermentative Lactobacillus.

PubMed

Cui, Shumao; Zhao, Jianxin; Liu, Xiaoming; Chen, Yong Q; Zhang, Hao; Chen, Wei

2016-07-01

Fed-batch and pH-controlled cultures have been widely used for industrial production of probiotics. The aim of this study was to systematically investigate the relationship between the maximum biomass of different homofermentative Lactobacillus and lactate accumulation, and to develop a prediction equation for the maximum biomass concentration in such cultures. The accumulation of the end products and the depletion of nutrients by various strains were evaluated. In addition, the minimum inhibitory concentrations (MICs) of acid anions for various strains at pH 7.0 were examined. The lactate concentration at the point of complete inhibition was not significantly different from the MIC of lactate for all of the strains, although the inhibition mechanism of lactate and acetate on Lactobacillus rhamnosus was different from the other strains which were inhibited by the osmotic pressure caused by acid anions at pH 7.0. When the lactate concentration accumulated to the MIC, the strains stopped growing. The maximum biomass was closely related to the biomass yield per unit of lactate produced (YX/P) and the MIC (C) of lactate for different homofermentative Lactobacillus. Based on the experimental data obtained using different homofermentative Lactobacillus, a prediction equation was established as follows: Xmax - X0 = (0.59 ± 0.02)·YX/P·C. Copyright © 2016. Published by Elsevier B.V.

Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum.

PubMed

Zhang, Xin; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

2018-05-03

L-Serine is widely used in the pharmaceutical, food, and cosmetics industries. Although direct fermentative production of L-serine from sugar in Corynebacterium glutamicum has been achieved, the L-serine yield remains relatively low. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the L-serine yield based on engineered C. glutamicum ΔSSAAI strain. Subsequently, we developed a novel high-throughput screening method using a biosensor constructed based on NCgl0581, a transcriptional factor specifically responsive to L-serine, so that L-serine concentration within single cell of C. glutamicum can be monitored via fluorescence-activated cell sorting (FACS). Novel L-serine-producing mutants were isolated from a large library of mutagenized cells. The mutant strain A36-pDser was screened from 1.2 × 10 5 cells, and the magnesium ion concentration in the medium was optimized specifically for this mutant. C. glutamicum A36-pDser accumulated 34.78 g/L L-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent C. glutamicum ΔSSAAI-pDser strain, respectively. The L-serine yield achieved in this mutant was the highest of all reported L-serine-producing strains of C. glutamicum. Moreover, the whole-genome sequencing identified 11 non-synonymous mutations of genes associated with metabolic and transport pathways, which might be responsible for the higher L-serine production and better cell growth in C. glutamicum A36-pDser. This study explored an effective mutagenesis strategy and reported a novel high-throughput screening method for the development of L-serine-producing strains.

Texture studies of hot compressed near alpha titanium alloy (IMI 834) at 1000°C with different strain rates

NASA Astrophysics Data System (ADS)

Kodli, B. K.; Saxena, K. K.; Dey, S. R.; Pancholi, V.; Bhattacharjee, A.

2015-04-01

IMI 834 Titanium alloy is a near alpha (hcp) titanium alloy used for high temperature applications with the service temperature up to 600°C. Generally, this alloy is widely used in gas turbine engine applications such as low pressure compressor discs. For these applications, good fatigue and creep properties are required, which have been noticed better in a bimodal microstructure, containing 15-20% volume fraction of primary alpha grains (αp) and remaining bcc beta (β) grains transformed secondary alpha laths (αs). The bimodal microstructure is achieved during processing of IMI 834 in the high temperature α+β region. The major issue of bimodal IMI 834 during utilization is its poor dwell fatigue life time caused by textured macrozones. Textured macrozone is the spatial accumulation of similar oriented grains in the microstructure generated during hot processing in the high temperature α+β region. Textured macrozone can be mitigated by controlling the hot deformation with certain strain rate under stable plastic conditions having β grains undergoing dynamic recrystallization. Hence, a comprehensive study is required to understand the deformation behavior of α and β grains at different strain rates in that region. Hot compression tests up to 5°% strain of the samples are performed with five different strain rates i.e. 10-3 s-1, 10-2 s-1, 10-1 s-1, 1 s-1 and 10 s-1 at 1000°C using Gleeble 3800. The resultant bimodal microstructure and the texture studies of primary alpha grains (αp) and secondary alpha laths (αs) are carried out using scanning electron microscopy (SEM)-electron back scattered diffraction (EBSD) method.

Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white rot fungi.

PubMed Central

Field, J A; de Jong, E; Feijoo Costa, G; de Bont, J A

1992-01-01

Eight rapid Poly R-478 dye-decolorizing isolates from The Netherlands were screened in this study for the biodegradation of polycyclic aromatic hydrocarbons (PAH) supplied at 10 mg liter(-1). Several well-known ligninolytic culture collection strains, Phanerochaete chrysosporium BKM-F-1767, Trametes versicolor Paprican 52, and Bjerkandera adusta CBS 595.78 were tested in parallel. All of the strains significantly removed anthracene, and nine of the strains significantly removed benzo(a)pyrene beyond the limited losses observed in sterile liquid and HgCl2-poisoned fungus controls. One of the new isolates, Bjerkandera sp. strain Bos 55, was the best degrader of both anthracene and benzo(a)pyrene, removing 99.2 and 83.1% of these compounds after 28 days, respectively. Half of the strains, exemplified by strains of the genera Bjerkandera and Phanerochaete, converted anthracene to anthraquinone, which was found to be a dead-end metabolite, in high yields. The extracellular fluids of selected strains were shown to be implicated in this conversion. In contrast, four Trametes strains removed anthracene without significant accumulation of the quinone. The ability of Trametes strains to degrade anthraquinone was confirmed in this study. None of the strains accumulated PAH quinones during benzo(a)pyrene degradation. Biodegradation of PAH by the various strains was highly correlated to the rate by which they decolorized Poly R-478 dye, demonstrating that ligninolytic indicators are useful in screening for promising PAH-degrading white rot fungal strains. PMID:1637159

Thraustochytrids as production organisms for docosahexaenoic acid (DHA), squalene, and carotenoids.

PubMed

Aasen, Inga Marie; Ertesvåg, Helga; Heggeset, Tonje Marita Bjerkan; Liu, Bin; Brautaset, Trygve; Vadstein, Olav; Ellingsen, Trond E

2016-05-01

Thraustochytrids have been applied for industrial production of the omega-3 fatty acid docosahexaenoic (DHA) since the 1990s. During more than 20 years of research on this group of marine, heterotrophic microorganisms, considerable increases in DHA productivities have been obtained by process and medium optimization. Strains of thraustochytrids also produce high levels of squalene and carotenoids, two other commercially interesting compounds with a rapidly growing market potential, but where yet few studies on process optimization have been reported. Thraustochytrids use two pathways for fatty acid synthesis. The saturated fatty acids are produced by the standard fatty acid synthesis, while DHA is synthesized by a polyketide synthase. However, fundamental knowledge about the relationship between the two pathways is still lacking. In the present review, we extract main findings from the high number of reports on process optimization for DHA production and interpret these in the light of the current knowledge of DHA synthesis in thraustochytrids and lipid accumulation in oleaginous microorganisms in general. We also summarize published reports on squalene and carotenoid production and review the current status on strain improvement, which has been hampered by the yet very few published genome sequences and the lack of tools for gene transfer to the organisms. As more sequences now are becoming available, targets for strain improvement can be identified and open for a system-level metabolic engineering for improved productivities.

Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria

PubMed Central

Kaper, Thijs; Lager, Ida; Looger, Loren L; Chermak, Diane; Frommer, Wolf B

2008-01-01

Background Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production. Results An Escherichia coli expression vector compatible with phage λ recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates. Conclusion The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation. PMID:18522753

Production and Assessment of Damaged High Energy Propellant Samples,

DTIC Science & Technology

1980-05-08

and (c) -69.8% ...... 14 3 Longitudinal Velocity one hour after Compressing Versus Applied Engineering Compressive Strain for Propellant Samples...LONGITUDINAL VELOCITY ONE HOUR AFTER COMPRESSING VERSUS APPLIED ENGINEERING COMPRESSIVE STRAIN FOR PROPELLANT SAMPLES (NOMINAL 40 mm DIA x 13 mm HIGH

Role of Cations in Accumulation and Release of Phosphate by Acinetobacter Strain 210A

PubMed Central

van Groenestijn, Johan W.; Vlekke, Gerard J. F. M.; Anink, Désirée M. E.; Deinema, Maria H.; Zehnder, Alexander J. B.

1988-01-01

Cells of the strictly aerobic Acinetobacter strain 210A, containing aerobically large amounts of polyphosphate (100 mg of phosphorus per g [dry weight] of biomass), released in the absence of oxygen 1.49 mmol of Pi, 0.77 meq of Mg2+, 0.48 meq of K+, 0.02 meq of Ca2+, and 0.14 meq of NH4+ per g (dry weight) of biomass. The drop in pH during this anaerobic phase was caused by the release of 1.8 protons per PO43− molecule. Cells of Acinetobacter strain 132, which do not accumulate polyphosphate aerobically, released only 0.33 mmol of Pi and 0.13 meq of Mg2+ per g (dry weight) of biomass but released K+ in amounts comparable to those released by strain 210A. Stationary-phase cultures of Acinetobacter strain 210A, in which polyphosphate could not be detected by Neisser staining, aerobically took up phosphate simultaneously with Mg2+, the most important counterion in polyphosphate. In the absence of dissolved phosphate in the medium, no Mg2+ was taken up. Cells containing polyphosphate granules were able to grow in a Mg-free medium, whereas cells without these granules were not. Mg2+ was not essential as a counterion because it could be replaced by Ca2+. The presence of small amounts of K+ was essential for polyphosphate formation in cells of strain 210A. During continuous cultivation under K+ limitation, cells of Acinetobacter strain 210A contained only 14 mg of phosphorus per g (dry weight) of biomass, whereas this element was accumulated in amounts of 59 mg/g under substrate limitation and 41 mg/g under Mg2+ limitation. For phosphate uptake in activated sludge, the presence of K+ seemed to be crucial. PMID:16347788

Phase-Change Memory Materials by Design: A Strain Engineering Approach.

PubMed

Zhou, Xilin; Kalikka, Janne; Ji, Xinglong; Wu, Liangcai; Song, Zhitang; Simpson, Robert E

2016-04-20

Van der Waals heterostructure superlattices of Sb2 Te1 and GeTe are strain-engineered to promote switchable atomic disordering, which is confined to the GeTe layer. Careful control of the strain in the structures presents a new degree of freedom to design the properties of functional superlattice structures for data storage and photonics applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increase of chromium tolerance in Scenedesmus acutus after sulfur starvation: Chromium uptake and compartmentalization in two strains with different sensitivities to Cr(VI).

PubMed

Marieschi, M; Gorbi, G; Zanni, C; Sardella, A; Torelli, A

2015-10-01

In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys. Hence, sulfur availability can modulate the capacity to cope with environmental stresses, a phenomenon known as SIR/SED (Sulfur Induced Resistance or Sulfur Enhanced Defence). Since chromate may compete for sulfate transport into the cells, in this study chromium accumulation and tolerance were investigated in relation to sulfur availability in two strains of the unicellular green alga Scenedesmus acutus with different Cr-sensitivities. Paradoxically, sulfur deprivation has been demonstrated to induce a transient increase of Cr-tolerance in both strains. Sulfur deprivation is known to enhance the sulfate uptake/assimilation pathway leading to important consequences on Cr-tolerance: (i) reduced chromate uptake due to the induction of high affinity sulfate transporters (ii) higher production of cysteine and GSH which can play a role both through the formation of unsoluble complexes and their sequestration in inert compartments. To investigate the role of the above mentioned mechanisms, Cr accumulation in total cells and in different cell compartments (cell wall, membranes, soluble and miscellaneous fractions) was analyzed in both sulfur-starved and unstarved cells. Both strains mainly accumulated chromium in the soluble fraction, but the uptake was higher in the wild-type. In this type a short period of sulfur starvation before Cr(VI) treatment lowered chromium accumulation to the level observed in the unstarved Cr-tolerant strain, in which Cr uptake seems instead less influenced by S-starvation, since no significant decrease was observed. The increase in Cr-tolerance following S-starvation seems thus to rely on different mechanisms in the two strains, suggesting the induction of a mechanism constitutively active in the Cr-tolerant strain, maybe a high affinity sulfate transporter also in the wild-type. Changes observed in the cell wall and membrane fractions suggest a strong involvement of these compartments in Cr-tolerance increase following S-starvation. Copyright © 2015 Elsevier B.V. All rights reserved.

Promoter engineering of the Saccharomyces cerevisiae RIM15 gene for improvement of alcoholic fermentation rates under stress conditions.

PubMed

Watanabe, Daisuke; Kaneko, Akie; Sugimoto, Yukiko; Ohnuki, Shinsuke; Takagi, Hiroshi; Ohya, Yoshikazu

2017-02-01

A loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-like protein kinase, is one of the major causes of the high alcoholic fermentation rates in Saccharomyces cerevisiae sake strains closely related to Kyokai no. 7 (K7). However, impairment of Rim15p may not be beneficial under more severe fermentation conditions, such as in the late fermentation stage, as it negatively affects stress responses. To balance stress tolerance and fermentation performance, we inserted the promoter of a gluconeogenic gene, PCK1, into the 5'-untranslated region (5'-UTR) of the RIM15 gene in a laboratory strain to achieve repression of RIM15 gene expression in the glucose-rich early stage with its induction in the stressful late stage of alcoholic fermentation. The promoter-engineered strain exhibited a fermentation rate comparable to that of the RIM15-deleted strain with no decrease in cell viability. The engineered strain achieved better alcoholic fermentation performance than the RIM15-deleted strain under repetitive and high-glucose fermentation conditions. These data demonstrated the validity of promoter engineering of the RIM15 gene that governs inhibitory control of alcoholic fermentation. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A study of elevated temperature testing techniques for the fatigue behavior of PMCS: Application to T650-35/AMB21

NASA Technical Reports Server (NTRS)

Gyekenyesi, Andrew L.; Gastelli, Michael G.; Ellis, John R.; Burke, Christopher S.

1995-01-01

An experimental study was conducted to investigate the mechanical behavior of a T650-35/AMB21 eight-harness satin weave polymer composite system. Emphasis was placed on the development and refinement of techniques used in elevated temperature uniaxial PMC testing. Issues such as specimen design, gripping, strain measurement, and temperature control and measurement were addressed. Quasi-static tensile and fatigue properties (R(sub sigma) = 0.1) were examined at room and elevated temperatures. Stiffness degradation and strain accumulation during fatigue cycling were recorded to monitor damage progression and provide insight for future analytical modeling efforts. Accomplishments included an untabbed dog-bone specimen design which consistently failed in the gage section, accurate temperature control and assessment, and continuous in-situ strain measurement capability during fatigue loading at elevated temperatures. Finally, strain accumulation and stiffness degradation during fatigue cycling appeared to be good indicators of damage progression.

Predicting cell viability within tissue scaffolds under equiaxial strain: multi-scale finite element model of collagen-cardiomyocytes constructs.

PubMed

Elsaadany, Mostafa; Yan, Karen Chang; Yildirim-Ayan, Eda

2017-06-01

Successful tissue engineering and regenerative therapy necessitate having extensive knowledge about mechanical milieu in engineered tissues and the resident cells. In this study, we have merged two powerful analysis tools, namely finite element analysis and stochastic analysis, to understand the mechanical strain within the tissue scaffold and residing cells and to predict the cell viability upon applying mechanical strains. A continuum-based multi-length scale finite element model (FEM) was created to simulate the physiologically relevant equiaxial strain exposure on cell-embedded tissue scaffold and to calculate strain transferred to the tissue scaffold (macro-scale) and residing cells (micro-scale) upon various equiaxial strains. The data from FEM were used to predict cell viability under various equiaxial strain magnitudes using stochastic damage criterion analysis. The model validation was conducted through mechanically straining the cardiomyocyte-encapsulated collagen constructs using a custom-built mechanical loading platform (EQUicycler). FEM quantified the strain gradients over the radial and longitudinal direction of the scaffolds and the cells residing in different areas of interest. With the use of the experimental viability data, stochastic damage criterion, and the average cellular strains obtained from multi-length scale models, cellular viability was predicted and successfully validated. This methodology can provide a great tool to characterize the mechanical stimulation of bioreactors used in tissue engineering applications in providing quantification of mechanical strain and predicting cellular viability variations due to applied mechanical strain.

From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

PubMed Central

Heinrich, Daniel; Andreessen, Björn; Madkour, Mohamed H.; Al-Ghamdi, Mansour A.; Shabbaj, Ibrahim I.

2013-01-01

In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process. PMID:23542629

From waste to plastic: synthesis of poly(3-hydroxypropionate) in Shimwellia blattae.

PubMed

Heinrich, Daniel; Andreessen, Björn; Madkour, Mohamed H; Al-Ghamdi, Mansour A; Shabbaj, Ibrahim I; Steinbüchel, Alexander

2013-06-01

In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.

Characterization and Functional Analyses of R-Specific Enoyl Coenzyme A Hydratases in Polyhydroxyalkanoate-Producing Ralstonia eutropha

PubMed Central

Kawashima, Yui; Cheng, Wen; Mifune, Jun; Orita, Izumi; Nakamura, Satoshi

2012-01-01

A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R-specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded by phaJ4 from Pseudomonas aeruginosa (phaJ4Pa). The recombinant forms of the three proteins, termed PhaJ4aRe to PhaJ4cRe, actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C4 to C8. PhaJ4aRe and PhaJ4bRe showed >10-fold-higher catalytic efficiency than PhaJ4cRe. The functions of the new PhaJ4 proteins were investigated using previously engineered R. eutropha strains as host strains; these strains are capable of synthesizing poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Deletion of phaJ4aRe from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4bRe or phaJ4cRe, indicating that only PhaJ4aRe was one of the major enzymes supplying the (R)-3HHx-CoA monomer through β-oxidation. Introduction of phaJ4aRe or phaJ4bRe into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4cRe did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co-3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially when phaJ4aRe or phaJ4bRe was tandemly introduced with phaJAc from Aeromonas caviae. PMID:22081565

Materials science. Modeling strain hardening the hard way.

PubMed

Gumbsch, Peter

2003-09-26

The plastic deformation of metals results in strain hardening, that is, an increase in the stress with increasing strain. Materials engineers can provide a simple approximate description of such deformation and hardening behavior. In his perspective, Gumbsch discusses work by Madec et al. who have undertaken the formidable task of computing the physical basis for the development of strain hardening by individually following the fate of all the dislocations involved. Their simulations show that the collinear dislocation interaction makes a substantial contribution to strain hardening. It is likely that such simulations will play an important role in guiding the development of future engineering descriptions of deformation and hardening.

Conversion of the high-yield salinomycin producer Streptomyces albus BK3-25 into a surrogate host for polyketide production.

PubMed

Zhang, Xiaojie; Lu, Chenyang; Bai, Linquan

2017-09-01

An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization, fast growth, abundant precursors and energy supply, and a pronounced gene expression. Streptomyces albus BK3-25 is a high-yield industrial strain producing type-I polyketide salinomycin, with a unique ability of bean oil utilization. Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein. Firstly, introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and salinomycin, and subsequent deletion of the salinomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors, including malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA. Secondly, the energy and reducing force were measured, and the improved accumulation of ATP and NADPH was observed in the mutant. Furthermore, the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases, and a strong constitutive promoter was identified, providing a useful tool for further engineered gene expression. Finally, the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor, and the heterologous production of actinorhodin was obtained. This work clearly indicated the potential of the high-yield salinomycin producer as a surrogate host for heterologous production of polyketides, although more genetic manipulation should be conducted to streamline its performance.

Strain Gage Measurements of Aft Nacelle Shock Absorbers.

DTIC Science & Technology

ENGINE NACELLES, SHOCK ABSORBERS ), (* SHOCK ABSORBERS , STRESSES), SURFACE TO SURFACE MISSILES, LAUNCHING, STRAIN GAGES, COMPRESSIVE PROPERTIES, CALIBRATION, STRAIN(MECHANICS), FAILURE, GROUND SUPPORT EQUIPMENT.

Suitability of a PLCL fibrous scaffold for soft tissue engineering applications: A combined biological and mechanical characterisation.

PubMed

Laurent, Cédric P; Vaquette, Cédryck; Liu, Xing; Schmitt, Jean-François; Rahouadj, Rachid

2018-04-01

Poly(lactide-co-ε-caprolactone) (PLCL) has been reported to be a good candidate for tissue engineering because of its good biocompatibility. Particularly, a braided PLCL scaffold (PLL/PCL ratio = 85/15) has been recently designed and partially validated for ligament tissue engineering. In the present study, we assessed the in vivo biocompatibility of acellular and cellularised scaffolds in a rat model. We then determined its in vitro biocompatibility using stem cells issued from both bone marrow and Wharton Jelly. From a biological point of view, the scaffold was shown to be suitable for tissue engineering in all these cases. Secondly, while the initial mechanical properties of this scaffold have been previously reported to be adapted to load-bearing applications, we studied the evolution in time of the mechanical properties of PLCL fibres due to hydrolytic degradation. Results for isolated PLCL fibres were extrapolated to the fibrous scaffold using a previously developed numerical model. It was shown that no accumulation of plastic strain was to be expected for a load-bearing application such as anterior cruciate ligament tissue engineering. However, PLCL fibres exhibited a non-expected brittle behaviour after two months. This may involve a potential risk of premature failure of the scaffold, unless tissue growth compensates this change in mechanical properties. This combined study emphasises the need to characterise the properties of biomaterials in a pluridisciplinary approach, since biological and mechanical characterisations led in this case to different conclusions concerning the suitability of this scaffold for load-bearing applications.

Imminent Engine Failure Probe Investigation.

DTIC Science & Technology

probe signature determination, development of data recording techniques, accumulation of data during durability testing of T56 or TF41 engines and...any other opportunistic gas turbine engine test. The electrostatic probe demonstrated some capability to detect engine distress in TF41 and T56 engines

Automotive Stirling Engine Development Program

NASA Technical Reports Server (NTRS)

Nightingale, N.; Ernst, W.; Richey, A.; Simetkosky, M.; Smith, G.; Rohdenburg, C.; Antonelli, M. (Editor)

1983-01-01

Program status and plans are discussed for component and technology development; reference engine system design, the upgraded Mod 1 engine; industry test and evaluation; and product assurance. Four current Mod 1 engines reached a total of 2523 operational hours, while two upgraded engines accumulated 166 hours.

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli.

PubMed

Chin, Wei-Chih; Lin, Kuo-Hsing; Liu, Chun-Chi; Tsuge, Kenji; Huang, Chieh-Chen

2017-04-11

N-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell. The host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis. These results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD + and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD + redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.

Retaining large and adjustable elastic strains of kilogram-scale Nb nanowires [Better Superconductor by Elastic Strain Engineering: Kilogram-scale Free-Standing Niobium Metal Composite with Large Retained Elastic Strains

DOE PAGES

Hao, Shijie; Cui, Lishan; Wang, Hua; ...

2016-02-10

Crystals held at ultrahigh elastic strains and stresses may exhibit exceptional physical and chemical properties. Individual metallic nanowires can sustain ultra-large elastic strains of 4-7%. However, retaining elastic strains of such magnitude in kilogram-scale nanowires is challenging. Here, we find that under active load, ~5.6% elastic strain can be achieved in Nb nanowires in a composite material. Moreover, large tensile (2.8%) and compressive (-2.4%) elastic strains can be retained in kilogram-scale Nb nanowires when the composite is unloaded to a free-standing condition. It is then demonstrated that the retained tensile elastic strains of Nb nanowires significantly increase their superconducting transitionmore » temperature and critical magnetic fields, corroborating ab initio calculations based on BCS theory. This free-standing nanocomposite design paradigm opens new avenues for retaining ultra-large elastic strains in great quantities of nanowires and elastic-strain-engineering at industrial scale.« less

Genome engineering and gene expression control for bacterial strain development.

PubMed

Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

2015-01-01

In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanisms of fatty acid synthesis in marine fungus-like protists.

PubMed

Xie, Yunxuan; Wang, Guangyi

2015-10-01

Thraustochytrids are unicellular fungus-like protists and are well known for their ability to produce interesting nutraceutical compounds. Significant efforts have been made to improve their efficient production of important fatty acids (FAs), mostly by optimizing fermentation conditions and selecting highly productive thraustochytrid strains. Furthermore, noticeable improvements have been made in understanding the mechanism of FA biosynthesis, allowing for a better understanding of how thraustochytrids assemble these unique metabolites and how their biosynthesis is coupled with other related pathways. This review summarizes recent achievements on two major FA biosynthesis pathways, the standard pathway and the polyketide synthase pathway, and detail features of individual enzymes involved in FA biosynthesis, biotechnological advances in pathway engineering and enzyme characterization, and the discovery of other pathways that affect the efficiency of FA accumulation. Perspectives of biotechnological potential application of thraustochytrids are also discussed.

Characterization of Clostridium thermocellum strains with disrupted fermentation end product pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Der Veen, Douwe; Lo, Jonathan; Brown, Steven D

2013-01-01

Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of Llactate ( ldh) and/or acetate ( pta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end products. Cellobiose-grown cultures of the ldh strain had identical biomass accumulation, fermentation end products, transcription profile and intracellular metabolite concentrations compared to its parent strain (DSM1313 hpt spo0A). The pta-deficient strain grew slower and had 30% lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol.more » A ldh pta double mutant strain evolved for faster growth had growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to pta. Free amino acids were secreted by all examined strains, with both pta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for ldh pta reached 5 mM by the end of growth, or 2.7% of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to 6-fold in the pta and 16-fold in the ldh pta strain. We hypothesize that the deletions in fermentation end product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP+ through increased production of amino acids.« less

Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Der Veen, Douwe; Lo, Jonathan; Brown, Steven D

2013-01-01

Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of L-lactate (Dldh) and/or acetate (Dpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Dldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Dhpt Dspo0A). The Dpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A Dldh Dptamore » double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to Dpta. Free amino acids were secreted by all examined strains, with both Dpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Dldh Dpta reached 5 mM by the end of growth, or 2.7 % of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to sixfold in the Dpta and 16-fold in the Dldh Dpta strain. We hypothesize that the deletions in fermentation end-product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP* through increased production of amino acids.« less

Environmental and High-Strain Rate effects on composites for engine applications

NASA Technical Reports Server (NTRS)

Chamis, C. C.; Smith, G. T.

1982-01-01

The Lewis Research Center is conducting a series of programs intended to investigate and develop the application of composite materials to structural components for turbojet engines. A significant part of that effort is directed to establishing resistance, defect growth, and strain rate characteristics of composite materials over the wide range of environmental and load conditions found in commercial turbojet engine operations. Both analytical and experimental efforts are involved.

The role of the Serratia marcescens SdeAB multidrug efflux pump and TolC homologue in fluoroquinolone resistance studied via gene-knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-02-01

Serratia marcescens is a prominent opportunistic nosocomial pathogen resistant to several classes of antibiotics. The major mechanism for fluoroquinolone resistance in various Gram-negative pathogens is active efflux. Our group previously identified SdeAB, a resistance-nodulation-cell division (RND) efflux pump complex, and a TolC-like outer-membrane protein (HasF), which together mediate energy-dependent fluoroquinolone efflux. In addition, a regulatory protein-encoding gene in the upstream region of sdeAB was identified (sdeR) and found to be 40 % homologous to MarA, an Escherichia coli transcriptional regulator. To provide conclusive evidence as to the role of these components in S. marcescens, sdeB, hasF and sdeR deletion mutants were constructed. Suicide vectors were created and introduced via triparental mating into S. marcescens UOC-67 (wild-type) and, for sdeB and hasF, T-861 (clinical isolate). We have analysed these genetically altered strains using minimal inhibitory concentration (MIC) assays for a wide range of compounds (fluoroquinolones, SDS, novobiocin, ethidium bromide and chloramphenicol). Intracellular accumulation of a variety of fluoroquinolones was measured fluorospectroscopically. The sdeB, hasF and sdeR knockout strains were consistently more susceptible to antibiotics than the parent strains, with the sdeB/hasF double knockout strain showing the highest susceptibility. A marked increase in fluoroquinolone (ciprofloxacin) accumulation was observed for strains deficient in either the sdeB or hasF genes when compared to the parental strains, with the highest ciprofloxacin accumulation observed for the sdeB/hasF double knockout. Antibiotic accumulation assays for the sdeB knockout mutant strains performed in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force inhibitor, demonstrated that SdeAB-mediated efflux is proton-motive-force dependent. Due to the comparable susceptibility of the sdeB and the hasF individual knockouts, we conclude that S. marcescens HasF is the sole outer-membrane component of the SdeAB pump. In addition, MIC data for sdeR-deficient and overexpressing strains confirm that SdeR is an activator of sdeAB and acts to enhance the overall multidrug resistance of S. marcescens.

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4.

PubMed

Herman, Nicolaus A; Li, Jeffrey; Bedi, Ripika; Turchi, Barbara; Liu, Xiaoji; Miller, Michael J; Zhang, Wenjun

2017-01-15

While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 × 10 6 CFU/μg DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombination-mediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5- and 11.8-fold increases (respectively) in batch ethanol production. This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals. Copyright © 2016 American Society for Microbiology.

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

PubMed Central

Herman, Nicolaus A.; Li, Jeffrey; Bedi, Ripika; Turchi, Barbara; Liu, Xiaoji

2016-01-01

ABSTRACT While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 × 106 CFU/μg DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombination-mediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5- and 11.8-fold increases (respectively) in batch ethanol production. IMPORTANCE This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals. PMID:27836845

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens

PubMed Central

Yan, Qing; Lopes, Lucas D.; Shaffer, Brenda T.; Kidarsa, Teresa A.; Vining, Oliver; Philmus, Benjamin; Song, Chunxu; Stockwell, Virginia O.; Raaijmakers, Jos M.; McPhail, Kerry L.; Andreote, Fernando D.; Chang, Jeff H.

2018-01-01

ABSTRACT Secondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. In Pseudomonas spp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations in gacS or gacA (Gac− mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac− mutants in Pseudomonas protegens strain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac− mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac− mutants: a reduced proportion of Gac− mutants accumulated when P. protegens Pf-5 was cocultured with Bacillus subtilis than in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution of P. protegens, assessed by the accumulation of Gac− mutants. PMID:29339425

Crustal strain accumulation on Southern Basin and Range Province faults modulated by distant plate boundary earthquakes? Evidence from geodesy, seismic imaging, and paleoseismology

NASA Astrophysics Data System (ADS)

Bennett, R. A.; Shirzaei, M.; Broermann, J.; Spinler, J. C.; Holland, A. A.; Pearthree, P.

2014-12-01

GPS in Arizona reveals a change in the pattern of crustal strain accumulation in 2010 and based on viscoelastic modeling appears to be associated with the distant M7.2 El Mayor-Cucapah (EMC) earthquake in Baja California, Mexico. GPS data collected between 1999 and 2009 near the Santa Rita normal fault in SE Arizona reveal a narrow zone of crustal deformation coincident with the fault trace, delineated by W-NW facing Pleistocene fault scarps of heights 1 to 7 m. The apparent deformation zone is also seen in a preliminary InSAR interferogram. Total motion across the zone inferred using an elastic block model constrained by the pre-2010 GPS measurements is ~1 mm/yr in a sense consistent with normal fault motion. However, continuous GPS measurements throughout Arizona reveal pronounced changes in crustal velocity following the EMC earthquake, such that the relative motion across the Santa Rita fault post-2010 is negligible. Paleoseismic evidence indicates that mapped Santa Rita fault scarps were formed by two or more large magnitude (M6.7 to M7.6) surface rupturing normal-faulting earthquakes 60 to 100 kyrs ago. Seismic refraction and reflection data constrained by deep (~800 m) well log data provide evidence of progressive, possibly intermittent, displacement on the fault through time. The rate of strain accumulation observed geodetically prior to 2010, if constant over the past 60 to 100 kyrs, would imply an untenable minimum slip rate deficit of 60 to 100 m since the most recent earthquake. One explanation for the available geodetic, seismic, and paleoseismic evidence is that strain accumulation is modulated by viscoelastic relaxation associated with frequent large magnitude earthquakes in the Salton Trough region, episodically inhibiting the accumulation of elastic strain required to generate large earthquakes on the Santa Rita and possibly other faults in the Southern Basin and Range. An important question is thus for how long the postseismic velocity changes will persist relative to the recurrence interval of large Salton Trough earthquakes. Understanding the influence of far-field postseismic deformation on the southern Arizona strain rate field could have implications for other regions of diffuse intracontinental deformation in proximity to frequently rupturing large magnitude plate boundary faults.

Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

PubMed Central

Röttig, Annika; Strittmatter, Carl Simon; Schauer, Jennifer; Hiessl, Sebastian; Daniel, Rolf

2016-01-01

ABSTRACT Recently, we isolated a novel Streptomyces strain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed in Escherichia coli. The purified enzyme AtfG25 showed acyltransferase activity with C12- or C16-acyl-CoA, C12 to C18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of AtfG25 on lipid accumulation, the respective gene, atfG25, was inactivated in Streptomyces sp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that AtfG25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study. IMPORTANCE A novel Streptomyces strain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected acyltransferase were studied. As discussed in this paper, and in contrast to many other bacteria, streptomycetes seem to possess a complex metabolic network to synthesize lipids, whereof crucial steps are still largely unknown. This paper therefore provides insights into a range of topics, including extremophile bacteria, the physiology of lipid accumulation, and the biotechnological production of bacterial lipids. PMID:27474711

An enhanced genome-scale metabolic reconstruction of Streptomyces clavuligerus identifies novel strain improvement strategies.

PubMed

Toro, León; Pinilla, Laura; Avignone-Rossa, Claudio; Ríos-Estepa, Rigoberto

2018-05-01

In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.

Microbial Enzyme Production Using Lignocellulosic Food Industry Wastes as Feedstock: A Review

PubMed Central

Ravindran, Rajeev; Jaiswal, Amit K.

2016-01-01

Enzymes are of great importance in the industry due to their substrate and product specificity, moderate reaction conditions, minimal by-product formation and high yield. They are important ingredients in several products and production processes. Up to 30% of the total production cost of enzymes is attributed to the raw materials costs. The food industry expels copious amounts of processing waste annually, which is mostly lignocellulosic in nature. Upon proper treatment, lignocellulose can replace conventional carbon sources in media preparations for industrial microbial processes, such as enzyme production. However, wild strains of microorganisms that produce industrially important enzymes show low yield and cannot thrive on artificial substrates. The application of recombinant DNA technology and metabolic engineering has enabled researchers to develop superior strains that can not only withstand harsh environmental conditions within a bioreactor but also ensure timely delivery of optimal results. This article gives an overview of the current complications encountered in enzyme production and how accumulating food processing waste can emerge as an environment-friendly and economically feasible solution for a choice of raw material. It also substantiates the latest techniques that have emerged in enzyme purification and recovery over the past four years. PMID:28952592

Enhanced Shewanella biofilm promotes bioelectricity generation.

PubMed

Liu, Ting; Yu, Yang-Yang; Deng, Xiao-Peng; Ng, Chun Kiat; Cao, Bin; Wang, Jing-Yuan; Rice, Scott A; Kjelleberg, Staffan; Song, Hao

2015-10-01

Electroactive biofilms play essential roles in determining the power output of microbial fuel cells (MFCs). To engineer the electroactive biofilm formation of Shewanella oneidensis MR-1, a model exoelectrogen, we herein heterologously overexpressed a c-di-GMP biosynthesis gene ydeH in S. oneidensis MR-1, constructing a mutant strain in which the expression of ydeH is under the control of IPTG-inducible promoter, and a strain in which ydeH is under the control of a constitutive promoter. Such engineered Shewanella strains had significantly enhanced biofilm formation and bioelectricity generation. The MFCs inoculated with these engineered strains accomplished a maximum power density of 167.6 ± 3.6 mW/m(2) , which was ∼ 2.8 times of that achieved by the wild-type MR-1 (61.0 ± 1.9 mW/m(2) ). In addition, the engineered strains in the bioelectrochemical system at poised potential of 0.2 V vs. saturated calomel electrode (SCE) generated a stable current density of 1100 mA/m(2) , ∼ 3.4 times of that by wild-type MR-1 (320 mA/m(2) ). © 2015 Wiley Periodicals, Inc.

Effect of crude and pure glycerol on biomass production and trehalose accumulation by Propionibacterium freudenreichii ssp. shermanii 1.

PubMed

Pawlicka-Kaczorowska, Joanna; Czaczyk, Katarzyna

2017-01-01

The dairy propionibacteria, which are traditionally used for the production of Swiss cheeses, are able to synthesize valuable biomolecules, e.g. B group vitamins, propionic acid, and trehalose with unique chemical and physical properties. Both, dairy propionibacteria cells and trehalose, have found many applications as attractive and effective components in food, beauty and health care products. This study confirmed the ability of several strains from the Propionibacterium genus to create trehalose from glycerol. The research aimed to investigate the effect of crude and pure glycerol on biomass production and on trehalose accumulation by Propionibacterium freudenreichii ssp. shermanii 1. The results indicated that the capacity for trehalose accumulation by Propionibacterium spp. was strain dependent. Propionibacterium freudenreichii ssp. shermanii 1 was able to grow on crude glycerol. For both, pure and crude glycerol, the highest amount of dry biomass leveled off at about 4 g/L. While the use of crude glycerol had no effect on the final concentration of biomass, it reduced the accumulation of trehalose in the cells. An increase in the concentration of carbon source (2-8%) resulted in more than a 5-fold rise in trehalose production. The highest trehalose concentration of 195.04 mg/L was obtained with cultures of the said strain supplemented to 8% with pure glycerol.

Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus.

PubMed Central

Chen, B; Choi, G H; Nuss, D L

1993-01-01

Transmissible hypovirulence is a novel form of biological control in which virulence of a fungal pathogen is attenuated by an endogenous RNA virus. The feasibility of engineering hypovirulence was recently demonstrated by transformation of the chestnut blight fungus, Cryphonectria parasitica, with a full-length cDNA copy of a hypovirulence-associated viral RNA. Engineered hypovirulent transformants were found to contain both a chromsomally integrated cDNA copy of the viral genome and a resurrected cytoplasmically replicating double-stranded RNA form. We now report stable maintenance of integrated viral cDNA through repeated rounds of asexual sporulation and passages on host plant tissue. We also demonstrate stable nuclear inheritance of the integrated viral cDNA and resurrection of the cytoplasmic viral double-stranded RNA form in progeny resulting from the mating of an engineered hypovirulent C. parasitica strain and a vegetatively incompatible virulent strain. Mitotic stability of the viral cDNA ensures highly efficient transmission of the hypovirulence phenotype through conidia. Meiotic transmission, a mode not observed for natural hypovirulent strains, introduces virus into ascospore progeny representing a spectrum of vegetative compatibility groups, thereby circumventing barriers to anastomosis-mediated transmission imposed by the fungal vegetative incompatibility system. These transmission properties significantly enhance the potential of engineered hypovirulent C. parasitica strains as effective biocontrol agents. Images PMID:8344241

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

PubMed Central

Parreiras, Lucas S.; Breuer, Rebecca J.; Avanasi Narasimhan, Ragothaman; Higbee, Alan J.; La Reau, Alex; Tremaine, Mary; Qin, Li; Willis, Laura B.; Bice, Benjamin D.; Bonfert, Brandi L.; Pinhancos, Rebeca C.; Balloon, Allison J.; Uppugundla, Nirmal; Liu, Tongjun; Li, Chenlin; Tanjore, Deepti; Ong, Irene M.; Li, Haibo; Pohlmann, Edward L.; Serate, Jose; Withers, Sydnor T.; Simmons, Blake A.; Hodge, David B.; Westphall, Michael S.; Coon, Joshua J.; Dale, Bruce E.; Balan, Venkatesh; Keating, David H.; Zhang, Yaoping; Landick, Robert; Gasch, Audrey P.; Sato, Trey K.

2014-01-01

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH. PMID:25222864

Users manual for program ADMIT: Admittance and pressure transfer function developed for use on a PC computer

NASA Technical Reports Server (NTRS)

Armstrong, Wilbur C.

1992-01-01

The piping in a liquid rocket can assume complex configurations due to multiple tanks, multiple engines, and structures that must be piped around. The capability to handle some of these complex configurations have been incorporated into the ADMIT code. The capability to modify the input on line has been implemented. The configurations allowed include multiple tanks, multiple engines, the splitting of a pipe into unequal segments going to different (or the same) engines. This program will handle the following type elements: straight pipes, bends, inline accumulators, tuned stub accumulators, Helmholtz resonators, parallel resonators, pumps, split pipes, multiple tanks, and multiple engines.

Users manual for program SSFREQ intermediate mode stability curves: Developed for use on a PC computer

NASA Technical Reports Server (NTRS)

Armstrong, Wilbur C.

1992-01-01

The piping in a liquid rocket can assume complex configurations due to multiple tanks, multiple engines, and structures that must be piped around. The capability to handle some of these complex configurations have been incorporated into the SSFREQ code. The capability to modify the input on line has been implemented. The configurations allowed include multiple tanks, multiple engines, the splitting of a pipe into equal segments going to different (or the same) engines. This program will handle the following type elements: straight pipes, bends, inline accumulators, tuned stub accumulators, Helmholtz resonators, parallel resonators, pumps, split pipes, multiple tanks, and multiple engines.

Improved eco-friendly recombinant Anabaena sp. strain PCC7120 with enhanced nitrogen biofertilizer potential.

PubMed

Chaurasia, Akhilesh Kumar; Apte, Shree Kumar

2011-01-01

Photosynthetic, nitrogen-fixing Anabaena strains are native to tropical paddy fields and contribute to the carbon and nitrogen economy of such soils. Genetic engineering was employed to improve the nitrogen biofertilizer potential of Anabaena sp. strain PCC7120. Constitutive enhanced expression of an additional integrated copy of the hetR gene from a light-inducible promoter elevated HetR protein expression and enhanced functional heterocyst frequency in the recombinant strain. The recombinant strain displayed consistently higher nitrogenase activity than the wild-type strain and appeared to be in homeostasis with compatible modulation of photosynthesis and respiration. The enhanced combined nitrogen availability from the recombinant strain positively catered to the nitrogen demand of rice seedlings in short-term hydroponic experiments and supported better growth. The engineered strain is stable, eco-friendly, and useful for environmental application as nitrogen biofertilizer in paddy fields.

Improved Eco-Friendly Recombinant Anabaena sp. Strain PCC7120 with Enhanced Nitrogen Biofertilizer Potential▿

PubMed Central

Chaurasia, Akhilesh Kumar; Apte, Shree Kumar

2011-01-01

Photosynthetic, nitrogen-fixing Anabaena strains are native to tropical paddy fields and contribute to the carbon and nitrogen economy of such soils. Genetic engineering was employed to improve the nitrogen biofertilizer potential of Anabaena sp. strain PCC7120. Constitutive enhanced expression of an additional integrated copy of the hetR gene from a light-inducible promoter elevated HetR protein expression and enhanced functional heterocyst frequency in the recombinant strain. The recombinant strain displayed consistently higher nitrogenase activity than the wild-type strain and appeared to be in homeostasis with compatible modulation of photosynthesis and respiration. The enhanced combined nitrogen availability from the recombinant strain positively catered to the nitrogen demand of rice seedlings in short-term hydroponic experiments and supported better growth. The engineered strain is stable, eco-friendly, and useful for environmental application as nitrogen biofertilizer in paddy fields. PMID:21057013

Absorption of Radionuclides from the Fukushima Nuclear Accident by a Novel Algal Strain

PubMed Central

Shimura, Hiroki; Itoh, Katsuhiko; Sugiyama, Atsushi; Ichijo, Sayaka; Ichijo, Masashi; Furuya, Fumihiko; Nakamura, Yuji; Kitahara, Ken; Kobayashi, Kazuhiko; Yukawa, Yasuhiro; Kobayashi, Tetsuro

2012-01-01

Large quantities of radionuclides have leaked from the Fukushima Daiichi Nuclear Power Plant into the surrounding environment. Effective prevention of health hazards resulting from radiation exposure will require the development of efficient and economical methods for decontaminating radioactive wastewater and aquatic ecosystems. Here we describe the accumulation of water-soluble radionuclides released by nuclear reactors by a novel strain of alga. The newly discovered green microalgae, Parachlorella sp. binos (Binos) has a thick alginate-containing extracellular matrix and abundant chloroplasts. When this strain was cultured with radioiodine, a light-dependent uptake of radioiodine was observed. In dark conditions, radioiodine uptake was induced by addition of hydrogen superoxide. High-resolution secondary ion mass spectrometry (SIMS) showed a localization of accumulated iodine in the cytosol. This alga also exhibited highly efficient incorporation of the radioactive isotopes strontium and cesium in a light-independent manner. SIMS analysis showed that strontium was distributed in the extracellular matrix of Binos. Finally we also showed the ability of this strain to accumulate radioactive nuclides from water and soil samples collected from a heavily contaminated area in Fukushima. Our results demonstrate that Binos could be applied to the decontamination of iodine, strontium and cesium radioisotopes, which are most commonly encountered after nuclear reactor accidents. PMID:22984475

Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast

PubMed Central

Brickwedde, Anja; van den Broek, Marcel; Geertman, Jan-Maarten A.; Magalhães, Frederico; Kuijpers, Niels G. A.; Gibson, Brian; Pronk, Jack T.; Daran, Jean-Marc G.

2017-01-01

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (“attenuation”) of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains. PMID:28943864

Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast.

PubMed

Brickwedde, Anja; van den Broek, Marcel; Geertman, Jan-Maarten A; Magalhães, Frederico; Kuijpers, Niels G A; Gibson, Brian; Pronk, Jack T; Daran, Jean-Marc G

2017-01-01

The lager brewing yeast Saccharomyces pastorianus , an interspecies hybrid of S. eubayanus and S. cerevisiae , ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion ("attenuation") of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14 C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.

Metabolic engineering of Schizosaccharomyces pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose.

PubMed

Ozaki, Aiko; Konishi, Rie; Otomo, Chisako; Kishida, Mayumi; Takayama, Seiya; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

2017-12-01

Modification of the Schizosaccharomyces pombe genome is often laborious, time consuming due to the lower efficiency of homologous recombination. Here, we constructed metabolically engineered S. pombe strains using a CRISPR-Cas9 system and also demonstrated D-lactic acid (D-LA) production from glucose and cellobiose. Genes encoding two separate pyruvate decarboxylases (PDCs), an L-lactic acid dehydrogenase (L-LDH), and a minor alcohol dehydrogenase (SPBC337.11) were disrupted, thereby attenuating ethanol production. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced genes encoding bacterial acetylating acetaldehyde dehydrogenase enzymes (Escherichia coli MhpF and EutE). D-LA production by the resulting strain was achieved by expressing a Lactobacillus plantarum gene encoding D-lactate dehydrogenase. The engineered strain efficiently consumed glucose and produced D-LA at 25.2 g/L from 35.5 g/L of consumed glucose with a yield of 0.71 g D-LA / g glucose. We further modified this strain by expressing beta-glucosidase by cell surface display; the resulting strain produced D-LA at 24.4 g/L from 30 g/L of cellobiose in minimal medium, with a yield of 0.68 g D-LA / g glucose. To our knowledge, this study represents the first report of a S. pombe strain that was metabolically engineered using a CRISPR-Cas9 system, and demonstrates the possibility of engineering S. pombe for the production of value-added chemicals.

Production of novel antibacterial liamocins by strains of Aureobasidium pullulans

USDA-ARS?s Scientific Manuscript database

Certain strains of Aureobasidium pullulans produce liamocins, heavier-than-water “oils” that accumulate in liquid cultures. Liamocins are surface active, and inhibit mammalian cancer cell lines. Recently, we discovered that liamocins have antibacterial activity with specificity against Streptococcus...

SYNTHETIC BIOLOGY. Emergent genetic oscillations in a synthetic microbial consortium.

PubMed

Chen, Ye; Kim, Jae Kyoung; Hirning, Andrew J; Josić, Krešimir; Bennett, Matthew R

2015-08-28

A challenge of synthetic biology is the creation of cooperative microbial systems that exhibit population-level behaviors. Such systems use cellular signaling mechanisms to regulate gene expression across multiple cell types. We describe the construction of a synthetic microbial consortium consisting of two distinct cell types—an "activator" strain and a "repressor" strain. These strains produced two orthogonal cell-signaling molecules that regulate gene expression within a synthetic circuit spanning both strains. The two strains generated emergent, population-level oscillations only when cultured together. Certain network topologies of the two-strain circuit were better at maintaining robust oscillations than others. The ability to program population-level dynamics through the genetic engineering of multiple cooperative strains points the way toward engineering complex synthetic tissues and organs with multiple cell types. Copyright © 2015, American Association for the Advancement of Science.

CO2-limitation-inducible Green Recovery of fatty acids from cyanobacterial biomass

PubMed Central

Liu, Xinyao; Fallon, Sarah; Sheng, Jie; Curtiss, Roy

2011-01-01

Using genetically modified cyanobacterial strains, we engineered a Green Recovery strategy to convert membrane lipids into fatty acids for economical and environmentally sustainable biofuel production. The Green Recovery strategy utilizes lipolytic enzymes under the control of promoters induced by CO2 limitation. Data indicate that strains of the cyanobacterium Synechocystis sp. PCC6803 engineered for Green Recovery underwent degradation of membrane diacylglycerols upon CO2 limitation, leading to release of fatty acids into the culture medium. Recovered fatty acid yields of 36.1 × 10-12 mg/cell were measured in one of the engineered strains (SD239). Green Recovery can be incorporated into previously constructed fatty-acid-secretion strains, enabling fatty acid recovery from the remaining cyanobacterial biomass that will be generated during fatty acid biofuel production in photobioreactors. PMID:21482802

Improving methionine and ATP availability by MET6 and SAM2 co-expression combined with sodium citrate feeding enhanced SAM accumulation in Saccharomyces cerevisiae.

PubMed

Chen, Hailong; Wang, Zhou; Wang, Zhilai; Dou, Jie; Zhou, Changlin

2016-04-01

S-adenosyl-L-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.

Lifetime prediction for the subsurface crack propagation using three-dimensional dynamic FEA model

NASA Astrophysics Data System (ADS)

Yin, Yuan; Chen, Yun-Xia; Liu, Le

2017-03-01

The subsurface crack propagation is one of the major interests for gear system research. The subsurface crack propagation lifetime is the number of cycles remaining for a spall to appear, which can be obtained through either stress intensity factor or accumulated plastic strain analysis. In this paper, the heavy loads are applied to the gear system. When choosing stress intensity factor, the high compressive stress suppresses Mode I stress intensities and severely reduces Mode II stress intensities in the heavily loaded lubricated contacts. Such that, the accumulated plastic strain is selected to calculate the subsurface crack propagation lifetime from the three-dimensional FEA model through ANSYS Workbench transient analysis. The three-dimensional gear FEA dynamic model with the subsurface crack is built through dividing the gears into several small elements. The calculation of the total cycles of the elements is proposed based on the time-varying accumulated plastic strain, which then will be used to calculate the subsurface crack propagation lifetime. During this process, the demonstration from a subsurface crack to a spall can be uncovered. In addition, different sizes of the elements around the subsurface crack are compared in this paper. The influences of the frictional coefficient and external torque on the crack propagation lifetime are also discussed. The results show that the lifetime of crack propagation decreases significantly when the external load T increasing from 100 N m to 150 N m. Given from the distributions of the accumulated plastic strain, the lifetime shares no significant difference when the frictional coefficient f ranging in 0.04-0.06.

Radio Frequency Sensing of Particulate Matter Accumulation on a Gasoline Particulate Filter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parks, James; Prikhodko, Vitaly Y.; Sappok, Alex

Filter Sensing Technology’s radio frequency (RF) sensor for particulate filter on-board diagnostics (OBD) was studied on a lean gasoline engine at the National Transportation Research Center (NTRC) at Oak Ridge National Laboratory (ORNL). The response of the RF sensor to particulate matter (PM) or “soot” accumulation on the gasoline particulate filter (GPF) installed in the engine exhaust was evaluated. In addition, end plugs of the GPF were purposely removed, and subsequent changes to the RF sensor measured soot loading on the GPF were characterized. Results from the study showed that the RF sensor can accurately measure soot accumulation on amore » GPF; furthermore, the predicted decreased soot accumulation due to plug removal was detected by the RF sensor. Overall, the studies were short and preliminary in nature; however, clearly, the RF sensor demonstrated the capability of measuring GPF soot loading at a level suitable for use in lean gasoline engine emission control OBD and control.« less

Thermally Strained Band Gap Engineering of Transition-Metal Dichalcogenide Bilayers with Enhanced Light-Matter Interaction toward Excellent Photodetectors.

PubMed

Wang, Sheng-Wen; Medina, Henry; Hong, Kuo-Bin; Wu, Chun-Chia; Qu, Yindong; Manikandan, Arumugam; Su, Teng-Yu; Lee, Po-Tsung; Huang, Zhi-Quan; Wang, Zhiming; Chuang, Feng-Chuan; Kuo, Hao-Chung; Chueh, Yu-Lun

2017-09-26

Integration of strain engineering of two-dimensional (2D) materials in order to enhance device performance is still a challenge. Here, we successfully demonstrated the thermally strained band gap engineering of transition-metal dichalcogenide bilayers by different thermal expansion coefficients between 2D materials and patterned sapphire structures, where MoS 2 bilayers were chosen as the demonstrated materials. In particular, a blue shift in the band gap of the MoS 2 bilayers can be tunable, displaying an extraordinary capability to drive electrons toward the electrode under the smaller driven bias, and the results were confirmed by simulation. A model to explain the thermal strain in the MoS 2 bilayers during the synthesis was proposed, which enables us to precisely predict the band gap-shifted behaviors on patterned sapphire structures with different angles. Furthermore, photodetectors with enhancement of 286% and 897% based on the strained MoS 2 on cone- and pyramid-patterned sapphire substrates were demonstrated, respectively.

Earthquake potential in California-Nevada implied by correlation of strain rate and seismicity

USGS Publications Warehouse

Zeng, Yuehua; Petersen, Mark D.; Shen, Zheng-Kang

2018-01-01

Rock mechanics studies and dynamic earthquake simulations show that patterns of seismicity evolve with time through (1) accumulation phase, (2) localization phase, and (3) rupture phase. We observe a similar pattern of changes in seismicity during the past century across California and Nevada. To quantify these changes, we correlate GPS strain rates with seismicity. Earthquakes of M > 6.5 are collocated with regions of highest strain rates. By contrast, smaller magnitude earthquakes of M ≥ 4 show clear spatiotemporal changes. From 1933 to the late 1980s, earthquakes of M ≥ 4 were more diffused and broadly distributed in both high and low strain rate regions (accumulation phase). From the late 1980s to 2016, earthquakes were more concentrated within the high strain rate areas focused on the major fault strands (localization phase). In the same time period, the rate of M > 6.5 events also increased significantly in the high strain rate areas. The strong correlation between current strain rate and the later period of seismicity indicates that seismicity is closely related to the strain rate. The spatial patterns suggest that before the late 1980s, the strain rate field was also broadly distributed because of the stress shadows from previous large earthquakes. As the deformation field evolved out of the shadow in the late 1980s, strain has refocused on the major fault systems and we are entering a period of increased risk for large earthquakes in California.

Earthquake Potential in California-Nevada Implied by Correlation of Strain Rate and Seismicity

NASA Astrophysics Data System (ADS)

Zeng, Yuehua; Petersen, Mark D.; Shen, Zheng-Kang

2018-02-01

Rock mechanics studies and dynamic earthquake simulations show that patterns of seismicity evolve with time through (1) accumulation phase, (2) localization phase, and (3) rupture phase. We observe a similar pattern of changes in seismicity during the past century across California and Nevada. To quantify these changes, we correlate GPS strain rates with seismicity. Earthquakes of M > 6.5 are collocated with regions of highest strain rates. By contrast, smaller magnitude earthquakes of M ≥ 4 show clear spatiotemporal changes. From 1933 to the late 1980s, earthquakes of M ≥ 4 were more diffused and broadly distributed in both high and low strain rate regions (accumulation phase). From the late 1980s to 2016, earthquakes were more concentrated within the high strain rate areas focused on the major fault strands (localization phase). In the same time period, the rate of M > 6.5 events also increased significantly in the high strain rate areas. The strong correlation between current strain rate and the later period of seismicity indicates that seismicity is closely related to the strain rate. The spatial patterns suggest that before the late 1980s, the strain rate field was also broadly distributed because of the stress shadows from previous large earthquakes. As the deformation field evolved out of the shadow in the late 1980s, strain has refocused on the major fault systems and we are entering a period of increased risk for large earthquakes in California.

Delay time and Hartman effect in strain engineered graphene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xi, E-mail: xchen@shu.edu.cn; Deng, Zhi-Yong; Ban, Yue, E-mail: yban@shu.edu.cn

2014-05-07

Tunneling times, including group delay and dwell time, are studied for massless Dirac electrons transmitting through a one-dimensional barrier in strain-engineered graphene. The Hartman effect, the independence of group delay on barrier length, is induced by the strain effect, and associated with the transmission gap and the evanescent mode. The influence of barrier height/length and strain modulus/direction on the group delay is also discussed, which provides the flexibility to control the group delay with applications in graphene-based devices. The relationship between group delay and dwell time is finally derived to clarify the nature of the Hartman effect.

Increasing β-catenin/Wnt3A activity levels drive mechanical strain-induced cell cycle progression through mitosis

PubMed Central

Benham-Pyle, Blair W; Sim, Joo Yong; Hart, Kevin C; Pruitt, Beth L; Nelson, William James

2016-01-01

Mechanical force and Wnt signaling activate β-catenin-mediated transcription to promote proliferation and tissue expansion. However, it is unknown whether mechanical force and Wnt signaling act independently or synergize to activate β-catenin signaling and cell division. We show that mechanical strain induced Src-dependent phosphorylation of Y654 β-catenin and increased β-catenin-mediated transcription in mammalian MDCK epithelial cells. Under these conditions, cells accumulated in S/G2 (independent of DNA damage) but did not divide. Activating β-catenin through Casein Kinase I inhibition or Wnt3A addition increased β-catenin-mediated transcription and strain-induced accumulation of cells in S/G2. Significantly, only the combination of mechanical strain and Wnt/β-catenin activation triggered cells in S/G2 to divide. These results indicate that strain-induced Src phosphorylation of β-catenin and Wnt-dependent β-catenin stabilization synergize to increase β-catenin-mediated transcription to levels required for mitosis. Thus, local Wnt signaling may fine-tune the effects of global mechanical strain to restrict cell divisions during tissue development and homeostasis. DOI: http://dx.doi.org/10.7554/eLife.19799.001 PMID:27782880

Strain analysis and microstructural evolution characteristic of neoproterozoic rocks associations of Wadi El Falek, centre Eastern Desert, Egypt

NASA Astrophysics Data System (ADS)

Kassem, Osama M. K.; Rahim, Said H. Abd El; Nashar, El Said R. El

2012-09-01

The estimation of finite strain in rocks is fundamental to a meaningful understanding of deformational processes and products on all scales from microscopic fabric development to regional structural analyses. The Rf/φ and Fry methods on feldspar porphyroclasts and mafic grains from 5 granite, 1 metavolcanic, 3 metasedimentary and 1 granodiorite samples were used in Wadi El Falek region. Finite-strain data shows that a high to moderate range of deformation of the granitic to metavolcano-sedimentary samples and axial ratios in the XZ section range from 1.60 to 4.10 for the Rf/φ method and from 2.80 to 4.90 for the Fry method. Furthermore, the short axes are subvertical associated with a subhorizontal foliation. We conclude that finite strain in the deformed granite rocks is of the same order of magnitude as that from metavolcano-sedimentary rocks. Furthermore, contacts formed during intrusion of plutons with some faults in the Wadi El Falek area under brittle to semi-ductile deformation conditions. In this case, finite strain accumulated during superimposed deformation on the already assembled nappe structure. It indicates that the nappe contacts formed during the accumulation of finite strain.

Developing cellulolytic Yarrowia lipolytica as a platform for the production of valuable products in consolidated bioprocessing of cellulose.

PubMed

Guo, Zhong-Peng; Robin, Julien; Duquesne, Sophie; O'Donohue, Michael Joseph; Marty, Alain; Bordes, Florence

2018-01-01

Both industrial biotechnology and the use of cellulosic biomass as feedstock for the manufacture of various commercial goods are prominent features of the bioeconomy. In previous work, with the aim of developing a consolidated bioprocess for cellulose bioconversion, we conferred cellulolytic activity of Yarrowia lipolytica , one of the most widely studied "nonconventional" oleaginous yeast species. However, further engineering this strain often leads to the loss of previously introduced heterologous genes due to the presence of multiple LoxP sites when using Cre -recombinase to remove previously employed selection markers. In the present study, we first optimized the strategy of expression of multiple cellulases and rescued selection makers to obtain an auxotrophic cellulolytic Y. lipolytica strain. Then we pursued the quest, exemplifying how this cellulolytic Y. lipolytica strain can be used as a CBP platform for the production of target products. Our results reveal that overexpression of SCD1 gene, encoding stearoyl-CoA desaturase, and DGA1 , encoding acyl-CoA:diacylglycerol acyltransferase, confers the obese phenotype to the cellulolytic Y. lipolytica . When grown in batch conditions and minimal medium, the resulting strain consumed 12 g/L cellulose and accumulated 14% (dry cell weight) lipids. Further enhancement of lipid production was achieved either by the addition of glucose or by enhancing cellulose consumption using a commercial cellulase cocktail. Regarding the latter option, although the addition of external cellulases is contrary to the concept of CBP, the amount of commercial cocktail used remained 50% lower than that used in a conventional process (i.e., without internalized production of cellulases). The introduction of the LIP2 gene into cellulolytic Y. lipolytica led to the production of a strain capable of producing lipase 2 while growing on cellulose. Remarkably, when the strain was grown on glucose, the expression of six cellulases did not alter the level of lipase production. When grown in batch conditions on cellulose, the engineered strain consumed 16 g/L cellulose and produced 9.0 U/mL lipase over a 96-h period. The lipase yield was 562 U lipase/g cellulose, which represents 60% of that obtained on glucose. Finally, expression of the hydroxylase from Claviceps purpurea (CpFAH12) in cellulolytic Y. lipolytica procured a strain that can produce ricinoleic acid (RA). Using this strain in batch cultures revealed that the consumption of 11 g/L cellulose sustained the production of 2.2 g/L RA in the decane phase, 69% of what was obtained on glucose. In summary, this study has further demonstrated the potential of cellulolytic Y. lipolytica as a microbial platform for the bioconversion of cellulose into target products. Its ability to be used in consolidated process designs has been exemplified and clues revealing how cellulose consumption can be further enhanced using commercial cellulolytic cocktails are provided.

Antibody responses to avian influenza viruses in wild birds broaden with age

PubMed Central

Manvell, Ruth J.; Schulenburg, Bodo; Shell, Wendy; Wikramaratna, Paul S.; Perrins, Christopher; Sheldon, Ben C.; Brown, Ian H.; Pybus, Oliver G.

2016-01-01

For viruses such as avian influenza, immunity within a host population can drive the emergence of new strains by selecting for viruses with novel antigens that avoid immune recognition. The accumulation of acquired immunity with age is hypothesized to affect how influenza viruses emerge and spread in species of different lifespans. Despite its importance for understanding the behaviour of avian influenza viruses, little is known about age-related accumulation of immunity in the virus's primary reservoir, wild birds. To address this, we studied the age structure of immune responses to avian influenza virus in a wild swan population (Cygnus olor), before and after the population experienced an outbreak of highly pathogenic H5N1 avian influenza in 2008. We performed haemagglutination inhibition assays on sampled sera for five avian influenza strains and show that breadth of response accumulates with age. The observed age-related distribution of antibody responses to avian influenza strains may explain the age-dependent mortality observed during the highly pathogenic H5N1 outbreak. Age structures and species lifespan are probably important determinants of viral epidemiology and virulence in birds. PMID:28003449

Local Mechanical Response of Superelastic NiTi Shape-Memory Alloy Under Uniaxial Loading

NASA Astrophysics Data System (ADS)

Xiao, Yao; Zeng, Pan; Lei, Liping; Du, Hongfei

2015-11-01

In this paper, we focus on the local mechanical response of superelastic NiTi SMA at different temperatures under uniaxial loading. In situ DIC is applied to measure the local strain of the specimen. Based on the experimental results, two types of mechanical response, which are characterized with localized phase transformation and homogenous phase transformation, are identified, respectively. Motivated by residual strain accumulation phenomenon of the superelastic mechanical response, we conduct controlled experiments, and infer that for a given material point, all (or most) of the irreversibility is accumulated when the transformation front is traversing the material point. A robust constitutive model is established to explain the experimental phenomena and we successfully simulate the evolution of local strain that agrees closely with the experimental results.

Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression

PubMed Central

Miyamoto, Kiyoko T.; Komatsu, Mamoru

2014-01-01

Mycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae. These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the order Actinomycetales, Actinosynnema mirum DSM 43827 and Pseudonocardia sp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture, Pseudonocardia sp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereas A. mirum did not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster of A. mirum was in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host, Streptomyces avermitilis SUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore, S. avermitilis SUKA22 transformants carrying the biosynthetic gene cluster for MAA of A. mirum accumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutes l-alanine for the l-serine of shinorine. PMID:24907338

Discovery of gene cluster for mycosporine-like amino acid biosynthesis from Actinomycetales microorganisms and production of a novel mycosporine-like amino acid by heterologous expression.

PubMed

Miyamoto, Kiyoko T; Komatsu, Mamoru; Ikeda, Haruo

2014-08-01

Mycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae. These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the order Actinomycetales, Actinosynnema mirum DSM 43827 and Pseudonocardia sp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture, Pseudonocardia sp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereas A. mirum did not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster of A. mirum was in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host, Streptomyces avermitilis SUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore, S. avermitilis SUKA22 transformants carrying the biosynthetic gene cluster for MAA of A. mirum accumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutes l-alanine for the l-serine of shinorine. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae.

PubMed

Dai, Zhubo; Liu, Yi; Huang, Luqi; Zhang, Xueli

2012-11-01

Metabolic engineering of microorganisms is an alternative and attractive route for production of valuable terpenoids that are usually extracted from plant sources. Tanshinones are the bioactive components of Salvia miltiorrhizha Bunge, which is a well-known traditional Chinese medicine widely used for treatment of many cardiovascular diseases. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase-like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2 mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over-expression of a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1) gene did improve supply of farnesyl diphosphate (FPP), production of miltiradiene was not increased while large amounts of squalene (78 mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8 mg/L by improving supply of GGPP through over-expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over-expressing ERG20-BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2 mg/L. Combinatorial over-expression of tHMGR-upc2.1 and ERG20-BTS1-SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8 mg/L. Finally, fed-batch fermentation was performed, and 488 mg/L miltiradiene was produced. The yeast strains engineered in this work provide a basis for creating an alternative way for production of tanshinones in place of extraction from plant sources. Copyright © 2012 Wiley Periodicals, Inc.

Enhanced nutrient transport improves the depth-dependent properties of tri-layered engineered cartilage constructs with zonal co-culture of chondrocytes and MSCs.

PubMed

Kim, Minwook; Farrell, Megan J; Steinberg, David R; Burdick, Jason A; Mauck, Robert L

2017-08-01

Biomimetic design in cartilage tissue engineering is a challenge given the complexity of the native tissue. While numerous studies have generated constructs with near-native bulk properties, recapitulating the depth-dependent features of native tissue remains a challenge. Furthermore, limitations in nutrient transport and matrix accumulation in engineered constructs hinders maturation within the central core of large constructs. To overcome these limitations, we fabricated tri-layered constructs that recapitulate the depth-dependent cellular organization and functional properties of native tissue using zonally derived chondrocytes co-cultured with MSCs. We also introduced porous hollow fibers (HFs) and HFs/cotton threads to enhance nutrient transport. Our results showed that tri-layered constructs with depth-dependent organization and properties could be fabricated. The addition of HFs or HFs/threads improved matrix accumulation in the central core region. With HF/threads, the local modulus in the deep region of tri-layered constructs nearly matched that of native tissue, though the properties in the central regions remained lower. These constructs reproduced the zonal organization and depth-dependent properties of native tissue, and demonstrate that a layer-by-layer fabrication scheme holds promise for the biomimetic repair of focal cartilage defects. Articular cartilage is a highly organized tissue driven by zonal heterogeneity of cells, extracellular matrix proteins and fibril orientations, resulting in depth-dependent mechanical properties. Therefore, the recapitulation of the functional properties of native cartilage in a tissue engineered construct requires such a biomimetic design of the morphological organization, and this has remained a challenge in cartilage tissue engineering. This study demonstrates that a layer-by-layer fabrication scheme, including co-cultures of zone-specific articular CHs and MSCs, can reproduce the depth-dependent characteristics and mechanical properties of native cartilage while minimizing the need for large numbers of chondrocytes. In addition, introduction of a porous hollow fiber (combined with a cotton thread) enhanced nutrient transport and depth-dependent properties of the tri-layered construct. Such a tri-layered construct may provide critical advantages for focal cartilage repair. These constructs hold promise for restoring native tissue structure and function, and may be beneficial in terms of zone-to-zone integration with adjacent host tissue and providing more appropriate strain transfer after implantation. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

PubMed

Wisselink, H Wouter; Mars, Astrid E; van der Meer, Pieter; Eggink, Gerrit; Hugenholtz, Jeroen

2004-07-01

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

Strain-controlled nonvolatile magnetization switching

NASA Astrophysics Data System (ADS)

Geprägs, S.; Brandlmaier, A.; Brandt, M. S.; Gross, R.; Goennenwein, S. T. B.

2014-11-01

We investigate different approaches towards a nonvolatile switching of the remanent magnetization in single-crystalline ferromagnets at room temperature via elastic strain using ferromagnetic thin film/piezoelectric actuator hybrids. The piezoelectric actuator induces a voltage-controllable strain along different crystalline directions of the ferromagnetic thin film, resulting in modifications of its magnetization by converse magnetoelastic effects. We quantify the magnetization changes in the hybrids via ferromagnetic resonance spectroscopy and superconducting quantum interference device magnetometry. These measurements demonstrate a significant strain-induced change of the magnetization, limited by an inefficient strain transfer and domain formation in the particular system studied. To overcome these obstacles, we address practicable engineering concepts and use a model to demonstrate that a strain-controlled, nonvolatile magnetization switching should be possible in appropriately engineered ferromagnetic/piezoelectric actuator hybrids.

Strain of Escherichia coli with a temperature-sensitive mutation affecting ribosomal ribonucleic acid accumulation.

PubMed Central

Frey, T; Newlin, L L; Atherly, A G

1975-01-01

A mutant of Escherichia coli has been isolated that has a temperature-sensitive mutation that results in specific loss of ribosomal ribonucleic acid (RNA) synthesis and some reduction in messenger RNA synthesis. When the strain was grown in glucose medium at a restrictive temperature, RNA accumulation ceased, but both messenger RNA and protein synthesis continued for an extended time. Because carbon metabolism was slowed drastically when strain AA-157 was placed at the restrictive temperature, this phenotype can be compared with carbon depletion conditions present during diauxic lag. However, the phenotype of mutant AA-157 differs from shift-down conditions in that guanosine-3',5'-tetraphosphate levels are unaffected; therefore, a different site is affected. This mutant strain (AA-157) thus shows many characteristics similar to an aldolase mutant previously reported (Böck and Neidhardt, 1966). However, the mutation occurred in a different position on the E. coli genetic map, and furthermore, aldolase was not temperature sensitive in strain AA-157. In this paper we present a study of macromolecular biosynthesis in this mutant. PMID:1090609

An ocean of stress? The relationship between psychosocial workload and mental strain among engine officers in the Swedish merchant fleet.

PubMed

Rydstedt, Leif W; Lundh, Monica

2010-01-01

The first purpose of this study was to compare the psychosocial working conditions and mental health of our sample of maritime engine officers with a sample of British shore-based professional engineers. The second purpose was to analyse the relationship between the psychosocial working conditions onboard and mental strain for the Swedish maritime engine officers. There were a total of 731 engine officers in the Swedish merchant fleet, almost all males with higher education. The British comparison sample consisted of 312 professional shore-based engineers. A questionnaire was distributed to the Swedish engine officers with a modified version of the JCQ for the DC-S model, the Role conflict and Ambiguity scale, and two items on family-work inter-role conflicts (WFI/FWI), as workload indicators. The General Health Questionnaire (GHQ12) and Perceived Stress Scale (PSS10) were used as strain indicators. There were no significant differences in perceived job stain or in WFI/FWI between the Swedish engine officers and the British professional engineers in perceived job strain. While the British shore-based engineers reported significantly higher role ambiguity the Swedish engine officers perceived a significantly higher degree of role conflict and higher perceived stress. Hierarchic linear regression analysis showed that the Role Stress was strongly related to perceived stress (R(2) = 0.319) as well as to mental health (R(2) = 0.222). When introduced in the second step the DC-S model was significantly related to the outcome measures, as was WFI/FWI when finally introduced. The main source of the high degree of perceived stress among the engine officers does not seem to be the job content but may rather be understood from an interactional perspective, where conflicting requirements are directed towards the individual officer. It can be assumed that the fast technological and organizational changes and the increased pressure for economic profitability that characterize the shipping industry have attenuated these role conflicts.

Impact and Penetration of Thin Aluminum 2024 Flat Panels at Oblique Angles of Incidence

NASA Technical Reports Server (NTRS)

Ruggeri, Charles R.; Revilock, Duane M.; Pereira, J. Michael; Emmerling, William; Queitzsch, Gilbert K., Jr.

2015-01-01

The U.S. Federal Aviation Administration (FAA) and the National Aeronautics and Space Administration (NASA) are actively involved in improving the predictive capabilities of transient finite element computational methods for application to safety issues involving unintended impacts on aircraft and aircraft engine structures. One aspect of this work involves the development of an improved deformation and failure model for metallic materials, known as the Tabulated Johnson-Cook model, or MAT224, which has been implemented in the LS-DYNA commercial transient finite element analysis code (LSTC Corp., Livermore, CA) (Ref. 1). In this model the yield stress is a function of strain, strain rate and temperature and the plastic failure strain is a function of the state of stress, temperature and strain rate. The failure criterion is based on the accumulation of plastic strain in an element. The model also incorporates a regularization scheme to account for the dependency of plastic failure strain on mesh size. For a given material the model requires a significant amount of testing to determine the yield stress and failure strain as a function of the three-dimensional state of stress, strain rate and temperature. In addition, experiments are required to validate the model. Currently the model has been developed for Aluminum 2024 and validated against a series of ballistic impact tests on flat plates of various thicknesses (Refs. 1 to 3). Full development of the model for Titanium 6Al-4V is being completed, and mechanical testing for Inconel 718 has begun. The validation testing for the models involves ballistic impact tests using cylindrical projectiles impacting flat plates at a normal incidence (Ref. 2). By varying the thickness of the plates, different stress states and resulting failure modes are induced, providing a range of conditions over which the model can be validated. The objective of the study reported here was to provide experimental data to evaluate the model under more extreme conditions, using a projectile with a more complex shape and sharp contacts, impacting flat panels at oblique angles of incidence.

40 CFR 92.508 - Calculation and reporting of test results.

Code of Federal Regulations, 2010 CFR

2010-07-01

... specified in § 92.506(a). These results must also include the green engine factor, if applicable. The... engine, including: (A) Configuration and engine family identification; (B) Year, make, and build date; (C... accumulated on locomotive or locomotive engine prior to testing; and (E) Description of green engine factor...

Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications.

PubMed

Takagi, Hiroshi

2008-11-01

Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T (m) of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive gamma-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production.

Rational selection and engineering of exogenous principal sigma factor (σ(HrdB)) to increase teicoplanin production in an industrial strain of Actinoplanes teichomyceticus.

PubMed

Wang, Haiyong; Yang, Liu; Wu, Kuo; Li, Guanghui

2014-01-16

Transcriptional engineering has presented a strong ability of phenotypic improvement in microorganisms. However, it could not be directly applied to Actinoplanes teichomyceticus L-27 because of the paucity of endogenous transcription factors in the strain. In this study, exogenous transcription factors were rationally selected and transcriptional engineering was carried out to increase the productivity of teicoplanin in L-27. It was illuminated that the σ(HrdB) molecules shared strong similarity of amino acid sequences among some genera of actinomycetes. Combining this advantage with the ability of transcriptional engineering, exogenous sigma factor σ(HrdB) molecules were rationally selected and engineered to improve L-27. hrdB genes from Actinoplanes missouriensis 431, Micromonospora aurantiaca ATCC 27029 and Salinispora arenicola CNS-205 were selected based on molecular evolutionary analysis. Random mutagenesis, DNA shuffling and point mutation were subsequently performed to generate diversified mutants. A recombinant was identified through screening program, yielding 5.3 mg/ml of teicoplanin, over 2-fold compared to that of L-27. More significantly, the engineered strain presented a good performance in 500-l pilot scale fermentation, which meant its valuable potential application in industry. Through rational selection and engineering of exogenous transcriptional factor, we have extended the application of transcriptional engineering. To our knowledge, it is the first time to focus on the related issue. In addition, possessing the advantage of efficient metabolic perturbation in transcription level, this strategy could be useful in analyzing metabolic and physiological mechanisms of strains, especially those with the only information on taxonomy.

Systems and synthetic metabolic engineering for amino acid production - the heartbeat of industrial strain development.

PubMed

Becker, Judith; Wittmann, Christoph

2012-10-01

With a world market of more than four million tons per year, l-amino acids are among the most important products in industrial biotechnology. The recent years have seen a tremendous progress in the development of tailor-made strains for such products, intensively driven from systems metabolic engineering, which upgrades strain engineering into a concept of optimization on a global scale. This concept seems especially valuable for efficient amino acid production, demanding for a global modification of pathway fluxes - a challenge with regard to the high complexity of the underlying metabolism, superimposed by various layers of metabolic and transcriptional control. Copyright © 2011 Elsevier Ltd. All rights reserved.

(13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

PubMed

Zhao, Lina; Zhang, Huaiyuan; Wang, Liping; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

2015-12-01

The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Accumulation of radionuclides from radioactive substrata by some micromycetes.

PubMed

Zhdanova, N N; Redchits, T I; Zheltonozhsky, V A; Sadovnikov, L V; Gerzabek, M H; Olsson, S; Strebl, F; Mück, K

2003-01-01

Overgrowing (interaction) and dissolution of intact and milled hot particles by various micromycetes were studied under laboratory conditions. Hot particles used for the investigation originated from the Chernobyl accident release and atomic bomb testing sites. The micromycetes investigated were mitosporic fungi mainly isolated from the Chernobyl site and vicinity. Most of the fungal species and strains showed a tendency to grow towards the hot particle, overgrow it and dissolve it after prolonged contact. The accumulation (absorption and adsorption) of radionuclides from intact hot particles was generally more intensive for (152)Eu than for (137)Cs by a factor of about 2.6-134, while in experiments with milled samples the (152)Eu and (137)Cs accumulation was similar, except for some fungal species, which showed higher (152)Eu than (137)Cs sorption. It could be shown that the main factors influencing Cs and Eu accumulation in fungi are: fungal species and strains and the size and composition of the hot particle.

Enhanced iron and zinc accumulation in genetically engineered pineapple plants using soybean ferritin gene.

PubMed

Mhatre, Minal; Srinivas, Lingam; Ganapathi, Thumballi R

2011-12-01

Pineapple (Ananas comosus L. Merr., cv. "Queen") leaf bases were transformed with Agrobacterium tumefaciens strain EHA 105 harboring the pSF and pEFESF plasmids with soybean ferritin cDNA. Four to eight percent of the co-cultivated leaf bases produced multiple shoots 6 weeks after transfer to Murashige and Skoog's medium supplemented with α-naphthalene acetic acid 1.8 mg/l, indole-3-butyric acid 2.0 mg/l, kinetin 2.0 mg/l, cefotaxime 400 mg/l, and kanamycin 50 mg/l. Putatively transformed shoots (1-2 cm) were selected and multiplied on medium of the same composition and elongated shoots (5 cm) were rooted on liquid rooting medium supplemented with cefotaxime 400 mg/l and kanamycin 100 mg/l. The rooted plants were analyzed through PCR, genomic Southern analysis, and reverse transcription PCR. The results clearly confirmed the integration and expression of soybean ferritin gene in the transformed plants. Atomic absorption spectroscopic analysis carried out with six independently transformed lines of pSF and pEFE-SF revealed a maximum of 5.03-fold increase in iron and 2.44-fold increase in zinc accumulation in the leaves of pSF-transformed plants. In pEFE-SF-transformed plants, a 3.65-fold increase in iron and 2.05-fold increase in zinc levels was observed. Few of the transgenic plants were hardened in the greenhouse and are being grown to maturity to determine the enhanced iron and zinc accumulation in the fruits. To the best of our knowledge this is the first report on the transformation of pineapple with soybean ferritin for enhanced accumulation of iron and zinc content in the transgenic plants.

Effects of strain rate, mixing ratio, and stress-strain definition on the mechanical behavior of the polydimethylsiloxane (PDMS) material as related to its biological applications.

PubMed

Khanafer, Khalil; Duprey, Ambroise; Schlicht, Marty; Berguer, Ramon

2009-04-01

Tensile tests on Polydimethylsiloxane (PDMS) materials were conducted to illustrate the effects of mixing ratio, definition of the stress-strain curve, and the strain rate on the elastic modulus and stress-strain curve. PDMS specimens were prepared according to the ASTM standards for elastic materials. Our results indicate that the physiological elastic modulus depends strongly on the definition of the stress-strain curve, mixing ratio, and the strain rate. For various mixing ratios and strain rates, true stress-strain definition results in higher stress and elastic modulus compared with engineering stress-strain and true stress-engineering strain definitions. The elastic modulus increases as the mixing ratio increases up-to 9:1 ratio after which the elastic modulus begins to decrease even as the mixing ratio continues to increase. The results presented in this study will be helpful to assist the design of in vitro experiments to mimic blood flow in arteries and to understand the complex interaction between blood flow and the walls of arteries using PDMS elastomer.

Finite-strain analysis of Metavolcano-sedimentary rocks at Gabel El Mayet area, Central Eastern Desert, Egypt

NASA Astrophysics Data System (ADS)

Kassem, Osama M. K.; Abd El Rahim, Said H.

2010-09-01

Finite strain was estimated in the metavolcano-sedimentary rocks, which surround by serpentinites of Gabel El Mayet area. Finite strain shows a relationship to nappe contacts between the metavolcano-sedimentary rocks and serpentinite and sheds light on the nature of the subhorizontal foliation typical for the Gable Mayet shear zone. We used the Rf/ ϕ and Fry methods on feldspar porphyroclasts and mafic grains from 10 metasedimentary and six metavolcanic samples in Gabel El Mayet region. Our finite-strain data show that the metavolcano-sedimentary rocks were moderately deformed and axial ratios in the XZ section range from 1.9 to 3.9. The long axes of the finite-strain ellipsoids trend W/WNW in the north and W/WSW in the south of the Gabel El Mayet shear zone. Furthermore, the short axes are subvertical to a subhorizontal foliation. The strain magnitudes increase towards the tectonic contacts between the metavolcano-sedimentary rocks and serpentinite. The data indicate oblate strain symmetry in the metavolcano-sedimentary rocks. Hence, our strain data also indicate flattening strain. We assume that the metasedimentary and metavolcanics rocks have similar deformation behaviour. The fact that finite strain accumulated during the metamorphism indicates that the nappe contacts formed during the accumulation of finite strain and thus during thrusting. We conclude that the nappe contacts formed during progressive thrusting under brittle to semi-brittle deformation conditions by simple shear and involved a component of vertical shortening, which caused the subhorizontal foliation in the Gabel El Mayet shear zone.

Overexpression of the Coq8 Kinase in Saccharomyces cerevisiae coq Null Mutants Allows for Accumulation of Diagnostic Intermediates of the Coenzyme Q6 Biosynthetic Pathway*

PubMed Central

Xie, Letian X.; Ozeir, Mohammad; Tang, Jeniffer Y.; Chen, Jia Y.; Jaquinod, Sylvie-Kieffer; Fontecave, Marc; Clarke, Catherine F.; Pierrel, Fabien

2012-01-01

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q6 biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q6 biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q6. Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q6 biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway. PMID:22593570

Overexpression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.

PubMed

Xie, Letian X; Ozeir, Mohammad; Tang, Jeniffer Y; Chen, Jia Y; Jaquinod, Sylvie-Kieffer; Fontecave, Marc; Clarke, Catherine F; Pierrel, Fabien

2012-07-06

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q(6) biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q(6) biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q(6). Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q(6) biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway.

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

PubMed Central

Flärdh, K; Axberg, T; Albertson, N H; Kjelleberg, S

1994-01-01

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14. PMID:7928955

Observations of strain accumulation across the San Andreas fault near Palmdale, California, with a two-color geodimeter

USGS Publications Warehouse

Langbein, J.O.; Linker, M.F.; McGarr, A.; Slater, L.E.

1982-01-01

Two-color laser ranging measurements during a 15-month period over a geodetic network spanning the San Andreas fault near Palmdale, California, indicate that the crust expands and contracts aseismically in episodes as short as 2 weeks. Shear strain parallel to the fault has accumulated monotonically since November 1980, but at a variable rate. Improvements in measurement precision and temporal resolution over those of previous geodetic studies near Palmdale have resulted in the definition of a time history of crustal deformation that is much more complex than formerly realized. Copyright ?? 1982 AAAS.

Regional Characteristics of Stress State of Main Seismic Active Faults in Mid-Northern Part of Sichuan-Yunnan Block

NASA Astrophysics Data System (ADS)

Weiwei, W.; Yaling, W.

2017-12-01

We restore the seismic source spectrums of 1012 earthquakes(2.0 ≤ ML ≤ 5.0) in the mid-northern part of Sichuan-Yunnan seismic block(26 ° N-33 ° N, 99 ° E-104 ° E),then calculate the source parameters.Based on the regional seismic tectonic background, the distribution of active faults and seismicity, the study area is divided into four statistical units (Z1 Jinshajiang and Litang fault zone, Z2 Xianshuihe fault zone, Z3 Anninghe-Zemuhe fault zone, Z4 Lijiang-Xiaojinhe fault zone). Seismic source stress drop results show the following, (1)The stress at the end of the Jinshajiang fault is low, strong earthquake activity rare.Stress-strain loading deceases gradually from northwest to southeast along Litang fault, the northwest section which is relatively locked is more likely to accumulate strain than southeast section. (2)Stress drop of Z2 is divided by Kangding, the southern section is low and northern section is high. Southern section (Kangding-Shimian) is difficult to accumulate higher strain in the short term, but in northern section (Garzê-Kangding), moderate and strong earthquakes have not filled the gaps of seismic moment release, there is still a high stress accumulation in partial section. (3)High stress-drop events were concentrated on Z3, strain accumulation of this unit is strong, and stress level is the highest, earthquake risk is high. (4)On Z4, stress drop characteristics of different magnitude earthquakes are not the same, which is related to complex tectonic setting, the specific reasons still need to be discussed deeply.The study also show that, (1)Stress drops display a systematic change with different faults and locations, high stress-drop events occurs mostly on the fault intersection area. Faults without locking condition and mainly creep, are mainly characterized by low stress drop. (2)Contrasting to what is commonly thought that "strike-slip faults are not easy to accumulate stress ", Z2 and Z3 all exhibit high stress levels, which may be due to that the magnitude and intensity of medium-strong earthquakes are not enough to release the accumulated energy. On the other hand, when the tectonic unit blocking fault movement and its contribution to accumulation of stress play a key role, the earthquake of same magnitude will release higher stress drop.

Metabolic engineering of Corynebacterium glutamicum for L-arginine production.

PubMed

Park, Seok Hyun; Kim, Hyun Uk; Kim, Tae Yong; Park, Jun Seok; Kim, Suok-Su; Lee, Sang Yup

2014-08-05

L-arginine is an important amino acid for diverse industrial and health product applications. Here we report the development of metabolically engineered Corynebacterium glutamicum ATCC 21831 for the production of L-arginine. Random mutagenesis is first performed to increase the tolerance of C. glutamicum to L-arginine analogues, followed by systems metabolic engineering for further strain improvement, involving removal of regulatory repressors of arginine operon, optimization of NADPH level, disruption of L-glutamate exporter to increase L-arginine precursor and flux optimization of rate-limiting L-arginine biosynthetic reactions. Fed-batch fermentation of the final strain in 5 l and large-scale 1,500 l bioreactors allows production of 92.5 and 81.2 g l(-1) of L-arginine with the yields of 0.40 and 0.35 g L-arginine per gram carbon source (glucose plus sucrose), respectively. The systems metabolic engineering strategy described here will be useful for engineering Corynebacteria strains for the industrial production of L-arginine and related products.

Microbial production of 1-octanol: A naturally excreted biofuel with diesel-like properties

PubMed Central

Akhtar, M. Kalim; Dandapani, Hariharan; Thiel, Kati; Jones, Patrik R.

2014-01-01

The development of sustainable, bio-based technologies to convert solar energy and carbon dioxide into fuels is a grand challenge. A core part of this challenge is to produce a fuel that is compatible with the existing transportation infrastructure. This task is further compounded by the commercial desire to separate the fuel from the biotechnological host. Based on its fuel characteristics, 1-octanol was identified as an attractive metabolic target with diesel-like properties. We therefore engineered a synthetic pathway specifically for the biosynthesis of 1-octanol in Escherichia coli BL21(DE3) by over-expression of three enzymes (thioesterase, carboxylic acid reductase and aldehyde reductase) and one maturation factor (phosphopantetheinyl transferase). Induction of this pathway in a shake flask resulted in 4.4 mg 1-octanol L−1 h−1 which exceeded the productivity of previously engineered strains. Furthermore, the majority (73%) of the fatty alcohol was localised within the media without the addition of detergent or solvent overlay. The deletion of acrA reduced the production and excretion of 1-octanol by 3-fold relative to the wild-type, suggesting that the AcrAB–TolC complex may be responsible for the majority of product efflux. This study presents 1-octanol as a potential fuel target that can be synthesised and naturally accumulated within the media using engineered microbes. PMID:27066394