Four-way regulation of mosquito yolk protein precursor genes by juvenile hormone-, ecdysone-, nutrient-, and insulin-like peptide signaling pathways.

PubMed

Hansen, Immo A; Attardo, Geoffrey M; Rodriguez, Stacy D; Drake, Lisa L

2014-01-01

Anautogenous mosquito females require a meal of vertebrate blood in order to initiate the production of yolk protein precursors by the fat body. Yolk protein precursor gene expression is tightly repressed in a state-of-arrest before blood meal-related signals activate it and expression levels rise rapidly. The best understood example of yolk protein precursor gene regulation is the vitellogenin-A gene (vg) of the yellow fever mosquito Aedes aegypti. Vg-A is regulated by (1) juvenile hormone signaling, (2) the ecdysone-signaling cascade, (3) the nutrient sensitive target-of-rapamycin signaling pathway, and (4) the insulin-like peptide (ILP) signaling pathway. A plethora of new studies have refined our understanding of the regulation of yolk protein precursor genes since the last review on this topic in 2005 (Attardo et al., 2005). This review summarizes the role of these four signaling pathways in the regulation of vg-A and focuses upon new findings regarding the interplay between them on an organismal level.

Four-way regulation of mosquito yolk protein precursor genes by juvenile hormone-, ecdysone-, nutrient-, and insulin-like peptide signaling pathways

PubMed Central

Hansen, Immo A.; Attardo, Geoffrey M.; Rodriguez, Stacy D.; Drake, Lisa L.

2014-01-01

Anautogenous mosquito females require a meal of vertebrate blood in order to initiate the production of yolk protein precursors by the fat body. Yolk protein precursor gene expression is tightly repressed in a state-of-arrest before blood meal-related signals activate it and expression levels rise rapidly. The best understood example of yolk protein precursor gene regulation is the vitellogenin-A gene (vg) of the yellow fever mosquito Aedes aegypti. Vg-A is regulated by (1) juvenile hormone signaling, (2) the ecdysone-signaling cascade, (3) the nutrient sensitive target-of-rapamycin signaling pathway, and (4) the insulin-like peptide (ILP) signaling pathway. A plethora of new studies have refined our understanding of the regulation of yolk protein precursor genes since the last review on this topic in 2005 (Attardo et al., 2005). This review summarizes the role of these four signaling pathways in the regulation of vg-A and focuses upon new findings regarding the interplay between them on an organismal level. PMID:24688471

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

PubMed Central

Li, Min; Zhang, Zhi-Jun; Kong, Xu-Dong; Yu, Hui-Lei

2017-01-01

ABSTRACT Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T5015, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C5015, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter−1 day−1. These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor. IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low

Construction and engineering of large biochemical pathways via DNA assembler

PubMed Central

Shao, Zengyi; Zhao, Huimin

2015-01-01

Summary DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated. PMID:23996442

RNase MRP is required for entry of 35S precursor rRNA into the canonical processing pathway.

PubMed

Lindahl, Lasse; Bommankanti, Ananth; Li, Xing; Hayden, Lauren; Jones, Adrienne; Khan, Miriam; Oni, Tolulope; Zengel, Janice M

2009-07-01

RNase MRP is a nucleolar RNA-protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.

The Engineering Development Array: A Low Frequency Radio Telescope Utilising SKA Precursor Technology

NASA Astrophysics Data System (ADS)

Wayth, Randall; Sokolowski, Marcin; Booler, Tom; Crosse, Brian; Emrich, David; Grootjans, Robert; Hall, Peter J.; Horsley, Luke; Juswardy, Budi; Kenney, David; Steele, Kim; Sutinjo, Adrian; Tingay, Steven J.; Ung, Daniel; Walker, Mia; Williams, Andrew; Beardsley, A.; Franzen, T. M. O.; Johnston-Hollitt, M.; Kaplan, D. L.; Morales, M. F.; Pallot, D.; Trott, C. M.; Wu, C.

2017-08-01

We describe the design and performance of the Engineering Development Array, which is a low-frequency radio telescope comprising 256 dual-polarisation dipole antennas working as a phased array. The Engineering Development Array was conceived of, developed, and deployed in just 18 months via re-use of Square Kilometre Array precursor technology and expertise, specifically from the Murchison Widefield Array radio telescope. Using drift scans and a model for the sky brightness temperature at low frequencies, we have derived the Engineering Development Array's receiver temperature as a function of frequency. The Engineering Development Array is shown to be sky-noise limited over most of the frequency range measured between 60 and 240 MHz. By using the Engineering Development Array in interferometric mode with the Murchison Widefield Array, we used calibrated visibilities to measure the absolute sensitivity of the array. The measured array sensitivity matches very well with a model based on the array layout and measured receiver temperature. The results demonstrate the practicality and feasibility of using Murchison Widefield Array-style precursor technology for Square Kilometre Array-scale stations. The modular architecture of the Engineering Development Array allows upgrades to the array to be rolled out in a staged approach. Future improvements to the Engineering Development Array include replacing the second stage beamformer with a fully digital system, and to transition to using RF-over-fibre for the signal output from first stage beamformers.

Applied evolutionary theories for engineering of secondary metabolic pathways.

PubMed

Bachmann, Brian O

2016-12-01

An expanded definition of 'secondary metabolism' is emerging. Once the exclusive provenance of naturally occurring organisms, evolved over geological time scales, secondary metabolism increasingly encompasses molecules generated via human engineered biocatalysts and biosynthetic pathways. Many of the tools and strategies for enzyme and pathway engineering can find origins in evolutionary theories. This perspective presents an overview of selected proposed evolutionary strategies in the context of engineering secondary metabolism. In addition to the wealth of biocatalysts provided via secondary metabolic pathways, improving the understanding of biosynthetic pathway evolution will provide rich resources for methods to adapt to applied laboratory evolution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pathway Design, Engineering, and Optimization.

PubMed

Garcia-Ruiz, Eva; HamediRad, Mohammad; Zhao, Huimin

The microbial metabolic versatility found in nature has inspired scientists to create microorganisms capable of producing value-added compounds. Many endeavors have been made to transfer and/or combine pathways, existing or even engineered enzymes with new function to tractable microorganisms to generate new metabolic routes for drug, biofuel, and specialty chemical production. However, the success of these pathways can be impeded by different complications from an inherent failure of the pathway to cell perturbations. Pursuing ways to overcome these shortcomings, a wide variety of strategies have been developed. This chapter will review the computational algorithms and experimental tools used to design efficient metabolic routes, and construct and optimize biochemical pathways to produce chemicals of high interest.

Structural basis for precursor protein-directed ribosomal peptide macrocyclization

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

Li, Kunhua; Condurso, Heather L.; Li, Gengnan

Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight intomore » the unique protein–protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.« less

Learning from the Parallel Pathways of Makers to Broaden Pathways to Engineering

ERIC Educational Resources Information Center

Foster, Christina; Wigner, Aubrey; Lande, Micah; Jordan, Shawn S.

2018-01-01

Background: Makers are a growing community of STEM-minded people who bridge technical and non-technical backgrounds to imagine, build and fabricate engineering systems. Some have engineering training, some do not. This paper presents a study to explore the educational pathways of adult Makers and how they intersect with engineering. This research…

Combinatorial pathway optimization in Escherichia coli by directed co-evolution of rate-limiting enzymes and modular pathway engineering.

PubMed

Lv, Xiaomei; Gu, Jiali; Wang, Fan; Xie, Wenping; Liu, Min; Ye, Lidan; Yu, Hongwei

2016-12-01

Metabolic engineering of microorganisms for heterologous biosynthesis is a promising route to sustainable chemical production which attracts increasing research and industrial interest. However, the efficiency of microbial biosynthesis is often restricted by insufficient activity of pathway enzymes and unbalanced utilization of metabolic intermediates. This work presents a combinatorial strategy integrating modification of multiple rate-limiting enzymes and modular pathway engineering to simultaneously improve intra- and inter-pathway balance, which might be applicable for a range of products, using isoprene as an example product. For intra-module engineering within the methylerythritol-phosphate (MEP) pathway, directed co-evolution of DXS/DXR/IDI was performed adopting a lycopene-indicated high-throughput screening method developed herein, leading to 60% improvement of isoprene production. In addition, inter-module engineering between the upstream MEP pathway and the downstream isoprene-forming pathway was conducted via promoter manipulation, which further increased isoprene production by 2.94-fold compared to the recombinant strain with solely protein engineering and 4.7-fold compared to the control strain containing wild-type enzymes. These results demonstrated the potential of pathway optimization in isoprene overproduction as well as the effectiveness of combining metabolic regulation and protein engineering in improvement of microbial biosynthesis. Biotechnol. Bioeng. 2016;113: 2661-2669. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity▿

PubMed Central

Withers, Sydnor T.; Gottlieb, Shayin S.; Lieu, Bonny; Newman, Jack D.; Keasling, Jay D.

2007-01-01

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol. PMID:17693564

Effects of metabolic pathway precursors and polydimethylsiloxane (PDMS) on poly-(gamma)-glutamic acid production by Bacillus subtilis BL53.

PubMed

de Cesaro, Alessandra; da Silva, Suse Botelho; Ayub, Marco Antônio Záchia

2014-09-01

The aims of this study were to evaluate the effects of the addition of metabolic precursors and polydimethylsiloxane (PDMS) as an oxygen carrier to cultures of Bacillus subtilis BL53 during the production of γ-PGA. Kinetics analyses of cultivations of different media showed that B. subtilis BL53 is an exogenous glutamic acid-dependent strain. When the metabolic pathway precursors of γ-PGA synthesis, L-glutamine and a-ketoglutaric acid, were added to the culture medium, production of the biopolymer was increased by 20 % considering the medium without these precursors. The addition of 10 % of the oxygen carrier PDMS to cultures caused a two-fold increase in the volumetric oxygen mass transfer coefficient (kLa), improving γ-PGA production and productivity. Finally, bioreactor cultures of B. subtilis BL53 adopting the combination of optimized medium E, added of glutamine, α-ketoglutaric acid, and PDMS, showed a productivity of 1 g L(-1) h(-1) of g-PGA after only 24 h of cultivation. Results of this study suggest that the use of metabolic pathway precursors glutamine and a-ketolgutaric acid, combined with the addition of PDMS as an oxygen carrier in bioreactors, can improve γ-PGA production and productivity by Bacillus strains .

The arginine deiminase pathway of koji bacteria is involved in ethyl carbamate precursor production in soy sauce.

PubMed

Zhang, Jiran; Fang, Fang; Chen, Jian; Du, Guocheng

2014-09-01

Ethyl carbamate (EC) is a group 2A carcinogen generated from a few precursors in many fermented foods and alcoholic beverages. Citrulline, urea, carbamoyl phosphate, and ethanol are common precursors detected in fermented foods. In this study, citrulline was proved to be the main EC precursor in soy sauce, which was found to be accumulated in moromi mash period and correlated with the utilization of arginine by koji bacteria. Six koji isolates belonging to three genera were identified to be able to accumulate citrulline via the arginine deiminase (ADI) pathway. Among these strains, only Pediococcus acidilactici retained high activities in synthesis and accumulation of citrulline in the presence of high concentration of sodium chloride. These results suggested that P. acidilactici is responsible for the accumulation of citrulline, one of the EC precursors, in the process of soy sauce fermentation. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Precise precursor rebalancing for isoprenoids production by fine control of gapA expression in Escherichia coli.

PubMed

Jung, Juyoung; Lim, Jae Hyung; Kim, Se Yeon; Im, Dae-Kyun; Seok, Joo Yeon; Lee, Seung-Jae V; Oh, Min-Kyu; Jung, Gyoo Yeol

2016-11-01

Biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway requires equimolar glyceraldehyde 3-phosphate and pyruvate to divert carbon flux toward the products of interest. Here, we demonstrate that precursor balancing is one of the critical steps for the production of isoprenoids in Escherichia coli. First, the implementation of the synthetic lycopene production pathway as a model system and the amplification of the native DXP pathway were accomplished using synthetic constitutive promoters and redesigned 5'-untranslated regions (5'-UTRs). Next, fine-controlled precursor balancing was investigated by tuning phosphoenolpyruvate synthase (PpsA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results showed that tuning-down of gapA improved the specific lycopene content by 45% compared to the overexpression of ppsA. The specific lycopene content in the strains with down-regulated gapA increased by 97% compared to that in the parental strain. Our results indicate that gapA is the best target for precursor balancing to increase biosynthesis of isoprenoids. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Combining chemoinformatics with bioinformatics: in silico prediction of bacterial flavor-forming pathways by a chemical systems biology approach "reverse pathway engineering".

PubMed

Liu, Mengjin; Bienfait, Bruno; Sacher, Oliver; Gasteiger, Johann; Siezen, Roland J; Nauta, Arjen; Geurts, Jan M W

2014-01-01

The incompleteness of genome-scale metabolic models is a major bottleneck for systems biology approaches, which are based on large numbers of metabolites as identified and quantified by metabolomics. Many of the revealed secondary metabolites and/or their derivatives, such as flavor compounds, are non-essential in metabolism, and many of their synthesis pathways are unknown. In this study, we describe a novel approach, Reverse Pathway Engineering (RPE), which combines chemoinformatics and bioinformatics analyses, to predict the "missing links" between compounds of interest and their possible metabolic precursors by providing plausible chemical and/or enzymatic reactions. We demonstrate the added-value of the approach by using flavor-forming pathways in lactic acid bacteria (LAB) as an example. Established metabolic routes leading to the formation of flavor compounds from leucine were successfully replicated. Novel reactions involved in flavor formation, i.e. the conversion of alpha-hydroxy-isocaproate to 3-methylbutanoic acid and the synthesis of dimethyl sulfide, as well as the involved enzymes were successfully predicted. These new insights into the flavor-formation mechanisms in LAB can have a significant impact on improving the control of aroma formation in fermented food products. Since the input reaction databases and compounds are highly flexible, the RPE approach can be easily extended to a broad spectrum of applications, amongst others health/disease biomarker discovery as well as synthetic biology.

The Hippo Pathway Regulates Homeostatic Growth of Stem Cell Niche Precursors in the Drosophila Ovary

PubMed Central

Sarikaya, Didem P.; Extavour, Cassandra G.

2015-01-01

The Hippo pathway regulates organ size, stem cell proliferation and tumorigenesis in adult organs. Whether the Hippo pathway influences establishment of stem cell niche size to accommodate changes in organ size, however, has received little attention. Here, we ask whether Hippo signaling influences the number of stem cell niches that are established during development of the Drosophila larval ovary, and whether it interacts with the same or different effector signaling pathways in different cell types. We demonstrate that canonical Hippo signaling regulates autonomous proliferation of the soma, while a novel hippo-independent activity of Yorkie regulates autonomous proliferation of the germ line. Moreover, we demonstrate that Hippo signaling mediates non-autonomous proliferation signals between germ cells and somatic cells, and contributes to maintaining the correct proportion of these niche precursors. Finally, we show that the Hippo pathway interacts with different growth pathways in distinct somatic cell types, and interacts with EGFR and JAK/STAT pathways to regulate non-autonomous proliferation of germ cells. We thus provide evidence for novel roles of the Hippo pathway in establishing the precise balance of soma and germ line, the appropriate number of stem cell niches, and ultimately regulating adult female reproductive capacity. PMID:25643260

In Situ Passivation for Efficient PbS Quantum Dot Solar Cells by Precursor Engineering.

PubMed

Wang, Yongjie; Lu, Kunyuan; Han, Lu; Liu, Zeke; Shi, Guozheng; Fang, Honghua; Chen, Si; Wu, Tian; Yang, Fan; Gu, Mengfan; Zhou, Sijie; Ling, Xufeng; Tang, Xun; Zheng, Jiawei; Loi, Maria Antonietta; Ma, Wanli

2018-04-01

Current efforts on lead sulfide quantum dot (PbS QD) solar cells are mostly paid to the device architecture engineering and postsynthetic surface modification, while very rare work regarding the optimization of PbS synthesis is reported. Here, PbS QDs are successfully synthesized using PbO and PbAc 2  · 3H 2 O as the lead sources. QD solar cells based on PbAc-PbS have demonstrated a high power conversion efficiency (PCE) of 10.82% (and independently certificated values of 10.62%), which is significantly higher than the PCE of 9.39% for PbO-PbS QD based ones. For the first time, systematic investigations are carried out on the effect of lead precursor engineering on the device performance. It is revealed that acetate can act as an efficient capping ligands together with oleic acid, providing better surface coverage and replace some of the harmful hydroxyl (OH) ligands during the synthesis. Then the acetate on the surface can be exchanged by iodide and lead to desired passivation. This work demonstrates that the precursor engineering has great potential in performance improvement. It is also pointed out that the initial synthesis is an often neglected but critical stage and has abundant room for optimization to further improve the quality of the resultant QDs, leading to breakthrough efficiency. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhancement of Naringenin Biosynthesis from Tyrosine by Metabolic Engineering of Saccharomyces cerevisiae.

PubMed

Lyu, Xiaomei; Ng, Kuan Rei; Lee, Jie Lin; Mark, Rita; Chen, Wei Ning

2017-08-09

Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine as the precursor. Our strategy to improve naringenin production comprised three modules. In module 1, we employed a modified GAL system to overexpress the genes of the naringenin biosynthesis pathway and investigated their synergistic action. In module 2, we simultaneously up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to eliminate the feedback inhibition of tyrosine and also down-regulated competing pathways. It was found that modules 1 and 3 played important roles in improving naringenin production. We succeeded in producing up to ∼90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared to the parental strain.

Microbial chemical factories: recent advances in pathway engineering for synthesis of value added chemicals.

PubMed

Dhamankar, Himanshu; Prather, Kristala L J

2011-08-01

The dwindling nature of petroleum and other fossil reserves has provided impetus towards microbial synthesis of fuels and value added chemicals from biomass-derived sugars as a renewable resource. Microbes have naturally evolved enzymes and pathways that can convert biomass into hundreds of unique chemical structures, a property that can be effectively exploited for their engineering into Microbial Chemical Factories (MCFs). De novo pathway engineering facilitates expansion of the repertoire of microbially synthesized compounds beyond natural products. In this review, we visit some recent successes in such novel pathway engineering and optimization, with particular emphasis on the selection and engineering of pathway enzymes and balancing of their accessory cofactors. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

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.

Engineered biofiltration for the removal of disinfection by-product precursors and genotoxicity.

PubMed

McKie, Michael J; Taylor-Edmonds, Liz; Andrews, Susan A; Andrews, Robert C

2015-09-15

Disinfection by-products (DBPs) are formed when naturally occurring organic matter reacts with chlorine used in drinking water treatment, and DBPs formed in chlorinated drinking water samples have been shown to cause a genotoxic response. The objective of the current study was to further understand the principles of biofiltration and the resulting impacts on the formation of DBPs and genotoxicity. Pilot-scale systems were utilized to assess the performance of engineered biofilters enhanced with hydrogen peroxide, in-line coagulants, and nutrients when compared to passively operated biofilters and conventional treatment (coagulation, flocculation, sedimentation, non-biological filtration). Organic fractionation was completed using liquid chromatography-organic carbon detection (LC-OCD). Water samples were chlorinated after collection and examined for the removal of trihalomethane (THM), haloacetic acid (HAA), and adsorbable organic halide (AOX) precursors. Additionally, the formation potential of two halogenated furanones, 3-chloro-4(dichloromethyl)-2(5H)-furanone (MX) and mucochloric acid (MCA), and genotoxicity was determined. Biofiltration was shown to preferentially remove more DBP precursors than dissolved organic carbon (DOC). Formation potential of the unregulated DBPs, including MX and MCA, and genotoxic response was shown to be correlated to THM formation. These results infer that monitoring for THMs and HAAs provide insight to the formation of more mutagenic DBPs such as halogenated furanones, and that biofiltration may preferentially remove precursors to DBPs at a rate exceeding the removal of DOC. Copyright © 2015 Elsevier Ltd. All rights reserved.

Advanced Precursor Reaction Processing for Cu(InGa)(SeS)2 Solar Cells

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

Shafarman, William N.

This project “Advanced Precursor Reaction Processing for Cu(InGa)(SeS)2 Solar Cells”, completed by the Institute of Energy Conversion (IEC) at the University of Delaware in collaboration with the Department of Chemical Engineering at the University of Florida, developed the fundamental understanding and technology to increase module efficiency and improve the manufacturability of Cu(InGa)(SeS)2 films using the precursor reaction approach currently being developed by a number of companies. Key results included: (1) development of a three-step H2Se/Ar/H2S reaction process to control Ga distribution through the film and minimizes back contact MoSe2 formation; (2) Ag-alloying to improve precursor homogeneity by avoiding In phasemore » agglomeration, faster reaction and improved adhesion to allow wider reaction process window; (3) addition of Sb, Bi, and Te interlayers at the Mo/precursor junction to produce more uniform precursor morphology and improve adhesion with reduced void formation in reacted films; (4) a precursor structure containing Se and a reaction process to reduce processing time to 5 minutes and eliminate H2Se usage, thereby increasing throughput and reducing costs. All these results were supported by detailed characterization of the film growth, reaction pathways, thermodynamic assessment and device behavior.« less

Engineering terpene biosynthesis in Streptomyces for production of the advanced biofuel precursor bisabolene.

PubMed

Phelan, Ryan M; Sekurova, Olga N; Keasling, Jay D; Zotchev, Sergey B

2015-04-17

The past decade has witnessed a large influx of research toward the creation of sustainable, biologically derived fuels. While significant effort has been exerted to improve production capacity in common hosts, such as Escherichia coli or Saccharomyces cerevisiae, studies concerning alternate microbes comparatively lag. In an effort to expand the breadth of characterized hosts for fuel production, we map the terpene biosynthetic pathway in a model actinobacterium, Streptomyces venezuelae, and further alter secondary metabolism to afford the advanced biofuel precursor bisabolene. Leveraging information gained from study of the native isoprenoid pathway, we were able to increase bisabolene titer nearly 5-fold over the base production strain, more than 2 orders of magnitude greater than the combined terpene yield in the wild-type host. We also explored production on carbon sources of varying complexity to, notably, define this host as one able to perform consolidated bioprocessing.

Identification of a novel amyloid precursor protein processing pathway that generates secreted N-terminal fragments.

PubMed

Vella, Laura J; Cappai, Roberto

2012-07-01

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system. The proteolytic processing of the amyloid precursor protein (APP) into the β-amyloid (Aβ) peptide is a central event in AD. While the pathway that generates Aβ is well described, many questions remain concerning general APP metabolism and its metabolites. It is becoming clear that the amino-terminal region of APP can be processed to release small N-terminal fragments (NTFs). The purpose of this study was to investigate the occurrence and generation of APP NTFs in vivo and in cell culture (SH-SY5Y) in order to delineate the cellular pathways implicated in their generation. We were able to detect 17- to 28-kDa APP NTFs in human and mouse brain tissue that are distinct from N-APP fragments previously reported. We show that the 17- to 28-kDa APP NTFs were highly expressed in mice from the age of 2 wk to adulthood. SH-SY5Y studies indicate the generation of APP NTFs involves a novel APP processing pathway, regulated by protein kinase C, but independent of α-secretase or β-secretase 1 (BACE) activity. These results identify a novel, developmentally regulated APP processing pathway that may play an important role in the physiological function of APP.

Fatty Aldehydes in Cyanobacteria Are a Metabolically Flexible Precursor for a Diversity of Biofuel Products

PubMed Central

Kaiser, Brett K.; Carleton, Michael; Hickman, Jason W.; Miller, Cameron; Lawson, David; Budde, Mark; Warrener, Paul; Paredes, Angel; Mullapudi, Srinivas; Navarro, Patricia; Cross, Fred; Roberts, James M.

2013-01-01

We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis. PMID:23505484

Pathway-engineering for highly-aligned block copolymer arrays

DOE PAGES

Choo, Youngwoo; Majewski, Paweł W.; Fukuto, Masafumi; ...

2017-12-06

While kinetic aspects of self-assembly can hinder ordering, non-equilibirum effects can also be exploited to enforce a particular kind of order. We develop a pathway-engineering approach, using it to select a particular arrangement of a block copolymer cylinder phase.

Pathway-engineering for highly-aligned block copolymer arrays

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

Choo, Youngwoo; Majewski, Paweł W.; Fukuto, Masafumi

While kinetic aspects of self-assembly can hinder ordering, non-equilibirum effects can also be exploited to enforce a particular kind of order. We develop a pathway-engineering approach, using it to select a particular arrangement of a block copolymer cylinder phase.

Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

PubMed

Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

2016-01-01

Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways. © 2016 Elsevier Inc. All rights reserved.

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors.

PubMed

Sugatani, T; Alvarez, U M; Hruska, K A

2003-09-01

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis. Copyright 2003 Wiley-Liss, Inc.

Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones.

PubMed

Ibdah, Mwafaq; Martens, Stefan; Gang, David R

2018-03-14

Dihydrochalcones are plant natural products containing the phenylpropanoid backbone and derived from the plant-specific phenylpropanoid pathway. Dihydrochalcone compounds are important in plant growth and response to stresses and, thus, can have large impacts on agricultural activity. In recent years, these compounds have also received increased attention from the biomedical community for their potential as anticancer treatments and other benefits for human health. However, they are typically produced at relatively low levels in plants. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts and to engineer the metabolic pathway/host to improve the production of these metabolites. In the present review, we discuss in detail the functions of genes and enzymes involved in the biosynthetic pathway of the dihydrochalcones and the recent strategies and achievements used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be able to attain higher amounts of desired dihydrochalcones.

Rational engineering of p-hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway.

PubMed

Chen, Zhenya; Shen, Xiaolin; Wang, Jian; Wang, Jia; Yuan, Qipeng; Yan, Yajun

2017-11-01

Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Biosensor-based engineering of biosynthetic pathways

DOE PAGES

Rogers, Jameson K.; Taylor, Noah D.; Church, George M.

2016-03-18

Biosynthetic pathways provide an enzymatic route from inexpensive renewable resources to valuable metabolic products such as pharmaceuticals and plastics. However, designing these pathways is challenging due to the complexities of biology. Advances in the design and construction of genetic variants has enabled billions of cells, each possessing a slightly different metabolic design, to be rapidly generated. However, our ability to measure the quality of these designs lags by several orders of magnitude. Recent research has enabled cells to report their own success in chemical production through the use of genetically encoded biosensors. A new engineering discipline is emerging around themore » creation and application of biosensors. Biosensors, implemented in selections and screens to identify productive cells, are paving the way for a new era of biotechnological progress.« less

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

PubMed

Halper, Sean M; Cetnar, Daniel P; Salis, Howard M

2018-01-01

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.

Pathway-engineering for highly-aligned block copolymer arrays.

PubMed

Choo, Youngwoo; Majewski, Paweł W; Fukuto, Masafumi; Osuji, Chinedum O; Yager, Kevin G

2017-12-21

While the ultimate driving force in self-assembly is energy minimization and the corresponding evolution towards equilibrium, kinetic effects can also play a very strong role. These kinetic effects, such as trapping in metastable states, slow coarsening kinetics, and pathway-dependent assembly, are often viewed as complications to be overcome. Here, we instead exploit these effects to engineer a desired final nano-structure in a block copolymer thin film, by selecting a particular ordering pathway through the self-assembly energy landscape. In particular, we combine photothermal shearing with high-temperature annealing to yield hexagonal arrays of block copolymer cylinders that are aligned in a single prescribed direction over macroscopic sample dimensions. Photothermal shearing is first used to generate a highly-aligned horizontal cylinder state, with subsequent thermal processing used to reorient the morphology to the vertical cylinder state in a templated manner. Finally, we demonstrate the successful transfer of engineered morphologies into inorganic replicas.

Exploring the Engineering Student Experience: Findings from the Academic Pathways of People Learning Engineering Survey (APPLES). TR-10-01

ERIC Educational Resources Information Center

Sheppard, Sheri; Gilmartin, Shannon; Chen, Helen L.; Donaldson, Krista; Lichtenstein, Gary; Eris, Ozgur; Lande, Micah; Toye, George

2010-01-01

This report is based on data from the Academic Pathways of People Learning Engineering Survey (APPLES), administered to engineering students at 21 U.S. engineering colleges and schools in the spring of 2008. The first comprehensive set of analyses completed on the APPLES dataset presented here looks at how engineering students experience their…

Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering.

PubMed

Pang, Yongzhen; Abeysinghe, I Sarath B; He, Ji; He, Xianzhi; Huhman, David; Mewan, K Mudith; Sumner, Lloyd W; Yun, Jianfei; Dixon, Richard A

2013-03-01

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

Engineered Photorespiratory Bypass Pathways Improve Photosynthetic Efficiency and Growth as Temperature Increases

NASA Astrophysics Data System (ADS)

Cavanagh, A. P.; South, P. F.; Ort, D. R.; Bernacchi, C.

2017-12-01

In C3 plants grown under ambient [CO2] at 25°C, 23% of the fixed carbon dioxide is lost to photorespiration, the energy expensive metabolic pathway that recycles toxic compounds produced by Rubisco oxygenation reactions. Furthermore, rates of photorespiration increase with rising temperature, as higher temperatures favor increased Rubisco oxygenation. Modelling suggests that the absence of photorespiration could improve gross photosynthesis by 12-55% under projected climate conditions; however, this is difficult to measure empirically, as photorespiration interacts with several metabolic pathways and is an essential process for all C3 plants grown at ambient [O2]. Introduced biochemical bypasses to the native photorespiration pathway hold promise as a strategy to mitigate the impact of temperature on photorespiratory losses. We grew tobacco containing engineered pathways to bypass photorespiration under ambient and elevated temperatures (+5°C) in the field to determine if bypassing photorespiration could mitigate high temperature induced losses in growth and physiology. Our preliminary results show that engineered plants have a higher quantum efficiency under heated conditions than do non-engineered plants, resulting in up to 20% lower yield losses under heated conditions compared to non-engineered plants. These results support the theoretical modelling of temperature impacts on photorespiratory losses, and suggest the bypassing photorespiration could be an important strategy to increase crop yields.

Creating metabolic demand as an engineering strategy in Pseudomonas putida - Rhamnolipid synthesis as an example.

PubMed

Tiso, Till; Sabelhaus, Petra; Behrens, Beate; Wittgens, Andreas; Rosenau, Frank; Hayen, Heiko; Blank, Lars Mathias

2016-12-01

Metabolic engineering of microbial cell factories for the production of heterologous secondary metabolites implicitly relies on the intensification of intracellular flux directed toward the product of choice. Apart from reactions following peripheral pathways, enzymes of the central carbon metabolism are usually targeted for the enhancement of precursor supply. In Pseudomonas putida , a Gram-negative soil bacterium, central carbon metabolism, i.e., the reactions required for the synthesis of all 12 biomass precursors, was shown to be regulated at the metabolic level and not at the transcriptional level. The bacterium's central carbon metabolism appears to be driven by demand to react rapidly to ever-changing environmental conditions. In contrast, peripheral pathways that are only required for growth under certain conditions are regulated transcriptionally. In this work, we show that this regulation regime can be exploited for metabolic engineering. We tested this driven-by-demand metabolic engineering strategy using rhamnolipid production as an example. Rhamnolipid synthesis relies on two pathways, i.e., fatty acid de novo synthesis and the rhamnose pathway, providing the required precursors hydroxyalkanoyloxy-alkanoic acid (HAA) and activated (dTDP-)rhamnose, respectively. In contrast to single-pathway molecules, rhamnolipid synthesis causes demand for two central carbon metabolism intermediates, i.e., acetyl-CoA for HAA and glucose-6-phosphate for rhamnose synthesis. Following the above-outlined strategy of driven by demand, a synthetic promoter library was developed to identify the optimal expression of the two essential genes ( rhlAB ) for rhamnolipid synthesis. The best rhamnolipid-synthesizing strain had a yield of 40% rhamnolipids on sugar [Cmol RL /Cmol Glc ], which is approximately 55% of the theoretical yield. The rate of rhamnolipid synthesis of this strain was also high. Compared to an exponentially growing wild type, the rhamnose pathway increased its

A portable expression resource for engineering cross-species genetic circuits and pathways

PubMed Central

Kushwaha, Manish; Salis, Howard M.

2015-01-01

Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals. However, their functions currently rely on organism-specific regulatory parts, fragmenting synthetic biology and metabolic engineering into host-specific domains. To unify efforts, here we have engineered a cross-species expression resource that enables circuits and pathways to reuse the same genetic parts, while functioning similarly across diverse organisms. Our engineered system combines mixed feedback control loops and cross-species translation signals to autonomously self-regulate expression of an orthogonal polymerase without host-specific promoters, achieving nontoxic and tuneable gene expression in diverse Gram-positive and Gram-negative bacteria. Combining 50 characterized system variants with mechanistic modelling, we show how the cross-species expression resource's dynamics, capacity and toxicity are controlled by the control loops' architecture and feedback strengths. We also demonstrate one application of the resource by reusing the same genetic parts to express a biosynthesis pathway in both model and non-model hosts. PMID:26184393

Metabolomics Analysis of the Toxic Effects of the Production of Lycopene and Its Precursors.

PubMed

Miguez, April M; McNerney, Monica P; Styczynski, Mark P

2018-01-01

Using cells as microbial factories enables highly specific production of chemicals with many advantages over chemical syntheses. A number of exciting new applications of this approach are in the area of precision metabolic engineering, which focuses on improving the specificity of target production. In recent work, we have used precision metabolic engineering to design lycopene-producing Escherichia coli for use as a low-cost diagnostic biosensor. To increase precursor availability and thus the rate of lycopene production, we heterologously expressed the mevalonate pathway. We found that simultaneous induction of these pathways increases lycopene production, but induction of the mevalonate pathway before induction of the lycopene pathway decreases both lycopene production and growth rate. Here, we aim to characterize the metabolic changes the cells may be undergoing during expression of either or both of these heterologous pathways. After establishing an improved method for quenching E. coli for metabolomics analysis, we used two-dimensional gas chromatography coupled to mass spectrometry (GCxGC-MS) to characterize the metabolomic profile of our lycopene-producing strains in growth conditions characteristic of our biosensor application. We found that the metabolic impacts of producing low, non-toxic levels of lycopene are of much smaller magnitude than the typical metabolic changes inherent to batch growth. We then used metabolomics to study differences in metabolism caused by the time of mevalonate pathway induction and the presence of the lycopene biosynthesis genes. We found that overnight induction of the mevalonate pathway was toxic to cells, but that the cells could recover if the lycopene pathway was not also heterologously expressed. The two pathways appeared to have an antagonistic metabolic effect that was clearly reflected in the cells' metabolic profiles. The metabolites homocysteine and homoserine exhibited particularly interesting behaviors and may be

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

PubMed Central

Liu, Huaiwei; Sun, Yuanzhang; Ramos, Kristine Rose M.; Nisola, Grace M.; Valdehuesa, Kris Niño G.; Lee, Won–Keun; Park, Si Jae; Chung, Wook-Jin

2013-01-01

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways. PMID:24376679

Volatile science? Metabolic engineering of terpenoids in plants.

PubMed

Aharoni, Asaph; Jongsma, Maarten A; Bouwmeester, Harro J

2005-12-01

Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that significant progress has been made over the past few years. Subcellular targeting of enzymes has demonstrated that terpenoid precursors in subcellular compartments are not as strictly separated as previously thought and that multistep pathway engineering is feasible, even across cell compartments. These engineered plants show that insect behavior is influenced by terpenoids. In the future, we expect rapid progress in the engineering of terpenoid production in plants. In addition to commercial applications, such transgenic plants should increase our understanding of the biological relevance of these volatile secondary metabolites.

Engineering dynamic pathway regulation using stress-response promoters.

PubMed

Dahl, Robert H; Zhang, Fuzhong; Alonso-Gutierrez, Jorge; Baidoo, Edward; Batth, Tanveer S; Redding-Johanson, Alyssa M; Petzold, Christopher J; Mukhopadhyay, Aindrila; Lee, Taek Soon; Adams, Paul D; Keasling, Jay D

2013-11-01

Heterologous pathways used in metabolic engineering may produce intermediates toxic to the cell. Dynamic control of pathway enzymes could prevent the accumulation of these metabolites, but such a strategy requires sensors, which are largely unknown, that can detect and respond to the metabolite. Here we applied whole-genome transcript arrays to identify promoters that respond to the accumulation of toxic intermediates, and then used these promoters to control accumulation of the intermediate and improve the final titers of a desired product. We apply this approach to regulate farnesyl pyrophosphate (FPP) production in the isoprenoid biosynthetic pathway in Escherichia coli. This strategy improved production of amorphadiene, the final product, by twofold over that from inducible or constitutive promoters, eliminated the need for expensive inducers, reduced acetate accumulation and improved growth. We extended this approach to another toxic intermediate to demonstrate the broad utility of identifying novel sensor-regulator systems for dynamic regulation.

Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli.

PubMed

Lin, Yuheng; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

2014-05-01

cis,cis-Muconic acid (MA) and salicylic acid (SA) are naturally-occurring organic acids having great commercial value. MA is a potential platform chemical for the manufacture of several widely-used consumer plastics; while SA is mainly used for producing pharmaceuticals (for example, aspirin and lamivudine) and skincare and haircare products. At present, MA and SA are commercially produced by organic chemical synthesis using petro-derived aromatic chemicals, such as benzene, as starting materials, which is not environmentally friendly. Here, we report a novel approach for efficient microbial production of MA via extending shikimate pathway by introducing the hybrid of an SA biosynthetic pathway with its partial degradation pathway. First, we engineered a well-developed phenylalanine producing Escherichia coli strain into an SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. The engineered strain is able to produce 1.2g/L of SA from simple carbon sources, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase and catechol 1,2-dioxygenase is established to achieve the conversion of SA to MA. Finally, a de novo MA biosynthetic pathway is assembled by integrating the established SA biosynthesis and degradation modules. Modular optimization enables the production of up to 1.5g/L MA within 48h in shake flasks. This study not only establishes an efficient microbial platform for the production of SA and MA, but also demonstrates a generalizable pathway design strategy for the de novo biosynthesis of valuable degradation metabolites. Copyright © 2014. Published by Elsevier Inc.

Engineering Emergency: African Americans and Hispanics Lack Pathways to Engineering. Vital Signs: Reports on the Condition of STEM Learning in the U.S.

ERIC Educational Resources Information Center

Change the Equation, 2014

2014-01-01

A quality education that leads to good jobs offers a reliable pathway to economic security, yet the first step on that pathway remains inaccessible to far too many Americans, especially Americans of color. Nowhere is this inequity more apparent than in engineering. On average, people with engineering bachelor's degrees earn higher salaries than…

Targeting Undergraduate Students for Surveys: Lessons from the Academic Pathways of People Learning Engineering Survey (APPLES). Research Brief

ERIC Educational Resources Information Center

Donaldson, Krista M.; Chen, Helen L.; Toye, George; Sheppard, Sheri D.

2007-01-01

The Academic Pathways of People Learning Engineering Survey (APPLES or APPLE survey) is a component of the Academic Pathways Study (APS) of the Center for the Advancement of Engineering Education (CAEE). The APS aims to provide a comprehensive account of how people become engineers by exploring key questions around the engineering learning…

Scaling up: Taking the Academic Pathways of People Learning Engineering Survey (APPLES) National. Research Brief

ERIC Educational Resources Information Center

Donaldson, Krista M.; Chen, Helen L.; Toye, George; Clark, Mia; Sheppard, Sheri D.

2008-01-01

The Academic Pathways of People Learning Engineering Survey (APPLES) was deployed for a second time in spring 2008 to undergraduate engineering students at 21 US universities. The goal of the second deployment of APPLES was to corroborate and extend findings from the Academic Pathways Study (APS; 2003-2007) and the first deployment of APPLES…

Thermal Studies of New Precursors to Indium-tin Oxides for Use as Sensor Materials in the Detection of NO(x)

NASA Technical Reports Server (NTRS)

Goldsby, J. C.; Kacik, T.; Hockensmith, C. M.

1999-01-01

Control of combustion product emissions in both sub and super-sonic jet engines can be facilitated by measurement of NO(x) levels with metal oxide sensors, In2O3, metal-doped SnO2, and SnO, (as well as other materials) show resistivity changes in the presence of NO(x), but often their sensitivity, stability, and selectivity are low. This study was designed to develop new synthetic pathways to precursors that produce high purity, two phase In2O3-SnO2. The precursors were formed by complexation of tin with any oxide ligands to give the ammonium salt (NH4). Thermal studies of these precursors were carried out by thermal gravimetry (TG) and differential scanning calorimetry (DSC). Further studies by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR) were also conducted.

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

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

Kirby, James; Dietzel, Kevin L.; Wichmann, Gale

2016-10-27

Isoprenoids are made by all free-living organisms and range from essential metabolites like sterols and quinones to more complex compounds like pinene and rubber. They are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages tomore » isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.« less

Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production.

PubMed

Tan, Zaigao; Chen, Jing; Zhang, Xueli

2016-01-01

Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production. Altering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge. In summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications.

PubMed

Sahoo, Sambit; Ang, Lay-Teng; Cho-Hong Goh, James; Toh, Siew-Lok

2010-02-01

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications. 2009 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production.

PubMed

Jo, Suah; Yoon, Jinkyung; Lee, Sun-Mi; Um, Youngsoon; Han, Sung Ok; Woo, Han Min

2017-09-20

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolic engineering of Escherichia coli for the production of phenylpyruvate derivatives.

PubMed

Liu, Shuang Ping; Zhang, Liang; Mao, Jian; Ding, Zhong Yang; Shi, Gui Yang

2015-11-01

Phenylpyruvate derivatives (PPD), such as phenylpropanoids, DL-phenylglycine, dl-phenylalanine, and styrene, are biosynthesized using phenylpyruvate as the precursor. They are widely used in human health and nutrition products. Recently, metabolic engineering provides effective strategies to develop PPD producers. Based on phenylpyruvate-producing chassis, genetically defined PPD producers have been successfully constructed. In this work, the most recent information on genetics and on the molecular mechanisms regulating phenylpyruvate synthesis pathways in Escherichia coli are summarized, and the engineering strategies to construct the PPD producers are also discussed. The enzymes and pathways are proposed for PPD-producer constructions, and potential difficulties in strain construction are also identified and discussed. With respect to recent advances in synthetic biology, future strategies to construct efficiently producers are discussed. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms

PubMed Central

Hossain, Gazi Sakir; Nadarajan, Saravanan Prabhu; Zhang, Lei; Ng, Tee-Kheang; Foo, Jee Loon; Ling, Hua; Choi, Won Jae; Chang, Matthew Wook

2018-01-01

Living organisms have evolved over millions of years to fine tune their metabolism to create efficient pathways for producing metabolites necessary for their survival. Advancement in the field of synthetic biology has enabled the exploitation of these metabolic pathways for the production of desired compounds by creating microbial cell factories through metabolic engineering, thus providing sustainable routes to obtain value-added chemicals. Following the past success in metabolic engineering, there is increasing interest in diversifying natural metabolic pathways to construct non-natural biosynthesis routes, thereby creating possibilities for producing novel valuable compounds that are non-natural or without elucidated biosynthesis pathways. Thus, the range of chemicals that can be produced by biological systems can be expanded to meet the demands of industries for compounds such as plastic precursors and new antibiotics, most of which can only be obtained through chemical synthesis currently. Herein, we review and discuss novel strategies that have been developed to rewrite natural metabolic blueprints in a bid to broaden the chemical repertoire achievable in microorganisms. This review aims to provide insights on recent approaches taken to open new avenues for achieving biochemical production that are beyond currently available inventions. PMID:29483901

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms.

PubMed

Hossain, Gazi Sakir; Nadarajan, Saravanan Prabhu; Zhang, Lei; Ng, Tee-Kheang; Foo, Jee Loon; Ling, Hua; Choi, Won Jae; Chang, Matthew Wook

2018-01-01

Living organisms have evolved over millions of years to fine tune their metabolism to create efficient pathways for producing metabolites necessary for their survival. Advancement in the field of synthetic biology has enabled the exploitation of these metabolic pathways for the production of desired compounds by creating microbial cell factories through metabolic engineering, thus providing sustainable routes to obtain value-added chemicals. Following the past success in metabolic engineering, there is increasing interest in diversifying natural metabolic pathways to construct non-natural biosynthesis routes, thereby creating possibilities for producing novel valuable compounds that are non-natural or without elucidated biosynthesis pathways. Thus, the range of chemicals that can be produced by biological systems can be expanded to meet the demands of industries for compounds such as plastic precursors and new antibiotics, most of which can only be obtained through chemical synthesis currently. Herein, we review and discuss novel strategies that have been developed to rewrite natural metabolic blueprints in a bid to broaden the chemical repertoire achievable in microorganisms. This review aims to provide insights on recent approaches taken to open new avenues for achieving biochemical production that are beyond currently available inventions.

Facilitated Protein Association via Engineered Target Search Pathways Visualized by Paramagnetic NMR Spectroscopy.

PubMed

An, So Young; Kim, Eun-Hee; Suh, Jeong-Yong

2018-06-05

Proteins assemble to form functional complexes via the progressive evolution of nonspecific complexes formed by transient encounters. This target search process generally involves multiple routes that lead the initial encounters to the final complex. In this study, we have employed NMR paramagnetic relaxation enhancement to visualize the encounter complexes between histidine-containing phosphocarrier protein and the N-terminal domain of enzyme I and demonstrate that protein association can be significantly enhanced by engineering on-pathways. Specifically, mutations in surface charges away from the binding interface can elicit new on-pathway encounter complexes, increasing their binding affinity by an order of magnitude. The structure of these encounter complexes indicates that such on-pathways extend the built-in target search process of the native protein complex. Furthermore, blocking on-pathways by countering mutations reverts their binding affinity. Our study thus illustrates that protein interactions can be engineered by rewiring the target search process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Metabolic engineering of biosynthetic pathway for production of renewable biofuels.

PubMed

Singh, Vijai; Mani, Indra; Chaudhary, Dharmendra Kumar; Dhar, Pawan Kumar

2014-02-01

Metabolic engineering is an important area of research that involves editing genetic networks to overproduce a certain substance by the cells. Using a combination of genetic, metabolic, and modeling methods, useful substances have been synthesized in the past at industrial scale and in a cost-effective manner. Currently, metabolic engineering is being used to produce sufficient, economical, and eco-friendly biofuels. In the recent past, a number of efforts have been made towards engineering biosynthetic pathways for large scale and efficient production of biofuels from biomass. Given the adoption of metabolic engineering approaches by the biofuel industry, this paper reviews various approaches towards the production and enhancement of renewable biofuels such as ethanol, butanol, isopropanol, hydrogen, and biodiesel. We have also identified specific areas where more work needs to be done in the future.

Engineering a monolignol 4- O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alchohol

DOE PAGES

Cai, Yuanheng; Shanklin, John; Mohammad -Wadud Bhuiya; ...

2015-09-16

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create anmore » enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. Lastly, the resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.« less

Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol*

PubMed Central

Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

2015-01-01

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. PMID:26378240

Requirement of zebrafish pcdh10a and pcdh10b in melanocyte precursor migration.

PubMed

Williams, Jason S; Hsu, Jessica Y; Rossi, Christy Cortez; Artinger, Kristin Bruk

2018-03-29

Melanocytes derive from neural crest cells, which are a highly migratory population of cells that play an important role in pigmentation of the skin and epidermal appendages. In most vertebrates, melanocyte precursor cells migrate solely along the dorsolateral pathway to populate the skin. However, zebrafish melanocyte precursors also migrate along the ventromedial pathway, in route to the yolk, where they interact with other neural crest derivative populations. Here, we demonstrate the requirement for zebrafish paralogs pcdh10a and pcdh10b in zebrafish melanocyte precursor migration. pcdh10a and pcdh10b are expressed in a subset of melanocyte precursor and somatic cells respectively, and knockdown and TALEN mediated gene disruption of pcdh10a results in aberrant migration of melanocyte precursors resulting in fully melanized melanocytes that differentiate precociously in the ventromedial pathway. Live cell imaging analysis demonstrates that loss of pchd10a results in a reduction of directed cell migration of melanocyte precursors, caused by both increased adhesion and a loss of cell-cell contact with other migratory neural crest cells. Also, we determined that the paralog pcdh10b is upregulated and can compensate for the genetic loss of pcdh10a. Disruption of pcdh10b alone by CRISPR mutagenesis results in somite defects, while the loss of both paralogs results in enhanced migratory melanocyte precursor phenotype and embryonic lethality. These results reveal a novel role for pcdh10a and pcdh10b in zebrafish melanocyte precursor migration and suggest that pcdh10 paralogs potentially interact for proper transient migration along the ventromedial pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

An engineered non-oxidative glycolysis pathway for acetone production in Escherichia coli.

PubMed

Yang, Xiaoyan; Yuan, Qianqian; Zheng, Yangyang; Ma, Hongwu; Chen, Tao; Zhao, Xueming

2016-08-01

To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli. Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain. Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.

A Decade of Molecular Understanding of Withanolide Biosynthesis and In vitro Studies in Withania somnifera (L.) Dunal: Prospects and Perspectives for Pathway Engineering

PubMed Central

Dhar, Niha; Razdan, Sumeer; Rana, Satiander; Bhat, Wajid W.; Vishwakarma, Ram; Lattoo, Surrinder K.

2015-01-01

Withania somnifera, a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids that are steroidal lactones known as withanolides. Though the plant has been well-characterized in terms of phytochemical profiles as well as pharmaceutical activities, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. This scenario limits biotechnological interventions for enhanced production of bioactive compounds. Nevertheless, recent emergent trends vis-à-vis, the exploration of genomic, transcriptomic, proteomic, metabolomics, and in vitro studies have opened new vistas regarding pathway engineering of withanolide production. During recent years, various strategic pathway genes have been characterized with significant amount of regulatory studies which allude toward development of molecular circuitries for production of key intermediates or end products in heterologous hosts. Another pivotal aspect covering redirection of metabolic flux for channelizing the precursor pool toward enhanced withanolide production has also been attained by deciphering decisive branch point(s) as robust targets for pathway modulation. With these perspectives, the current review provides a detailed overview of various studies undertaken by the authors and collated literature related to molecular and in vitro approaches employed in W. somnifera for understanding various molecular network interactions in entirety. PMID:26640469

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering1[W][OA

PubMed Central

Pang, Yongzhen; Abeysinghe, I. Sarath B.; He, Ji; He, Xianzhi; Huhman, David; Mewan, K. Mudith; Sumner, Lloyd W.; Yun, Jianfei; Dixon, Richard A.

2013-01-01

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (−)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (−)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols. PMID:23288883

Protein design and engineering of a de novo pathway for microbial production of 1,3-propanediol from glucose.

PubMed

Chen, Zhen; Geng, Feng; Zeng, An-Ping

2015-02-01

Protein engineering to expand the substrate spectrum of native enzymes opens new possibilities for bioproduction of valuable chemicals from non-natural pathways. No natural microorganism can directly use sugars to produce 1,3-propanediol (PDO). Here, we present a de novo route for the biosynthesis of PDO from sugar, which may overcome the mentioned limitations by expanding the homoserine synthesis pathway. The accomplishment of pathway from homoserine to PDO is achieved by protein engineering of glutamate dehydrogenase (GDH) and pyruvate decarboxylase to sequentially convert homoserine to 4-hydroxy-2-ketobutyrate and 3-hydroxypropionaldehyde. The latter is finally converted to PDO by using a native alcohol dehydrogenase. In this work, we report on experimental accomplishment of this non-natural pathway, especially by protein engineering of GDH for the key step of converting homoserine to 4-hydroxy-2-ketobutyrate. These results show the feasibility and significance of protein engineering for de novo pathway design and overproduction of desired industrial products. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alcohol.

PubMed

Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

2015-10-30

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

STEM Career Cluster Engineering and Technology Education pathway in Georgia: Perceptions of Georgia engineering and technology education high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education survey

NASA Astrophysics Data System (ADS)

Crenshaw, Mark VanBuren

This study examined the perceptions held by Georgia Science, Technology, Engineering, and Mathematics (STEM) Career Cluster Engineering and Technology Education (ETE) high school pathway teachers and Georgia's Career, Technical and Agriculture Education (CTAE) administrators regarding the ETE pathway and its effect on implementation within their district and schools. It provides strategies for ETE teaching methods, curriculum content, STEM integration, and how to improve the ETE pathway program of study. Current teaching and curricular trends were examined in ETE as well as the role ETE should play as related to STEM education. The study, using the Characteristics of Engineering and Technology Education Survey, was conducted to answer the following research questions: (a) Is there a significant difference in the perception of ETE teaching methodology between Georgia ETE high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education Survey? (b) Is there a significant difference in the perception of ETE curriculum content between Georgia ETE high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education Survey? (c) Is there a significant difference in the perception of STEM integration in the ETE high school pathway between Georgia ETE high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education Survey? and (d) Is there a significant difference in the perception of how to improve the ETE high school pathway between Georgia ETE high school teachers and CTAE administrators as measured by the Characteristics of Engineering and Technology Education Survey? Suggestions for further research also were offered.

The Scaling of Loss Pathways and Heat Transfer in Small Scale Internal Combustion Engines

DTIC Science & Technology

2016-09-16

less than 5%. Two factors drove the high short-circuiting observed in the 405 studied engines: excess fresh charge delivered to the engines beyond...losses do not begin to increase substantially until engine displacement decreases below 10 cm3. The objective concluded with a parametric study ...81 4.1. Why Loss Pathways in ICEs Scale ................................................................ 82 4.2. Scaling Studies

Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.).

PubMed

Ramírez Rivera, Naty G; García-Salinas, Carolina; Aragão, Francisco J L; Díaz de la Garza, Rocío Isabel

2016-10-01

Folate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed-specific overexpression of AtGCHI caused significant increases of up to 150-fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 μg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Effect of temperature on the reaction pathway of calcium carbonate formation via precursor phases

NASA Astrophysics Data System (ADS)

Purgstaller, Bettina; Mavromatis, Vasileios; Konrad, Florian; Dietzel, Martin

2016-04-01

It has been earlier postulated that some biogenic and sedimentary calcium carbonate (CaCO3) minerals (e.g. calcite and aragonite) are secondary in origin and have originally formed via a metastable calcium carbonate precursor phase (e.g. amorphous CaCO3, [1-2]). Such formation pathways are likely affected by various physicochemical parameters including aqueous Mg and temperature. In an effort to improve our understanding on the formation mechanism of CaCO3 minerals, precipitation experiments were carried out by the addition of a 0.6 M (Ca,Mg)Cl2 solution at distinct Mg/Ca ratios (1/4 and 1/8) into a 1 M NaHCO3 solution under constant pH conditions(8.3 ±0.1). The formation of CaCO3 was systematically examined as a function of temperature (6, 12, 18 and 25 ±0.3° C). During the experimental runs mineral precipitation was monitored by in situ Raman spectroscopy as well as by continuous sampling and analyzing of precipitates and reactive solutions. The results revealed two pathways of CaCO3 formation depending on the initial Mg/Ca ratio and temperature: (i) In experiments with a Mg/Ca ratio of 1/4 at ≤ 12° C as well as in experiments with a Mg/Ca ratio of 1/8 at ≤ 18° C, ikaite (CaCO3 6H2O) acts as a precursor phase for aragonite formation. (ii) In contrast higher temperatures induced the formation of Mg-rich amorphous CaCO3 (Mg-ACC) which was subsequently transformed to Mg-rich calcite. In situ Raman spectra showed that the transformation of Mg-ACC to Mg-calcite occurs at a higher rate (˜ 8 min) compared to that of ikaite to aragonite (> 2 h). Thus, the formation of aragonite rather than of Mg-calcite occurs due to the slower release of Ca2+and CO32- ions into the Mg-rich reactive solution during retarded ikaite dissolution. This behavior is generally consistent with the observation that calcite precipitation is inhibited at elevated aqueous Mg/Ca ratios. [1] Addadi L., Raz S. and Weiner S. (2003) Advanced Materials 15, 959-970. [2] Rodriguez-Blanco J. D

Engineering Heteromaterials to Control Lithium Ion Transport Pathways

DOE PAGES

Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; ...

2015-12-21

Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Some recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. We utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through themore » systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Furthermore, our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries.« less

Engineering Heteromaterials to Control Lithium Ion Transport Pathways

PubMed Central

Liu, Yang; Vishniakou, Siarhei; Yoo, Jinkyoung; Dayeh, Shadi A.

2015-01-01

Safe and efficient operation of lithium ion batteries requires precisely directed flow of lithium ions and electrons to control the first directional volume changes in anode and cathode materials. Understanding and controlling the lithium ion transport in battery electrodes becomes crucial to the design of high performance and durable batteries. Recent work revealed that the chemical potential barriers encountered at the surfaces of heteromaterials play an important role in directing lithium ion transport at nanoscale. Here, we utilize in situ transmission electron microscopy to demonstrate that we can switch lithiation pathways from radial to axial to grain-by-grain lithiation through the systematic creation of heteromaterial combinations in the Si-Ge nanowire system. Our systematic studies show that engineered materials at nanoscale can overcome the intrinsic orientation-dependent lithiation, and open new pathways to aid in the development of compact, safe, and efficient batteries. PMID:26686655

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa.

PubMed

Nag, Ambarish; Karpinets, Tatiana V; Chang, Christopher H; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

PubMed Central

Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway.

PubMed

Yan, Jinyong; Yan, Yunjun; Madzak, Catherine; Han, Bingnan

2017-02-01

Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.

Programmable genetic circuits for pathway engineering.

PubMed

Hoynes-O'Connor, Allison; Moon, Tae Seok

2015-12-01

Synthetic biology has the potential to provide decisive advances in genetic control of metabolic pathways. However, there are several challenges that synthetic biologists must overcome before this vision becomes a reality. First, a library of diverse and well-characterized sensors, such as metabolite-sensing or condition-sensing promoters, must be constructed. Second, robust programmable circuits that link input conditions with a specific gene regulation response must be developed. Finally, multi-gene targeting strategies must be integrated with metabolically relevant sensors and complex, robust logic. Achievements in each of these areas, which employ the CRISPR/Cas system, in silico modeling, and dynamic sensor-regulators, among other tools, provide a strong basis for future research. Overall, the future for synthetic biology approaches in metabolic engineering holds immense promise. Copyright © 2015 Elsevier Ltd. All rights reserved.

Carotenoid Biosynthesis in Arabidopsis: A Colorful Pathway

PubMed Central

Ruiz-Sola, M. Águila; Rodríguez-Concepción, Manuel

2012-01-01

Plant carotenoids are a family of pigments that participate in light harvesting and are essential for photoprotection against excess light. Furthermore, they act as precursors for the production of apocarotenoid hormones such as abscisic acid and strigolactones. In this review, we summarize the current knowledge on the genes and enzymes of the carotenoid biosynthetic pathway (which is now almost completely elucidated) and on the regulation of carotenoid biosynthesis at both transcriptional and post-transcriptional levels. We also discuss the relevance of Arabidopsis as a model system for the study of carotenogenesis and how metabolic engineering approaches in this plant have taught important lessons for carotenoid biotechnology. PMID:22582030

Current advance in biological production of malic acid using wild type and metabolic engineered strains.

PubMed

Dai, Zhongxue; Zhou, Huiyuan; Zhang, Shangjie; Gu, Honglian; Yang, Qiao; Zhang, Wenming; Dong, Weiliang; Ma, Jiangfeng; Fang, Yan; Jiang, Min; Xin, Fengxue

2018-06-01

Malic acid (2-hydroxybutanedioic acid) is a four-carbon dicarboxylic acid, which has attracted great interest due to its wide usage as a precursor of many industrially important chemicals in the food, chemicals, and pharmaceutical industries. Several mature routes for malic acid production have been developed, such as chemical synthesis, enzymatic conversion and biological fermentation. With depletion of fossil fuels and concerns regarding environmental issues, biological production of malic acid has attracted more attention, which mainly consists of three pathways, namely non-oxidative pathway, oxidative pathway and glyoxylate cycle. In recent decades, metabolic engineering of model strains, and process optimization for malic acid production have been rapidly developed. Hence, this review comprehensively introduces an overview of malic acid producers and highlight some of the successful metabolic engineering approaches. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cross-reactions between engineered xylose and galactose pathways in recombinant Saccharomyces cerevisiae.

PubMed

Garcia Sanchez, Rosa; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

2010-09-01

Overexpression of the PGM2 gene encoding phosphoglucomutase (Pgm2p) has been shown to improve galactose utilization both under aerobic and under anaerobic conditions. Similarly, xylose utilization has been improved by overexpression of genes encoding xylulokinase (XK), enzymes from the non-oxidative pentose phosphate pathway (non-ox PPP) and deletion of the endogenous aldose reductase GRE3 gene in engineered Saccharomyces cerevisiae strains carrying either fungal or bacterial xylose pathways. In the present study, we investigated how the combination of these traits affect xylose and galactose utilization in the presence or absence of glucose in S. cerevisiae strains engineered with the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. In the absence of PGM2 overexpression, the combined overexpression of XK, the non-ox PPP and deletion of the GRE3 gene significantly delayed aerobic growth on galactose, whereas no difference was observed between the control strain and the xylose-engineered strain when the PGM2 gene was overexpressed. Under anaerobic conditions, the overexpression of the PGM2 gene increased the ethanol yield and the xylose consumption rate in medium containing xylose as the only carbon source. The possibility of Pgm2p acting as a xylose isomerase (XI) could be excluded by measuring the XI activity in both strains. The additional copy of the PGM2 gene also resulted in a shorter fermentation time during the co-consumption of galactose and xylose. However, the effect was lost upon addition of glucose to the growth medium. PGM2 overexpression was shown to benefit xylose and galactose fermentation, alone and in combination. In contrast, galactose fermentation was impaired in the engineered xylose-utilizing strain harbouring extra copies of the non-ox PPP genes and a deletion of the GRE3 gene, unless PGM2 was overexpressed. These cross-reactions are of particular relevance for the fermentation of mixed sugars from lignocellulosic feedstock.

Genome engineering with TALENs and ZFNs: repair pathways and donor design.

PubMed

Carroll, Dana; Beumer, Kelly J

2014-09-01

Genome engineering with targetable nucleases depends on cellular pathways of DNA repair after target cleavage. Knowledge of how those pathways work, their requirements and their active factors, can guide experimental design and improve outcomes. While many aspects of both homologous recombination (HR) and nonhomologous end joining (NHEJ) are shared by a broad range of cells and organisms, some features are specific to individual situations. This article reviews the influence of repair mechanisms on the results of gene targeting experiments, with an emphasis on lessons learned from experiments with Drosophila. Copyright © 2014 Elsevier Inc. All rights reserved.

Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

PubMed

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

2011-05-11

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.

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 �

Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme

PubMed Central

Ben Aissa, Manel; April, Marie-Claude; Bergeron, Lucien-Junior; Perreault, Jean-Pierre; Levesque, Georges

2012-01-01

Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40–42 residue peptide (Aβ 40−42) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD. PMID:22482079

Computer-assisted engineering of the synthetic pathway for biodegradation of a toxic persistent pollutant.

PubMed

Kurumbang, Nagendra Prasad; Dvorak, Pavel; Bendl, Jaroslav; Brezovsky, Jan; Prokop, Zbynek; Damborsky, Jiri

2014-03-21

Anthropogenic halogenated compounds were unknown to nature until the industrial revolution, and microorganisms have not had sufficient time to evolve enzymes for their degradation. The lack of efficient enzymes and natural pathways can be addressed through a combination of protein and metabolic engineering. We have assembled a synthetic route for conversion of the highly toxic and recalcitrant 1,2,3-trichloropropane to glycerol in Escherichia coli, and used it for a systematic study of pathway bottlenecks. Optimal ratios of enzymes for the maximal production of glycerol, and minimal toxicity of metabolites were predicted using a mathematical model. The strains containing the expected optimal ratios of enzymes were constructed and characterized for their viability and degradation efficiency. Excellent agreement between predicted and experimental data was observed. The validated model was used to quantitatively describe the kinetic limitations of currently available enzyme variants and predict improvements required for further pathway optimization. This highlights the potential of forward engineering of microorganisms for the degradation of toxic anthropogenic compounds.

Pathway Construction in Corynebacterium glutamicum and Strain Engineering To Produce Rare Sugars from Glycerol.

PubMed

Yang, Jiangang; Zhu, Yueming; Men, Yan; Sun, Shangshang; Zeng, Yan; Zhang, Ying; Sun, Yuanxia; Ma, Yanhe

2016-12-21

Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered. Next, three pathways were assembled and the resulting strains synthesized 5-deoxypsicose, 5-deoxysorbose, and 5-deoxyfructose from glucose and glycerol and produce l-fructose, l-tagatose, l-sorbose, and l-psicose with glycerol as the only carbon source. To achieve higher product titer and yield, the recombinant strains were further engineered and fermentation conditions were optimized. Fed-batch culture of engineered strains obtained 38.1 g/L 5-deoxypsicose with a yield of 0.91 ± 0.04 mol product per mol of glycerol and synthesized 20.8 g/L l-fructose, 10.3 g/L l-tagatose, 1.2 g/L l-sorbose, and 0.95 g/L l-psicose.

A novel MVA-mediated pathway for isoprene production in engineered E. coli.

PubMed

Yang, Jianming; Nie, Qingjuan; Liu, Hui; Xian, Mo; Liu, Huizhou

2016-01-20

To deal with the increasingly severe energy crisis and environmental consequences, biofuels and biochemicals generated from renewable resources could serve as a promising alternative for replacing petroleum as a source of fuel and chemicals, among which isoprene (2-methyl-1,3-butadiene) in particular is of great significance in that it is an important platform chemical, which has been used in industrial production of synthetic rubber for tires and coatings or aviation fuel. We firstly introduced fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species into E. coli to directly convert MVA(mevalonate) into 3-methy-3-buten-1-ol. And then to transform 3-methy-3-buten-1-ol to isoprene, oleate hydratase (OhyAEM) from Elizabethkingia meningoseptica was overexpressed in E. coli. A novel biosynthetic pathway of isoprene in E. coli was established by co-expressing the heterologous mvaE gene encoding acetyl-CoA acetyltransferase/HMG-CoA reductase and mvaS gene encoding HMG-CoA synthase from Enterococcus faecalis, fatty acid decarboxylase (OleTJE) and oleate hydratase (OhyAEM). Furthermore, to enhance isoprene production, a further optimization of expression level of OleTJE, OhyAEM was carried out by using different promoters and copy numbers of plasmids. Thereafter, the fermentation process was also optimized to improve the production of isoprene. The final engineered strain, YJM33, bearing the innovative biosynthetic pathway of isoprene, was found to produce isoprene up to 2.2 mg/L and 620 mg/L under flask and fed-batch fermentation conditions, respectively. In this study, by using metabolic engineering techniques, the novel MVA-mediated biosynthetic pathway of isoprene was successfully assembled in E. coli BL21(DE3) with the heterologous MVA upper pathway, OleTJE from Jeotgalicoccus species and OhyAEM from Elizabethkingia meningoseptica. Compared with traditional MVA pathway, the novel pathway is shortened by 3 steps. In addition, this is the first report on the

Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology.

PubMed

Pandey, Ramesh Prasad; Parajuli, Prakash; Koffas, Mattheos A G; Sohng, Jae Kyung

2016-01-01

In this review, we address recent advances made in pathway engineering, directed evolution, and systems/synthetic biology approaches employed in the production and modification of flavonoids from microbial cells. The review is divided into two major parts. In the first, various metabolic engineering and system/synthetic biology approaches used for production of flavonoids and derivatives are discussed broadly. All the manipulations/engineering accomplished on the microorganisms since 2000 are described in detail along with the biosynthetic pathway enzymes, their sources, structures of the compounds, and yield of each product. In the second part of the review, post-modifications of flavonoids by four major reactions, namely glycosylations, methylations, hydroxylations and prenylations using recombinant strains are described. Copyright © 2016 Elsevier Inc. All rights reserved.

[Study on Precursors for Synthesis of Anthraquinone Metabolites from Rheum tanguticum].

PubMed

Hasi, Qi-mei-ge; Lj, Hai-ling; Cheng, Yan; Menggen, Qi-qi-ge; Zhang, Yang

2015-01-01

To explore the potential precursors of the anthraquinone metabolites from Rheum tanguticum and preliminanly identify the synthesis pathway thereof. Sterile seedlings sprouted from the seeds of Rheum tanguticum were chosen as materials for inducing callus. The effects of different precursors and feeding duration on the callus of Rheum tanguticum and the anthraquinone yield in adult rheum were studied. The greatest improvement of anthraquinone yield was achieved by acetic acid, increasing 43. 9% for the callus and 45. 8% in the adult rheum; the second greatest improvement was achieved by malonic acid, increasing 15. 8% for the callus and only 3. 6% in the adult rheum. The yield of anthraquinone was not influenced significantly by benzoic acid and p-benzoquinone, and in contrast, was inhibited in some degree by shikimic acid and α-ketoglutaric acid. A suitable feeding duration was 36 h, which worked well for the effects of precursors. The precursor for synthesis of anthraquinone metabolites from Rheum tan- guticum is acetic acid, which improves the yields of callus and anthraquinone in adult rheum, concluding that the anthraquinone metabolites are synthesized via polyketone pathway.

Growth of organic crystals via attachment and transformation of nanoscopic precursors

NASA Astrophysics Data System (ADS)

Jiang, Yuan; Kellermeier, Matthias; Gebaue, Denis; Lu, Zihao; Rosenberg, Rose; Moise, Adrian; Przybylski, Michael; Cölfen, Helmut

2017-06-01

A key requirement for the understanding of crystal growth is to detect how new layers form and grow at the nanoscale. Multistage crystallization pathways involving liquid-like, amorphous or metastable crystalline precursors have been predicted by theoretical work and have been observed experimentally. Nevertheless, there is no clear evidence that any of these precursors can also be relevant for the growth of crystals of organic compounds. Herein, we present a new growth mode for crystals of DL-glutamic acid monohydrate that proceeds through the attachment of preformed nanoscopic species from solution, their subsequent decrease in height at the surface and final transformation into crystalline 2D nuclei that eventually build new molecular layers by further monomer incorporation. This alternative mechanism provides a direct proof for the existence of multistage pathways in the crystallization of molecular compounds and the relevance of precursor units larger than the monomeric constituents in the actual stage of growth.

Engineering biosynthesis of high-value compounds in photosynthetic organisms.

PubMed

O'Neill, Ellis C; Kelly, Steven

2017-09-01

The photosynthetic, autotrophic lifestyle of plants and algae position them as ideal platform organisms for sustainable production of biomolecules. However, their use in industrial biotechnology is limited in comparison to heterotrophic organisms, such as bacteria and yeast. This usage gap is in part due to the challenges in generating genetically modified plants and algae and in part due to the difficulty in the development of synthetic biology tools for manipulating gene expression in these systems. Plant and algal metabolism, pre-installed with multiple biosynthetic modules for precursor compounds, bypasses the requirement to install these pathways in conventional production organisms, and creates new opportunities for the industrial production of complex molecules. This review provides a broad overview of the successes, challenges and future prospects for genetic engineering in plants and algae for enhanced or de novo production of biomolecules. The toolbox of technologies and strategies that have been used to engineer metabolism are discussed, and the potential use of engineered plants for industrial manufacturing of large quantities of high-value compounds is explored. This review also discusses the routes that have been taken to modify the profiles of primary metabolites for increasing the nutritional quality of foods as well as the production of specialized metabolites, cosmetics, pharmaceuticals and industrial chemicals. As the universe of high-value biosynthetic pathways continues to expand, and the tools to engineer these pathways continue to develop, it is likely plants and algae will become increasingly valuable for the biomanufacturing of high-value compounds.

The role of amorphous precursors in the crystallization of La and Nd carbonates

NASA Astrophysics Data System (ADS)

Vallina, Beatriz; Rodriguez-Blanco, Juan Diego; Brown, Andrew P.; Blanco, Jesus A.; Benning, Liane G.

2015-07-01

Crystalline La and Nd carbonates can be formed from poorly-ordered nanoparticulate precursors, termed amorphous lanthanum carbonate (ALC) and amorphous neodymium carbonate (ANC). When reacted in air or in aqueous solutions these precursors show highly variable lifetimes and crystallization pathways. We have characterized these precursors and the crystallization pathways and products with solid-state, spectroscopic and microscopic techniques to explain the differences in crystallization mechanisms between the La and Nd systems. ALC and ANC consist of highly hydrated, 10-20 nm spherical nanoparticles with a general formula of REE2(CO3)3.5H2O (REE = La, Nd). The stabilities differ by ~2 orders of magnitude, with ANC being far more stable than ALC. This difference is due to the Nd3+ ion having a far higher hydration energy compared to the La3+ ion. This, together with temperature and reaction times, leads to clear differences not only in the kinetics and mechanisms of crystallization of the amorphous precursor La- and Nd-carbonate phases but also in the resulting crystallite sizes and morphologies of the end products. All crystalline La and Nd carbonates developed spherulitic morphologies when crystallization occurred from hydrous phases in solution at temperatures above 60 °C (La system) and 95 °C (Nd system). We suggest that spherulitic growth occurs due to a rapid breakdown of the amorphous precursors and a concurrent rapid increase in supersaturation levels in the aqueous solution. The kinetic data show that the crystallization pathway for both La and Nd carbonate systems is dependent on the reaction temperature and the ionic potential of the REE3+ ion.Crystalline La and Nd carbonates can be formed from poorly-ordered nanoparticulate precursors, termed amorphous lanthanum carbonate (ALC) and amorphous neodymium carbonate (ANC). When reacted in air or in aqueous solutions these precursors show highly variable lifetimes and crystallization pathways. We have

Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli.

PubMed

Menendez-Bravo, Simón; Comba, Santiago; Sabatini, Martín; Arabolaza, Ana; Gramajo, Hugo

2014-07-01

Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Perfluoroalkyl Acids (PFAAs) and Selected Precursors in the Baltic Sea Environment: Do Precursors Play a Role in Food Web Accumulation of PFAAs?

PubMed

Gebbink, Wouter A; Bignert, Anders; Berger, Urs

2016-06-21

The present study examined the presence of perfluoroalkyl acids (PFAAs) and selected precursors in the Baltic Sea abiotic environment and guillemot food web, and investigated the relative importance of precursors in food web accumulation of PFAAs. Sediment, water, zooplankton, herring, sprat, and guillemot eggs were analyzed for perfluoroalkane sulfonic acids (PFSAs; C4,6,8,10) and perfluoroalkyl carboxylic acids (PFCAs; C6-15) along with six perfluoro-octane sulfonic acid (PFOS) precursors and 11 polyfluoroalkyl phosphoric acid diesters (diPAPs). FOSA, FOSAA and its methyl and ethyl derivatives (Me- and EtFOSAA), and 6:2/6:2 diPAP were detected in sediment and water. While FOSA and the three FOSAAs were detected in all biota, a total of nine diPAPs were only detected in zooplankton. Concentrations of PFOS precursors and diPAPs exceeded PFOS and PFCA concentrations, respectively, in zooplankton, but not in fish and guillemot eggs. Although PFOS precursors were present at all trophic levels, they appear to play a minor role in food web accumulation of PFOS based on PFOS precursor/PFOS ratios and PFOS and FOSA isomer patterns. The PFCA pattern in fish could not be explained by the intake pattern based on PFCAs and analyzed precursors, that is, diPAPs. Exposure to additional precursors might therefore be a dominant exposure pathway compared to direct PFCA exposure for fish.

Biosynthesis of cis,cis-muconic acid and its aromatic precursors, catechol and protocatechuic acid, from renewable feedstocks by Saccharomyces cerevisiae.

PubMed

Weber, Christian; Brückner, Christine; Weinreb, Sheila; Lehr, Claudia; Essl, Christine; Boles, Eckhard

2012-12-01

Adipic acid is a high-value compound used primarily as a precursor for the synthesis of nylon, coatings, and plastics. Today it is produced mainly in chemical processes from petrochemicals like benzene. Because of the strong environmental impact of the production processes and the dependence on fossil resources, biotechnological production processes would provide an interesting alternative. Here we describe the first engineered Saccharomyces cerevisiae strain expressing a heterologous biosynthetic pathway converting the intermediate 3-dehydroshikimate of the aromatic amino acid biosynthesis pathway via protocatechuic acid and catechol into cis,cis-muconic acid, which can be chemically dehydrogenated to adipic acid. The pathway consists of three heterologous microbial enzymes, 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase composed of three different subunits, and catechol 1,2-dioxygenase. For each heterologous reaction step, we analyzed several potential candidates for their expression and activity in yeast to compose a functional cis,cis-muconic acid synthesis pathway. Carbon flow into the heterologous pathway was optimized by increasing the flux through selected steps of the common aromatic amino acid biosynthesis pathway and by blocking the conversion of 3-dehydroshikimate into shikimate. The recombinant yeast cells finally produced about 1.56 mg/liter cis,cis-muconic acid.

Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production

PubMed Central

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L.; Evans, Charles R.; Brenner, Charles

2011-01-01

NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins. PMID:21589930

Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production.

PubMed

Masakapalli, Shyam K; Ritala, Anneli; Dong, Lemeng; van der Krol, Alexander R; Oksman-Caldentey, Kirsi-Marja; Ratcliffe, R George; Sweetlove, Lee J

2014-03-01

The goal of this study was to characterise the metabolic flux phenotype of transgenic tobacco (Nicotiana tabacum) hairy roots engineered for increased biosynthesis of geraniol, an intermediate of the terpenoid indole alkaloid pathway. Steady state, stable isotope labelling was used to determine flux maps of central carbon metabolism for transgenic lines over-expressing (i) plastid-targeted geraniol synthase (pGES) from Valeriana officinalis, and (ii) pGES in combination with plastid-targeted geranyl pyrophosphate synthase from Arabidopsis thaliana (pGES+pGPPS), as well as for wild type and control-vector-transformed roots. Fluxes were constrained by the redistribution of label from [1-¹³C]-, [2-¹³C]- or [¹³C6]glucose into amino acids, sugars and organic acids at isotopic steady state, and by biomass output fluxes determined from the fractionation of [U-¹⁴C]glucose into insoluble polymers. No significant differences in growth and biomass composition were observed between the lines. The pGES line accumulated significant amounts of geraniol/geraniol glycosides (151±24 ng/mg dry weight) and the de novo synthesis of geraniol in pGES was confirmed by ¹³C labelling analysis. The pGES+pGPPS also accumulated geraniol and geraniol glycosides, but to lower levels than the pGES line. Although there was a distinct impact of the transgenes at the level of geraniol synthesis, other network fluxes were unaffected, reflecting the capacity of central metabolism to meet the relatively modest demand for increased precursors in the transgenic lines. It is concluded that re-engineering of the terpenoid indole alkaloid pathway will only require simultaneous manipulation of the steps producing the pathway precursors that originate in central metabolism in tissues engineered to produce at least an order of magnitude more geraniol than has been achieved so far. Copyright © 2013 Elsevier Ltd. All rights reserved.

Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria.

PubMed

Xiong, Wei; Lee, Tai-Chi; Rommelfanger, Sarah; Gjersing, Erica; Cano, Melissa; Maness, Pin-Ching; Ghirardi, Maria; Yu, Jianping

2015-12-07

Central carbon metabolism in cyanobacteria comprises the Calvin-Benson-Bassham (CBB) cycle, glycolysis, the pentose phosphate (PP) pathway and the tricarboxylic acid (TCA) cycle. Redundancy in this complex metabolic network renders the rational engineering of cyanobacterial metabolism for the generation of biomass, biofuels and chemicals a challenge. Here we report the presence of a functional phosphoketolase pathway, which splits xylulose-5-phosphate (or fructose-6-phosphate) to acetate precursor acetyl phosphate, in an engineered strain of the model cyanobacterium Synechocystis (ΔglgC/xylAB), in which glycogen synthesis is blocked, and xylose catabolism enabled through the introduction of xylose isomerase and xylulokinase. We show that this mutant strain is able to metabolise xylose to acetate on nitrogen starvation. To see whether acetate production in the mutant is linked to the activity of phosphoketolase, we disrupted a putative phosphoketolase gene (slr0453) in the ΔglgC/xylAB strain, and monitored metabolic flux using (13)C labelling; acetate and 2-oxoglutarate production was reduced in the light. A metabolic flux analysis, based on isotopic data, suggests that the phosphoketolase pathway metabolises over 30% of the carbon consumed by ΔglgC/xylAB during photomixotrophic growth on xylose and CO2. Disruption of the putative phosphoketolase gene in wild-type Synechocystis also led to a deficiency in acetate production in the dark, indicative of a contribution of the phosphoketolase pathway to heterotrophic metabolism. We suggest that the phosphoketolase pathway, previously uncharacterized in photosynthetic organisms, confers flexibility in energy and carbon metabolism in cyanobacteria, and could be exploited to increase the efficiency of cyanobacterial carbon metabolism and photosynthetic productivity.

Men's and Women's Pathways to Adulthood and Their Adolescent Precursors

ERIC Educational Resources Information Center

Oesterle, Sabrina; Hawkins, J. David; Hill, Karl G.; Bailey, Jennifer A.

2010-01-01

This study compared men's and women's pathways to adulthood by examining how role transitions in education, work, marriage, and parenthood intersect and form developmental pathways from ages 18-30. The study investigated how sociodemographic factors and adolescent experiences were associated with these pathways. We used latent class analysis to…

Genetically engineered mouse models of human B-cell precursor leukemias.

PubMed

Hauer, Julia; Borkhardt, Arndt; Sánchez-García, Isidro; Cobaleda, César

2014-01-01

B-cell precursor acute lymphoblastic leukemias (pB-ALLs) are the most frequent type of malignancies of the childhood, and also affect an important proportion of adult patients. In spite of their apparent homogeneity, pB-ALL comprises a group of diseases very different both clinically and pathologically, and with very diverse outcomes as a consequence of their biology, and underlying molecular alterations. Their understanding (as a prerequisite for their cure) will require a sustained multidisciplinary effort from professionals coming from many different fields. Among all the available tools for pB-ALL research, the use of animal models stands, as of today, as the most powerful approach, not only for the understanding of the origin and evolution of the disease, but also for the development of new therapies. In this review we go over the most relevant (historically, technically or biologically) genetically engineered mouse models (GEMMs) of human pB-ALLs that have been generated over the last 20 years. Our final aim is to outline the most relevant guidelines that should be followed to generate an "ideal" animal model that could become a standard for the study of human pB-ALL leukemia, and which could be shared among research groups and drug development companies in order to unify criteria for studies like drug testing, analysis of the influence of environmental risk factors, or studying the role of both low-penetrance mutations and cancer susceptibility alterations.

Modeling of coupled differential equations for cellular chemical signaling pathways: Implications for assay protocols utilized in cellular engineering.

PubMed

O'Clock, George D

2016-08-01

Cellular engineering involves modification and control of cell properties, and requires an understanding of fundamentals and mechanisms of action for cellular derived product development. One of the keys to success in cellular engineering involves the quality and validity of results obtained from cell chemical signaling pathway assays. The accuracy of the assay data cannot be verified or assured if the effect of positive feedback, nonlinearities, and interrelationships between cell chemical signaling pathway elements are not understood, modeled, and simulated. Nonlinearities and positive feedback in the cell chemical signaling pathway can produce significant aberrations in assay data collection. Simulating the pathway can reveal potential instability problems that will affect assay results. A simulation, using an electrical analog for the coupled differential equations representing each segment of the pathway, provides an excellent tool for assay validation purposes. With this approach, voltages represent pathway enzyme concentrations and operational amplifier feedback resistance and input resistance values determine pathway gain and rate constants. The understanding provided by pathway modeling and simulation is strategically important in order to establish experimental controls for assay protocol structure, time frames specified between assays, and assay concentration variation limits; to ensure accuracy and reproducibility of results.

Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit

PubMed Central

Gupta, Apoorv; Brockman Reizman, Irene M.; Reisch, Christopher R.; Prather, Kristala L. J.

2017-01-01

Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We applied our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in 5.5-fold increased titres of myo-inositol and titers of glucaric acid that improved from unmeasurable quantities to >0.8 g/L. Scaled-up production in benchtop bioreactors resulted in almost 10-fold and 5-fold increases in titers of myo-inositol and glucaric acid. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable quantities to >100 mg/L. PMID:28191902

Precise metabolic engineering of carotenoid biosynthesis in Escherichia coli towards a low-cost biosensor.

PubMed

Watstein, Daniel M; McNerney, Monica P; Styczynski, Mark P

2015-09-01

Micronutrient deficiencies, including zinc deficiency, are responsible for hundreds of thousands of deaths annually. A key obstacle to allocating scarce treatment resources is the ability to measure population blood micronutrient status inexpensively and quickly enough to identify those who most need treatment. This paper develops a metabolically engineered strain of Escherichia coli to produce different colored pigments (violacein, lycopene, and β-carotene) in response to different extracellular zinc levels, for eventual use in an inexpensive blood zinc diagnostic test. However, obtaining discrete color states in the carotenoid pathway required precise engineering of metabolism to prevent reaction at low zinc concentrations but allow complete reaction at higher concentrations, and all under the constraints of natural regulator limitations. Hence, the metabolic engineering challenge was not to improve titer, but to enable precise control of pathway state. A combination of gene dosage, post-transcriptional, and post-translational regulation was necessary to allow visible color change over physiologically relevant ranges representing a small fraction of the regulator's dynamic response range, with further tuning possible by modulation of precursor availability. As metabolic engineering expands its applications and develops more complex systems, tight control of system components will likely become increasingly necessary, and the approach presented here can be generalized to other natural sensing systems for precise control of pathway state. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Genetic engineering of Escherichia coli to improve L-phenylalanine production.

PubMed

Liu, Yongfei; Xu, Yiran; Ding, Dongqin; Wen, Jianping; Zhu, Beiwei; Zhang, Dawei

2018-01-30

L-phenylalanine (L-Phe) is an essential amino acid for mammals and applications expand into human health and nutritional products. In this study, a system level engineering was conducted to enhance L-Phe biosynthesis in Escherichia coli. We inactivated the PTS system and recruited glucose uptake via combinatorial modulation of galP and glk to increase PEP supply in the Xllp01 strain. In addition, the HTH domain of the transcription factor TyrR was engineered to decrease the repression on the transcriptional levels of L-Phe pathway enzymes. Finally, proteomics analysis demonstrated the third step of the SHIK pathway (catalyzed via AroD) as the rate-limiting step for L-Phe production. After optimization of the aroD promoter strength, the titer of L-Phe increased by 13.3%. Analysis of the transcriptional level of genes involved in the central metabolic pathways and L-Phe biosynthesis via RT-PCR showed that the recombinant L-Phe producer exhibited a great capability in the glucose utilization and precursor (PEP and E4P) generation. Via systems level engineering, the L-Phe titer of Xllp21 strain reached 72.9 g/L in a 5 L fermenter under the non-optimized fermentation conditions, which was 1.62-times that of the original strain Xllp01. The metabolic engineering strategy reported here can be broadly employed for developing genetically defined organisms for the efficient production of other aromatic amino acids and derived compounds.

Validation of RetroPath, a computer-aided design tool for metabolic pathway engineering.

PubMed

Fehér, Tamás; Planson, Anne-Gaëlle; Carbonell, Pablo; Fernández-Castané, Alfred; Grigoras, Ioana; Dariy, Ekaterina; Perret, Alain; Faulon, Jean-Loup

2014-11-01

Metabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer-aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top-ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top-ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17-fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

PubMed Central

Averesch, Nils J. H.; Krömer, Jens O.

2018-01-01

The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations. PMID:29632862

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.

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae

PubMed Central

Feng, Quanzhou; Weber, Scott A.; Li, Shizhong

2018-01-01

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production. PMID:29621349

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae.

PubMed

Feng, Quanzhou; Liu, Z Lewis; Weber, Scott A; Li, Shizhong

2018-01-01

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production.

Precursors of Young Women's Family Formation Pathways

ERIC Educational Resources Information Center

Amato, Paul R.; Landale, Nancy S.; Havasevich-Brooks, Tara C.; Booth, Alan; Eggebeen, David J.; Schoen, Robert; McHale, Susan M.

2008-01-01

We used latent class analysis to create family formation pathways for women between the ages of 18 and 23. Input variables included cohabitation, marriage, parenthood, full-time employment, and attending school. Data (n = 2,290) came from Waves I and III of the National Longitudinal Study of Adolescent Health (Add Health). The analysis revealed…

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived hydrocarbons.

PubMed

Zhang, Yiming; Nielsen, Jens; Liu, Zihe

2018-06-05

Fatty acid-derived hydrocarbons attract increasing attention as biofuels due to their immiscibility with water, high-energy content, low freezing point, and high compatibility with existing refineries and end-user infrastructures. Yeast Saccharomyces cerevisiae has advantages for production of fatty acid-derived hydrocarbons as its native routes toward fatty acid synthesis involve only a few reactions that allow more efficient conversion of carbon substrates. Here we describe major biosynthetic pathways of fatty acid-derived hydrocarbons in yeast, and summarize key metabolic engineering strategies, including enhancing precursor supply, eliminating competing pathways, and expressing heterologous pathways. With recent advances in yeast production of fatty acid-derived hydrocarbons, our review identifies key research challenges and opportunities for future optimization, and concludes with perspectives and outlooks for further research directions. © 2018 Wiley Periodicals, Inc.

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli

PubMed Central

Bokinsky, Gregory; Peralta-Yahya, Pamela P.; George, Anthe; Holmes, Bradley M.; Steen, Eric J.; Dietrich, Jeffrey; Soon Lee, Taek; Tullman-Ercek, Danielle; Voigt, Christopher A.; Simmons, Blake A.; Keasling, Jay D.

2011-01-01

One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels. PMID:22123987

Per- and polyfluoroalkyl substances (PFASs) in San Francisco Bay wildlife: Temporal trends, exposure pathways, and notable presence of precursor compounds.

PubMed

Sedlak, Margaret D; Benskin, Jonathan P; Wong, Adam; Grace, Richard; Greig, Denise J

2017-10-01

Concentrations of perfluorooctane sulfonate (PFOS) in San Francisco Bay (SF Bay) wildlife have historically been among the highest reported globally. To track continuing exposures to PFASs and assess the impact of the 2002 phase-out of production of PFOS and related chemicals in the US, nine perfluoroalkyl carboxylic acids (PFCAs; C4-C12), three perfluoroalkyl sulfonic acids (PFSAs; C4, C6, C8) and perfluorooctane sulfonamide (PFOSA, a PFOS precursor) were measured in SF Bay cormorant eggs in 2012 and harbor seal serum sampled between 2009 and 2014. PFOS remained the dominant perfluoroalkyl acid (PFAA) in both cormorant eggs (36.1-466 ng/g) and seals (12.6-796 ng/g) from 2012 and 2014, respectively. Concentrations in seal and bird eggs from the South Bay have declined approximately 70% in both matrices. To elucidate potential pathways of exposure, prey fish, sediments and wastewater effluent were analyzed for PFASs, and in the case of sediment and effluent, a suite of PFAA precursors. PFOS was the dominant PFAA in prey fish and sediment. In effluent, different mixtures of PFAAs were measured, with PFOS, PFHxA, and PFOA detected in the highest concentrations. Polyfluoroalkyl phosphate diesters (PFCA-precursors) were observed at concentrations over an order of magnitude higher than PFCAs in sediment, highlighting their importance as a potential, on-going source of PFCAs to SF Bay wildlife. These findings suggest that the PFOS phase-out has resulted in reduced burdens to wildlife in SF Bay, but that exposure to diverse and incompletely characterized PFASs continues. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bio-based production of monomers and polymers by metabolically engineered microorganisms.

PubMed

Chung, Hannah; Yang, Jung Eun; Ha, Ji Yeon; Chae, Tong Un; Shin, Jae Ho; Gustavsson, Martin; Lee, Sang Yup

2015-12-01

Recent metabolic engineering strategies for bio-based production of monomers and polymers are reviewed. In the case of monomers, we describe strategies for producing polyamide precursors, namely diamines (putrescine, cadaverine, 1,6-diaminohexane), dicarboxylic acids (succinic, glutaric, adipic, and sebacic acids), and ω-amino acids (γ-aminobutyric, 5-aminovaleric, and 6-aminocaproic acids). Also, strategies for producing diols (monoethylene glycol, 1,3-propanediol, and 1,4-butanediol) and hydroxy acids (3-hydroxypropionic and 4-hydroxybutyric acids) used for polyesters are reviewed. Furthermore, we review strategies for producing aromatic monomers, including styrene, p-hydroxystyrene, p-hydroxybenzoic acid, and phenol, and propose pathways to aromatic polyurethane precursors. Finally, in vivo production of polyhydroxyalkanoates and recombinant structural proteins having interesting applications are showcased. Copyright © 2015 Elsevier Ltd. All rights reserved.

Estimating human exposure to PFOS isomers and PFCA homologues: the relative importance of direct and indirect (precursor) exposure.

PubMed

Gebbink, Wouter A; Berger, Urs; Cousins, Ian T

2015-01-01

Contributions of direct and indirect (via precursors) pathways of human exposure to perfluorooctane sulfonic acid (PFOS) isomers and perfluoroalkyl carboxylic acids (PFCAs) are estimated using a Scenario-Based Risk Assessment (SceBRA) modelling approach. Monitoring data published since 2008 (including samples from 2007) are used. The estimated daily exposures (resulting from both direct and precursor intake) for the general adult population are highest for PFOS and perfluorooctanoic acid (PFOA), followed by perfluorohexanoic acid (PFHxA) and perfluorodecanoic acid (PFDA), while lower daily exposures are estimated for perfluorobutanoic acid (PFBA) and perfluorododecanoic acid (PFDoDA). The precursor contributions to the individual perfluoroalkyl acid (PFAA) daily exposures are estimated to be 11-33% for PFOS, 0.1-2.5% for PFBA, 3.7-34% for PFHxA, 13-64% for PFOA, 5.2-66% for PFDA, and 0.7-25% for PFDoDA (ranges represent estimated precursor contributions in a low- and high-exposure scenario). For PFOS, direct intake via diet is the major exposure pathway regardless of exposure scenario. For PFCAs, the dominant exposure pathway is dependent on perfluoroalkyl chain length and exposure scenario. Modelled PFOS and PFOA concentrations in human serum using the estimated intakes from an intermediate-exposure scenario are in agreement with measured concentrations in different populations. The isomer pattern of PFOS resulting from total intakes (direct and via precursors) is estimated to be enriched with linear PFOS (84%) relative to technical PFOS (70% linear). This finding appears to be contradictory to the observed enrichment of branched PFOS isomers in recent human serum monitoring studies and suggests that either external exposure is not fully understood (e.g. there are unknown precursors, missing or poorly quantified exposure pathways) and/or that there is an incomplete understanding of the isomer-specific human pharmacokinetic processes of PFOS, its precursors and

Assimilation of NAD(+) precursors in Candida glabrata.

PubMed

Ma, Biao; Pan, Shih-Jung; Zupancic, Margaret L; Cormack, Brendan P

2007-10-01

The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection.

A Survey of Career Pathways of Engineering Deans in the United States: Strategies for Leadership Development

ERIC Educational Resources Information Center

Hargrove, S. Keith

2015-01-01

The career pathways of deans in higher education seem to follow the traditional model in academia from a senior faculty position and/or department chair. This however may be different from deans in engineering education. The goal of this survey research is to assess the career paths of current Deans of Colleges/Schools of Engineering in the United…

Engineering Escherichia coli to overproduce aromatic amino acids and derived compounds.

PubMed

Rodriguez, Alberto; Martínez, Juan A; Flores, Noemí; Escalante, Adelfo; Gosset, Guillermo; Bolivar, Francisco

2014-09-09

The production of aromatic amino acids using fermentation processes with recombinant microorganisms can be an advantageous approach to reach their global demands. In addition, a large array of compounds with alimentary and pharmaceutical applications can potentially be synthesized from intermediates of this metabolic pathway. However, contrary to other amino acids and primary metabolites, the artificial channelling of building blocks from central metabolism towards the aromatic amino acid pathway is complicated to achieve in an efficient manner. The length and complex regulation of this pathway have progressively called for the employment of more integral approaches, promoting the merge of complementary tools and techniques in order to surpass metabolic and regulatory bottlenecks. As a result, relevant insights on the subject have been obtained during the last years, especially with genetically modified strains of Escherichia coli. By combining metabolic engineering strategies with developments in synthetic biology, systems biology and bioprocess engineering, notable advances were achieved regarding the generation, characterization and optimization of E. coli strains for the overproduction of aromatic amino acids, some of their precursors and related compounds. In this paper we review and compare recent successful reports dealing with the modification of metabolic traits to attain these objectives.

Tomato Fruits-A Platform for Metabolic Engineering of Terpenes.

PubMed

Gutensohn, M; Dudareva, N

2016-01-01

Terpenoids are a large and diverse class of plant metabolites including mono-, sesqui-, and diterpenes. They have numerous functions in basic physiological processes as well as the interaction of plants with their biotic and abiotic environment. Due to the tight regulation of biosynthetic pathways and the resulting limited natural availability of terpenes, there is a strong interest in increasing their production in plants by metabolic engineering for agricultural, pharmaceutical, and industrial applications. The tomato fruit system was developed as a platform for metabolic engineering of terpenes to overcome detrimental effects on overall plant growth and photosynthesis traits, which are affected when terpenoid engineering is performed in vegetative tissues. Here we describe how the use of fruit-specific promoters for transgene expression can avoid these unwanted effects. In addition, targeting the expression of the introduced terpene biosynthetic gene to fruit tissue can take advantage of the large precursor pool provided by the methylerythritol-phosphate (MEP) pathway, which is highly active during tomato fruit ripening to facilitate the accumulation of carotenoids. We also discuss how the production of high levels of target terpene compounds can be achieved in fruits by the expression of individual or a combination of (i) the MEP or mevalonic acid pathway enzymes, (ii) prenyltransferases, and/or (iii) terpene synthases. Finally, we provide a brief outline of how the emitted as well as internal pools of terpenes can be analyzed in transgenic tomato fruits. © 2016 Elsevier Inc. All rights reserved.

Tetrahydroxystilbene glucoside modulates amyloid precursor protein processing via activation of AKT-GSK3β pathway in cells and in APP/PS1 transgenic mice.

PubMed

Yin, Xiaomin; Chen, Chen; Xu, Ting; Li, Lin; Zhang, Lan

2018-01-01

Alternative splicing of amyloid precursor protein (APP) exon 7 generates the isoforms containing a Kunitz protease inhibitor (KPI) domain. APP-KPI levels in the brain are correlated with amyloid beta (Aβ) production. Here, we determined the effect of Tetrahydroxystilbene glucoside (TSG) on the AKT-GSK3β pathway. We found GSK3β increased APP-KPI inclusion level and interacted with the splicing factor ASF. TSG was intragastrically administered to 5-month-old APP/PS1 transgenic mice for 12 months. We found that the activated the AKT-GSK3β signaling pathway suppressed APP-KPI inclusion. Moreover, TSG treatment attenuated amyloid deposition in APP/PS1 mice. This study demonstrates the neuroprotective effect of TSG on APP expression, suggesting that TSG may be beneficial for AD prevention and treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway.

PubMed

Höfer, René; Dong, Lemeng; André, François; Ginglinger, Jean-François; Lugan, Raphael; Gavira, Carole; Grec, Sebastien; Lang, Gerhard; Memelink, Johan; Van der Krol, Sander; Bouwmeester, Harro; Werck-Reichhart, Danièle

2013-11-01

The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway. Copyright © 2013 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Kinetic analysis of aerobic biotransformation pathways of a perfluorooctane sulfonate (PFOS) precursor in distinctly different soils.

PubMed

Zhang, Lilan; Lee, Linda S; Niu, Junfeng; Liu, Jinxia

2017-10-01

With the phaseout of perfluorooctane sulfonate (PFOS) production in most countries and its well known recalcitrance, there is a need to quantify the potential release of PFOS from precursors previously or currently being emitted into the environment. Aerobic biodegradation of N-ethyl perfluorooctane sulfonamidoethanol (EtFOSE) was monitored in two soils from Indiana, USA: an acidic forest silt loam (FRST-48, pH = 5.5) and a high pH agricultural loam (PSF-49, pH = 7.8) with similar organic carbon contents (2.4 and 2.6%) for 210 d and 180 d, respectively. At designated times, triplicate samples were sacrificed for which headspace samples were taken followed by three sequential extractions. Extracts were analyzed using HPLC-tandem mass spectrometry. Measured profiles of EtFOSE degradation and generation/degradation of subsequent metabolites were fitted to the Indiana soils data as well as to a previously published data set for a Canadian soil using an R-based model (KinGUII) to explore pathways and estimate half-lives (t 1/2 ) for EtFOSE and metabolites. EtFOSE degradation ranged from a few days to up to a month. PFOS yields ranged form 1.06-5.49 mol% with the alkaline soils being four to five times higher than the acidic soil. In addition, a direct pathway to PFOS had to be invoked to describe the early generation of PFOS in the Canadian soil. Of all metabolites, the sulfonamidoacetic acids were the most persistent (t 1/2  ≥ 3 months) in all soils. We hypothesized that while pH-pK a dependent speciation may have impacted rates, differences in microbial communities between the 3 soils arising from varied soil properties including pH, nutrient levels, soil management, and climatic regions are likely the major factors affecting pathways, rates, and PFOS yields. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pathways to Mathematics: Longitudinal Predictors of Performance

ERIC Educational Resources Information Center

LeFevre, Jo-Anne; Fast, Lisa; Skwarchuk, Sheri-Lynn; Smith-Chant, Brenda L.; Bisanz, Jeffrey; Kamawar, Deepthi; Penner-Wilger, Marcie

2010-01-01

A model of the relations among cognitive precursors, early numeracy skill, and mathematical outcomes was tested for 182 children from 4.5 to 7.5 years of age. The model integrates research from neuroimaging, clinical populations, and normal development in children and adults. It includes 3 precursor pathways: quantitative, linguistic, and spatial…

Systems metabolic engineering of Escherichia coli for L-threonine production.

PubMed

Lee, Kwang Ho; Park, Jin Hwan; Kim, Tae Yong; Kim, Hyun Uk; Lee, Sang Yup

2007-01-01

Amino-acid producers have traditionally been developed by repeated random mutagenesis owing to the difficulty in rationally engineering the complex and highly regulated metabolic network. Here, we report the development of the genetically defined L-threonine overproducing Escherichia coli strain by systems metabolic engineering. Feedback inhibitions of aspartokinase I and III (encoded by thrA and lysC, respectively) and transcriptional attenuation regulations (located in thrL) were removed. Pathways for Thr degradation were removed by deleting tdh and mutating ilvA. The metA and lysA genes were deleted to make more precursors available for Thr biosynthesis. Further target genes to be engineered were identified by transcriptome profiling combined with in silico flux response analysis, and their expression levels were manipulated accordingly. The final engineered E. coli strain was able to produce Thr with a high yield of 0.393 g per gram of glucose, and 82.4 g/l Thr by fed-batch culture. The systems metabolic engineering strategy reported here may be broadly employed for developing genetically defined organisms for the efficient production of various bioproducts.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

PubMed

Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

2015-11-06

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells*

PubMed Central

Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E.; Ziegler, Mathias; Nikiforov, Andrey

2015-01-01

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5′-nucleotidases (5′-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5′-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5′-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. PMID:26385918

Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.

PubMed

Kildegaard, Kanchana R; Jensen, Niels B; Schneider, Konstantin; Czarnotta, Eik; Özdemir, Emre; Klein, Tobias; Maury, Jérôme; Ebert, Birgitta E; Christensen, Hanne B; Chen, Yun; Kim, Il-Kwon; Herrgård, Markus J; Blank, Lars M; Forster, Jochen; Nielsen, Jens; Borodina, Irina

2016-03-15

In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacs (L641P). Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L(-1) 3HP with a yield of 13% C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by (13)C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes.

PubMed

Vargas-Tah, Alejandra; Gosset, Guillermo

2015-01-01

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement.

Reverse engineering the mechanical and molecular pathways in stem cell morphogenesis.

PubMed

Lu, Kai; Gordon, Richard; Cao, Tong

2015-03-01

The formation of relevant biological structures poses a challenge for regenerative medicine. During embryogenesis, embryonic cells differentiate into somatic tissues and undergo morphogenesis to produce three-dimensional organs. Using stem cells, we can recapitulate this process and create biological constructs for therapeutic transplantation. However, imperfect imitation of nature sometimes results in in vitro artifacts that fail to recapitulate the function of native organs. It has been hypothesized that developing cells may self-organize into tissue-specific structures given a correct in vitro environment. This proposition is supported by the generation of neo-organoids from stem cells. We suggest that morphogenesis may be reverse engineered to uncover its interacting mechanical pathway and molecular circuitry. By harnessing the latent architecture of stem cells, novel tissue-engineering strategies may be conceptualized for generating self-organizing transplants. Copyright © 2013 John Wiley & Sons, Ltd.

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors.

PubMed

Bormann, Sebastian; Baer, Zachary C; Sreekumar, Sanil; Kuchenreuther, Jon M; Dean Toste, F; Blanch, Harvey W; Clark, Douglas S

2014-09-01

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a blend with nearly 50wt%≤C9 products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9gL(-1) while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80wt% of catalysis products having a carbon chain length≥C11 which amounts to 9.8gL(-1) of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was

Microbial isoprenoid production: an example of green chemistry through metabolic engineering.

PubMed

Maury, Jérôme; Asadollahi, Mohammad A; Møller, Kasper; Clark, Anthony; Nielsen, Jens

2005-01-01

Saving energy, cost efficiency, producing less waste, improving the biodegradability of products, potential for producing novel and complex molecules with improved properties, and reducing the dependency on fossil fuels as raw materials are the main advantages of using biotechnological processes to produce chemicals. Such processes are often referred to as green chemistry or white biotechnology. Metabolic engineering, which permits the rational design of cell factories using directed genetic modifications, is an indispensable strategy for expanding green chemistry. In this chapter, the benefits of using metabolic engineering approaches for the development of green chemistry are illustrated by the recent advances in microbial production of isoprenoids, a diverse and important group of natural compounds with numerous existing and potential commercial applications. Accumulated knowledge on the metabolic pathways leading to the synthesis of the principal precursors of isoprenoids is reviewed, and recent investigations into isoprenoid production using engineered cell factories are described.

Proteome Profiling Reveals Potential Toxicity and Detoxification Pathways Following Exposure of BEAS-2B Cells to Engineered Titanium Dioxide Nanoparticles

EPA Science Inventory

Oxidative stress is known to play important roles in engineered nanomaterial induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial mediated oxidative stress and toxicity are largely unknown. To identify these toxicity ...

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

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.

Impacts of Lowered Urban Air Temperatures on Precursor Emission and Ozone Air Quality.

PubMed

Taha, Haider; Konopacki, Steven; Akbari, Hashem

1998-09-01

Meteorological, photochemical, building-energy, and power plant simulations were performed to assess the possible precursor emission and ozone air quality impacts of decreased air temperatures that could result from implementing the "cool communities" concept in California's South Coast Air Basin (SoCAB). Two pathways are considered. In the direct pathway, a reduction in cooling energy use translates into reduced demand for generation capacity and, thus, reduced precursor emissions from electric utility power plants. In the indirect pathway, reduced air temperatures can slow the atmospheric production of ozone as well as precursor emission from anthropogenic and biogenic sources. The simulations suggest small impacts on emissions following implementation of cool communities in the SoCAB. In summer, for example, there can be reductions of up to 3% in NO x emissions from in-basin power plants. The photochemical simulations suggest that the air quality impacts of these direct emission reductions are small. However, the indirect atmospheric effects of cool communities can be significant. For example, ozone peak concentrations can decrease by up to 11% in summer and population-weighted exceedance exposure to ozone above the California and National Ambient Air Quality Standards can decrease by up to 11 and 17%, respectively. The modeling suggests that if these strategies are combined with others, such as mobile-source emission control, the improvements in ozone air quality can be substantial.

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans

PubMed Central

Gorrepati, Lakshmi; Krause, Michael W.; Chen, Weiping; Brodigan, Thomas M.; Correa-Mendez, Margarita; Eisenmann, David M.

2015-01-01

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type–specific "mRNA tagging" to enrich for VPC and seam cell–specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type–specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. PMID:26048561

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

PubMed

Gorrepati, Lakshmi; Krause, Michael W; Chen, Weiping; Brodigan, Thomas M; Correa-Mendez, Margarita; Eisenmann, David M

2015-06-05

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. Copyright © 2015 Gorrepati et al.

Metabolic pathways for lipid synthesis under nitrogen stress in Chlamydomonas and Nannochloropsis.

PubMed

Banerjee, Avik; Maiti, Subodh K; Guria, Chandan; Banerjee, Chiranjib

2017-01-01

Microalgae are currently being considered as a clean, sustainable and renewable energy source. Enzymes that catalyse the metabolic pathways for biofuel production are specific and require strict regulation and co-ordination. Thorough knowledge of these key enzymes along with their regulatory molecules is essential to enable rational metabolic engineering, to drive the metabolic flux towards the desired metabolites of importance. This paper reviews two key enzymes that play their role in production of bio-oil: DGAT (acyl-CoA:diacylglycerol acyltransferase) and PDAT (phospholipid:diacylglycerol acyltransferase). It also deals with the transcription factors that control the enzymes while cell undergoes a metabolic shift under stress. The paper also discusses the association of other enzymes and pathways that provide substrates and precursors for oil accumulation. Finally a futuristic solution has been proposed about a synthetic algal cell platform that would be committed towards biofuel synthesis.

Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli.

PubMed

Watts, Kevin T; Lee, Pyung C; Schmidt-Dannert, Claudia

2006-03-21

Phenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells. A pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure. Phenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently

JAK/Stat signaling regulates heart precursor diversification in Drosophila

PubMed Central

Johnson, Aaron N.; Mokalled, Mayssa H.; Haden, Tom N.; Olson, Eric N.

2011-01-01

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain. PMID:21965617

Engineering Sialic Acid Synthesis Ability in Insect Cells.

PubMed

Viswanathan, Karthik; Narang, Someet; Betenbaugh, Michael J

2015-01-01

Insect cells lack the ability to synthesize the sialic acid donor molecule CMP-sialic acid or its precursor, sialic acid. In this chapter, we describe a method to engineer CMP-sialic acid synthesis capability into Spodoptera frugiperda (Sf9) cells, a prototypical insect cell line, by recombinant expression of sialic acid synthesis pathway genes using baculovirus technology. Co-expression of a sialuria mutant UDP-GlcNAc-2-epimerase/ManNAc kinase (EKR263L), wild-type sialic acid 9-phosphate synthase (SAS), and wild-type CMP-sialic acid synthetase (CSAS) in the presence of GlcNAc leads to synthesis of CMP-sialic acids synthesis to support sialylation of N-glycans on glycoproteins.

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes

PubMed Central

Vargas-Tah, Alejandra; Gosset, Guillermo

2015-01-01

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement. PMID:26347861

SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae.

PubMed

Vanegas, Katherina García; Lehka, Beata Joanna; Mortensen, Uffe Hasbro

2017-02-08

The yeast Saccharomyces cerevisiae is increasingly used as a cell factory. However, cell factory construction time is a major obstacle towards using yeast for bio-production. Hence, tools to speed up cell factory construction are desirable. In this study, we have developed a new Cas9/dCas9 based system, SWITCH, which allows Saccharomyces cerevisiae strains to iteratively alternate between a genetic engineering state and a pathway control state. Since Cas9 induced recombination events are crucial for SWITCH efficiency, we first developed a technique TAPE, which we have successfully used to address protospacer efficiency. As proof of concept of the use of SWITCH in cell factory construction, we have exploited the genetic engineering state of a SWITCH strain to insert the five genes necessary for naringenin production. Next, the naringenin cell factory was switched to the pathway control state where production was optimized by downregulating an essential gene TSC13, hence, reducing formation of a byproduct. We have successfully integrated two CRISPR tools, one for genetic engineering and one for pathway control, into one system and successfully used it for cell factory construction.

Modules for in vitro metabolic engineering: Pathway assembly for bio-based production of value-added chemicals.

PubMed

Taniguchi, Hironori; Okano, Kenji; Honda, Kohsuke

2017-06-01

Bio-based chemical production has drawn attention regarding the realization of a sustainable society. In vitro metabolic engineering is one of the methods used for the bio-based production of value-added chemicals. This method involves the reconstitution of natural or artificial metabolic pathways by assembling purified/semi-purified enzymes in vitro . Enzymes from distinct sources can be combined to construct desired reaction cascades with fewer biological constraints in one vessel, enabling easier pathway design with high modularity. Multiple modules have been designed, built, tested, and improved by different groups for different purpose. In this review, we focus on these in vitro metabolic engineering modules, especially focusing on the carbon metabolism, and present an overview of input modules, output modules, and other modules related to cofactor management.

Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

PubMed

Kozak, Barbara U; van Rossum, Harmen M; Luttik, Marijke A H; Akeroyd, Michiel; Benjamin, Kirsten R; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

2014-10-21

The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy

Role of Nicotinamide Adenine Dinucleotide and Related Precursors as Therapeutic Targets for Age-Related Degenerative Diseases: Rationale, Biochemistry, Pharmacokinetics, and Outcomes.

PubMed

Braidy, Nady; Berg, Jade; Clement, James; Khorshidi, Fatemeh; Poljak, Anne; Jayasena, Tharusha; Grant, Ross; Sachdev, Perminder

2018-05-11

Nicotinamide adenine dinucleotide (NAD + ) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD + depletion may occur in response to either excessive DNA damage due to free radical or ultraviolet attack, resulting in significant poly(ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD + , and/or chronic immune activation and inflammatory cytokine production resulting in accelerated CD38 activity and decline in NAD + levels. Recent studies have shown that enhancing NAD + levels can profoundly reduce oxidative cell damage in catabolic tissue, including the brain. Therefore, promotion of intracellular NAD + anabolism represents a promising therapeutic strategy for age-associated degenerative diseases in general, and is essential to the effective realization of multiple benefits of healthy sirtuin activity. The kynurenine pathway represents the de novo NAD + synthesis pathway in mammalian cells. NAD + can also be produced by the NAD + salvage pathway. Recent Advances: In this review, we describe and discuss recent insights regarding the efficacy and benefits of the NAD + precursors, nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), in attenuating NAD + decline in degenerative disease states and physiological aging. Results obtained in recent years have shown that NAD + precursors can play important protective roles in several diseases. However, in some cases, these precursors may vary in their ability to enhance NAD + synthesis via their location in the NAD + anabolic pathway. Increased synthesis of NAD + promotes protective cell responses, further demonstrating that NAD + is a regulatory molecule associated with several

Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin-Fatty Acid Biosynthetic Pathway.

PubMed

Haushalter, Robert W; Phelan, Ryan M; Hoh, Kristina M; Su, Cindy; Wang, George; Baidoo, Edward E K; Keasling, Jay D

2017-04-05

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway.

PubMed

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-Lin; Zhang, Xiao-Gang; Dawe, Gavin S; Xiao, Zhi-Cheng

2016-12-23

Amyloid precursor protein (APP), commonly associated with Alzheimer's disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Na v 1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway

PubMed Central

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-lin; Zhang, Xiao-Gang; Dawe, Gavin S.; Xiao, Zhi-Cheng

2016-01-01

Amyloid precursor protein (APP), commonly associated with Alzheimer’s disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Nav1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668. PMID:28008944

Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk.

PubMed

Trammell, Samuel Aj; Yu, Liping; Redpath, Philip; Migaud, Marie E; Brenner, Charles

2016-05-01

Nicotinamide riboside (NR) is a recently discovered NAD(+) precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD(+) precursors was unknown. We aimed to determine NAD(+) precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk. LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD(+) precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk. Cow milk typically contained ∼12 μmol NAD(+) precursor vitamins/L, of which 60% was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD(+) metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = -0.58, P = 0.014), and NR was degraded by S. aureus Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk. NR is a major NAD(+) precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD(+) precursor vitamin concentration of milk. © 2016 American Society for Nutrition.

Engineering of Microbial Cell Factories for the Production of Plant Polyphenols with Health-Beneficial Properties.

PubMed

Dudnik, Alexey; Gaspar, Paula; Neves, Ana Rute; Forster, Jochen

2018-05-15

Polyphenols form a group of important natural bioactive compounds with numerous ascribed health-beneficial attributes (e.g. antioxidant, anti-inflammatory, anti-microbial and tumor-suppressing properties). Some polyphenols can also be used as natural dyes or plastic precursors. Notwithstanding their relevance, production of most of these compounds still relies on extraction from plant material, which for most of it is a costly and an inefficient procedure. The use of microbial cell factories for this purpose is an emerging alternative that could allow a more efficient and sustainable production. The most recent advances in molecular biology and genetic engineering, combined with the ever-growing understanding of microbial physiology have led to multiple success stories. Production of multiple polyphenolic compounds or their direct precursors has been achieved not only in the common production hosts, such as Escherichia coli and Saccharomyces cerevisiae but also in Corynebacterium glutamicum and Lactococcus lactis. However, boosting production of native compounds or introduction of heterologous biosynthetic pathways also brings certain challenges, such as the need to express, balance and maintain efficient precursor supply. This review will discuss the most recent advances in the field of metabolic engineering of microorganisms for polyphenol biosynthesis and its future perspectives, as well as outlines their potential health benefits and current production methods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Recent advances in the metabolic engineering of lignan biosynthesis pathways for the production of transgenic plant-based foods and supplements.

PubMed

Satake, Honoo; Ono, Eiichiro; Murata, Jun

2013-12-04

Plant physiological, epidemiological, and food science studies have shed light on lignans as healthy diets for the reduction of the risk of lifestyle-related noncommunicable diseases and, thus, the demand for lignans has been rapidly increasing. However, the low efficiency and instability of lignan production via extraction from plant resources remain to be resolved, indicating the requirement for the development of new procedures for lignan production. The metabolic engineering of lignan-biosynthesizing plants is expected to be most promising for efficient, sustainable, and stable lignan production. This is supported by the recent verification of biosynthetic pathways of major dietary lignans and the exploration of lignan production via metabolic engineering using transiently gene-transfected or transgenic plants. The aim of this review is to present an overview of the biosynthetic pathways, biological activities, and metabolic engineering of lignans and also perspectives in metabolic engineering-based lignan production using transgenic plants for practical application.

Phospholipid biosynthesis in Candida albicans: Regulation by the precursors inositol and choline

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

Klig, L.S.; Friedli, L.; Schmid, E.

1990-08-01

Phospholipid metabolism in the pathogenic fungus Candida albicans was examined. The phospholipid biosynthetic pathways of C. albicans were elucidated and were shown to be similar to those of Saccharomyces cerevisiae. However, marked differences were seen between these two fungi in the regulation of the pathways in response to exogenously provided precursors inositol and choline. In S. cerevisiae, the biosynthesis of phosphatidylcholine via methylation of phosphatidylethanolamine appears to be regulated in response to inositol and choline; provision of choline alone does not repress the activity of this pathway. The same pathway in C. albicans responds to the exogenous provision of choline.more » Possible explanations for the observed differences in regulation are discussed.« less

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

PubMed

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D; Schulz, Stefan; Fleißner, André

2016-10-18

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion

PubMed Central

Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D.; Schulz, Stefan; Fleißner, André

2016-01-01

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion. PMID:27708165

In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments.

PubMed

Deshmukh, Amit T; Verheijen, Peter J T; Maleki Seifar, Reza; Heijnen, Joseph J; van Gulik, Walter M

2015-11-01

In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product. After the stepwise perturbation with PAA, the pathway produced PenG within seconds. From the extra- and intracellular metabolite measurements, hypotheses for the secretion mechanisms of penicillin pathway metabolites were derived. A dynamic model of the penicillin biosynthesis pathway was then constructed that included the formation and transport over the cytoplasmic membrane of pathway intermediates, PAA and the product penicillin-G (PenG). The model parameters and changes in the enzyme levels of the penicillin biosynthesis pathway under in vivo conditions were simultaneously estimated using experimental data obtained at three different timescales (seconds, minutes, hours). The model was applied to determine changes in the penicillin pathway enzymes in time, calculate fluxes and analyze the flux control of the pathway. This led to a reassessment of the in vivo behavior of the pathway enzymes and in particular Acyl-CoA:Isopenicillin N Acyltransferase (AT). Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Identifying precursors and aqueous organic aerosol formation pathways during the SOAS campaign

NASA Astrophysics Data System (ADS)

Sareen, Neha; Carlton, Annmarie G.; Surratt, Jason D.; Gold, Avram; Lee, Ben; Lopez-Hilfiker, Felipe D.; Mohr, Claudia; Thornton, Joel A.; Zhang, Zhenfa; Lim, Yong B.; Turpin, Barbara J.

2016-11-01

Aqueous multiphase chemistry in the atmosphere can lead to rapid transformation of organic compounds, forming highly oxidized, low-volatility organic aerosol and, in some cases, light-absorbing (brown) carbon. Because liquid water is globally abundant, this chemistry could substantially impact climate, air quality, and health. Gas-phase precursors released from biogenic and anthropogenic sources are oxidized and fragmented, forming water-soluble gases that can undergo reactions in the aqueous phase (in clouds, fogs, and wet aerosols), leading to the formation of secondary organic aerosol (SOAAQ). Recent studies have highlighted the role of certain precursors like glyoxal, methylglyoxal, glycolaldehyde, acetic acid, acetone, and epoxides in the formation of SOAAQ. The goal of this work is to identify additional precursors and products that may be atmospherically important. In this study, ambient mixtures of water-soluble gases were scrubbed from the atmosphere into water at Brent, Alabama, during the 2013 Southern Oxidant and Aerosol Study (SOAS). Hydroxyl (OH⚫) radical oxidation experiments were conducted with the aqueous mixtures collected from SOAS to better understand the formation of SOA through gas-phase followed by aqueous-phase chemistry. Total aqueous-phase organic carbon concentrations for these mixtures ranged from 92 to 179 µM-C, relevant for cloud and fog waters. Aqueous OH-reactive compounds were primarily observed as odd ions in the positive ion mode by electrospray ionization mass spectrometry (ESI-MS). Ultra high-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) spectra and tandem MS (MS-MS) fragmentation of these ions were consistent with the presence of carbonyls and tetrols. Products were observed in the negative ion mode and included pyruvate and oxalate, which were confirmed by ion chromatography. Pyruvate and oxalate have been found in the particle phase in many locations (as salts and complexes). Thus

A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop.

PubMed

Fuentes, Paulina; Zhou, Fei; Erban, Alexander; Karcher, Daniel; Kopka, Joachim; Bock, Ralph

2016-06-14

Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output.

A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop

PubMed Central

Fuentes, Paulina; Zhou, Fei; Erban, Alexander; Karcher, Daniel; Kopka, Joachim; Bock, Ralph

2016-01-01

Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output. DOI: http://dx.doi.org/10.7554/eLife.13664.001 PMID:27296645

Analysis and identification of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous by high-performance liquid chromatography.

PubMed

Lu, Mingbo; Zhang, Yang'e; Zhao, Chunfang; Zhou, Pengpeng; Yu, Longjiang

2010-01-01

This study presents an HPLC method for simultaneous analysis of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous. The HPLC method is accomplished by employing a C18 column and the mobile phase methanol/water/acetonitrile/ dichloromethane (70:4:13:13, v/v/v/v). Astaxanthin is quantified by detection at 480 nm. The carotenoid precursors are identified by LC-APCI-MS and UV-vis absorption spectra. Peaks showed in the HPLC chromatogram are identified as carotenoids in the monocyclic biosynthetic pathway or their derivatives. In the monocyclic carotenoid pathway, 3,3'-dihydroxy-beta,psi-carotene-4,4'-dione (DCD) is produced through gamma-carotene and torulene.

Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

ERIC Educational Resources Information Center

Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

2015-01-01

Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum.

PubMed

Zhang, Chuanzhi; Zhang, Junli; Kang, Zhen; Du, Guocheng; Chen, Jian

2015-05-01

Microbial production of L-phenylalanine (L-Phe) from renewable sources has attracted much attention recently. In the present study, Corynebacterium glutamicum 13032 was rationally engineered to produce L-Phe from inexpensive glucose. First, all the L-Phe biosynthesis pathway genes were investigated and the results demonstrated that in addition to AroF and PheA, the native PpsA, TktA, AroE and AroA, and the heterologous AroL and TyrB were also the key enzymes for L-Phe biosynthesis. Through combinational expression of these key enzymes, the L-Phe production was increased to 6.33 ± 0.13 g l(-1) which was about 1.48-fold of that of the parent strain C. glutamicum (pXM-pheA (fbr)-aroF (fbr)) (fbr, feedback-inhibition resistance). Furthermore, the production of L-Phe was improved to 9.14 ± 0.21 g l(-1) by modifying the glucose and L-Phe transport systems and blocking the acetate and lactate biosynthesis pathways. Eventually, the titer of L-Phe was enhanced to 15.76 ± 0.23 g l(-1) with a fed-batch fermentation strategy. To the best of our knowledge, this was the highest value reported in rationally engineered C. glutamicum 13032 strains. The results obtained will also contribute to rational engineering of C. glutamicum for production of other valuable aromatic compounds.

Pathways to space: A mission to foster the next generation of scientists and engineers

NASA Astrophysics Data System (ADS)

Dougherty, Kerrie; Oliver, Carol; Fergusson, Jennifer

2014-06-01

The first education project funded under the Australian Government's Australian Space Research Program (ASRP), Pathways to Space was a unique project combining education, science communication research and research in astrobiology and robotics. It drew upon the challenges of space exploration to inspire students to consider study and careers in science and engineering. A multi-faceted program, Pathways to Space provided hands-on opportunities for high school and university students to participate in realistic simulations of a robotic Mars exploration mission for astrobiology. Its development was a collaboration between the Australian Centre for Astrobiology (University of New South Wales), the Australian Centre for Field Robotics (University of Sydney), the Powerhouse Museum and industry partner, Cisco. Focused on students in Years 9-10 (15-16 years of age), this program provided them with the opportunity to engage directly with space engineers and astrobiologists, while carrying out a simulated Mars mission using the digital learning facilities available at the Powerhouse Museum. As a part of their program, the students operated robotic mini-rovers in the Powerhouse Museum's “Mars Yard”, a highly accurate simulation of the Martian surface, where university students also carry out the development and testing of experimental Mars roving vehicles. This aspect of the program has brought real science and engineering research into the public space of the museum. As they undertook the education program, the students participated in a research study aimed at understanding the effectiveness of the project in achieving its key objective - encouraging students to consider space related courses and careers. This paper outlines the development and operation of the Pathways to Space project over its 3-year funding period, during which it met and exceeded all the requirements of its ASRP grant. It will look at the goals of the project, the rationale behind the education and

Production of taxadiene by engineering of mevalonate pathway in Escherichia coli and endophytic fungus Alternaria alternata TPF6.

PubMed

Bian, Guangkai; Yuan, Yujie; Tao, Hui; Shi, Xiaofei; Zhong, Xiaofang; Han, Yichao; Fu, Shuai; Fang, Chengxiang; Deng, Zixin; Liu, Tiangang

2017-04-01

Taxol (paclitaxel) is a diterpenoid compound with significant and extensive applications in the treatment of cancer. The production of Taxol and relevant intermediates by engineered microbes is an attractive alternative to the semichemical synthesis of Taxol. In this study, based on a previously developed platform, the authors first established taxadiene production in mutant E. coli T2 and T4 by engineering of the mevalonate (MVA) pathway. The authors then developed an Agrobacterium tumefaciens-mediated transformation (ATMT) method and verified the strength of heterologous promoters in Alternaria alternata TPF6. The authors next transformed the taxadiene-producing platform into A. alternata TPF6, and the MVA pathway was engineered, with introduction of the plant taxadiene-forming gene. Notably, by co-overexpression of isopentenyl diphosphate isomerase (Idi), a truncated version of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1), and taxadiene synthase (TS), the authors could detect 61.9 ± 6.3 μg/L taxadiene in the engineered strain GB127. This is the first demonstration of taxadiene production in filamentous fungi, and the approach presented in this study provides a new method for microbial production of Taxol. The well-established ATMT method and the known promoter strengths facilitated further engineering of taxaenes in this fungus. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome Engineering of the 2,3-Butanediol Biosynthetic Pathway for Tight Regulation in Cyanobacteria.

PubMed

Nozzi, Nicole E; Atsumi, Shota

2015-11-20

Cyanobacteria have gained popularity among the metabolic engineering community as a tractable photosynthetic host for renewable chemical production. However, though a number of successfully engineered production systems have been reported, long-term genetic stability remains an issue for cyanobacterial systems. The genetic engineering toolbox for cyanobacteria is largely lacking inducible systems for expression control. The characterization of tight regulation systems for use in cyanobacteria may help to alleviate this problem. In this work we explore the function of the IPTG inducible promoter P(L)lacO1 in the model cyanobacterium Synechococcus elongatus PCC 7942 as well as the effect of gene order within an operon on pathway expression. According to our experiments, P(L)lacO1 functions well as an inducible promoter in S. elongatus. Additionally, we found that gene order within an operon can strongly influence control of expression of each gene.

Spinel formation for stabilizing simulated nickel-laden sludge with aluminum-rich ceramic precursors.

PubMed

Shih, Kaimin; White, Tim; Leckie, James O

2006-08-15

The feasibility of stabilizing nickel-laden sludge from commonly available Al-rich ceramic precursors was investigated and accomplished with high nickel incorporation efficiency. To simulate the process, nickel oxide was mixed alternatively with gamma-alumina, corundum, kaolinite, and mullite and was sintered from 800 to 1480 degrees C. The nickel aluminate spinel (NiAl2O4) was confirmed as the stabilization phase for nickel and crystallized with efficiencies greater than 90% for all precursors above 1250 degrees C and 3-h sintering. The nickel-incorporation reaction pathways with these precursors were identified, and the microstructure and spinel yield were investigated as a function of sintering temperature with fixed sintering time. This study has demonstrated a promising process for forming nickel spinel to stabilize nickel-laden sludge from a wide range of inexpensive ceramic precursors, which may provide an avenue for economically blending waste metal sludges via the building industry processes to reduce the environmental hazards of toxic metals. The correlation of product textures and nickel incorporation efficiencies through selection of different precursors also provides the option of tailoring property-specific products.

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.

Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin–Fatty Acid Biosynthetic Pathway

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

Haushalter, Robert W.; Phelan, Ryan M.; Hoh, Kristina M.

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here in this paper we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotinmore » and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.« less

Lu2O3-SiO2-ZrO2 Coatings for Environmental Barrier Application by Solution Precursor Plasma Spraying and Influence of Precursor Chemistry

NASA Astrophysics Data System (ADS)

Darthout, Émilien; Quet, Aurélie; Braidy, Nadi; Gitzhofer, François

2014-02-01

As environmental barrier coatings are subjected to thermal stress in gas turbine engines, the introduction of a secondary phase as zircon (ZrSiO4) is likely to increase the stress resistance of Lu2Si2O7 coatings generated by induction plasma spraying using liquid precursors. In a first step, precursor chemistry effect is investigated by the synthesis of ZrO2-SiO2 nanopowders by induction plasma nanopowder synthesis technique. Tetraethyl orthosilicate (TEOS) as silicon precursor and zirconium oxynitrate and zirconium ethoxide as zirconium precursors are mixed in ethanol and produce a mixture of tetragonal zirconia and amorphous silica nanoparticles. The use of zirconium ethoxide precursor results in zirconia particles with diameter below 50 nm because of exothermic thermal decomposition of the ethoxide and its high boiling point with respect to solvent, while larger particles are formed when zirconium oxynitrate is employed. The formation temperature of zircon from zirconia and silica oxides is found at 1425 °C. Second, coatings are synthesized in Lu2O3-ZrO2-SiO2 system. After heat treatment, the doping effect of lutetium on zirconia grains totally inhibits the zircon formation. Dense coatings are obtained with the use of zirconium ethoxide because denser particles with a homogeneous diameter distribution constitute the coating.

Aniline Is an Inducer, and Not a Precursor, for Indole Derivatives in Rubrivivax benzoatilyticus JA2

PubMed Central

Mohammed, Mujahid; Ch, Sasikala; Ch, Ramana V.

2014-01-01

Rubrivivax benzoatilyticus JA2 and other anoxygenic photosynthetic bacteria produce indole derivatives when exposed to aniline, a xenobiotic compound. Though this phenomenon has been reported previously, the role of aniline in the production of indoles is still a biochemical riddle. The present study aims at understanding the specific role of aniline (as precursor or stimulator) in the production of indoles and elucidating the biochemical pathway of indoles in aniline-exposed cells by using stable isotope approaches. Metabolic profiling revealed tryptophan accumulation only in aniline exposed cells along with indole 3-acetic acid (IAA) and indole 3-aldehyde (IAld), the two major catabolites of tryptophan. Deuterium labelled aniline feeding studies revealed that aniline is not a precursor of indoles in strain JA2. Further, production of indoles only in aniline-exposed cells suggests that aniline is an indoles stimulator. In addition, production of indoles depended on the presence of a carbon source, and production enhanced when carbon sources were added to the culture. Isotope labelled fumarate feeding identified, fumarate as the precursor of indole, indicating de novo synthesis of indoles. Glyphosate (shikimate pathway inhibitor) inhibited the indoles production, accumulation of tryptophan, IAA and IAld indicating that indoles synthesis in strain JA2 occurs via the de novo shikimate pathway. The up-regulation of anthranilate synthase gene and induction of anthranilate synthase activity correlated well with tryptophan production in strain JA2. Induction of tryptophan aminotransferase and tryptophan 2-monooxygenase activities corroborated well with IAA levels, suggesting that tryptophan catabolism occurs simultaneously in aniline exposed cells. Our study demonstrates that aniline (stress) stimulates tryptophan/indoles synthesis via the shikimate pathway by possibly modulating the metabolic pathway. PMID:24533057

Aniline is an inducer, and not a precursor, for indole derivatives in Rubrivivax benzoatilyticus JA2.

PubMed

Mujahid, Mohammed; Sasikala, Ch; Ramana, Ch V

2014-01-01

Rubrivivax benzoatilyticus JA2 and other anoxygenic photosynthetic bacteria produce indole derivatives when exposed to aniline, a xenobiotic compound. Though this phenomenon has been reported previously, the role of aniline in the production of indoles is still a biochemical riddle. The present study aims at understanding the specific role of aniline (as precursor or stimulator) in the production of indoles and elucidating the biochemical pathway of indoles in aniline-exposed cells by using stable isotope approaches. Metabolic profiling revealed tryptophan accumulation only in aniline exposed cells along with indole 3-acetic acid (IAA) and indole 3-aldehyde (IAld), the two major catabolites of tryptophan. Deuterium labelled aniline feeding studies revealed that aniline is not a precursor of indoles in strain JA2. Further, production of indoles only in aniline-exposed cells suggests that aniline is an indoles stimulator. In addition, production of indoles depended on the presence of a carbon source, and production enhanced when carbon sources were added to the culture. Isotope labelled fumarate feeding identified, fumarate as the precursor of indole, indicating de novo synthesis of indoles. Glyphosate (shikimate pathway inhibitor) inhibited the indoles production, accumulation of tryptophan, IAA and IAld indicating that indoles synthesis in strain JA2 occurs via the de novo shikimate pathway. The up-regulation of anthranilate synthase gene and induction of anthranilate synthase activity correlated well with tryptophan production in strain JA2. Induction of tryptophan aminotransferase and tryptophan 2-monooxygenase activities corroborated well with IAA levels, suggesting that tryptophan catabolism occurs simultaneously in aniline exposed cells. Our study demonstrates that aniline (stress) stimulates tryptophan/indoles synthesis via the shikimate pathway by possibly modulating the metabolic pathway.

Genetic and metabolic engineering for microbial production of poly-γ-glutamic acid.

PubMed

Cao, Mingfeng; Feng, Jun; Sirisansaneeyakul, Sarote; Song, Cunjiang; Chisti, Yusuf

2018-05-28

Poly-γ-glutamic acid (γ-PGA) is a natural biopolymer of glutamic acid. The repeating units of γ-PGA may be derived exclusively from d-glutamic acid, or l-glutamic acid, or both. The monomer units are linked by amide bonds between the α-amino group and the γ-carboxylic acid group. γ-PGA is biodegradable, edible and water-soluble. It has numerous existing and emerging applications in processing of foods, medicines and cosmetics. This review focuses on microbial production of γ-PGA via genetically and metabolically engineered recombinant bacteria. Strategies for improving production of γ-PGA include modification of its biosynthesis pathway, enhancing the production of its precursor (glutamic acid), and preventing loss of the precursor to competing byproducts. These and other strategies are discussed. Heterologous synthesis of γ-PGA in industrial bacterial hosts that do not naturally produce γ-PGA is discussed. Emerging trends and the challenges affecting the production of γ-PGA are reviewed. Copyright © 2018. Published by Elsevier Inc.

Phospholipid biosynthesis in Candida albicans: regulation by the precursors inositol and choline.

PubMed Central

Klig, L S; Friedli, L; Schmid, E

1990-01-01

Phospholipid metabolism in the pathogenic fungus Candida albicans was examined. The phospholipid biosynthetic pathways of C. albicans were elucidated and were shown to be similar to those of Saccharomyces cerevisiae. However, marked differences were seen between these two fungi in the regulation of the pathways in response to exogenously provided precursors inositol and choline. In S. cerevisiae, the biosynthesis of phosphatidylcholine via methylation of phosphatidylethanolamine appears to be regulated in response to inositol and choline; provision of choline alone does not repress the activity of this pathway (G. M. Carman and S. A. Henry, Annu. Rev. Biochem. 58:636-669, 1989). The same pathway in C. albicans responds to the exogenous provision of choline. Possible explanations for the observed differences in regulation are discussed. Images PMID:2198258

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering.

PubMed

Zhai, Yafei; Han, Donglei; Pan, Ying; Wang, Shuaishuai; Fang, Junqiang; Wang, Peng; Liu, Xian-wei

2015-02-01

Guanosine 5'-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5'-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue. Copyright © 2014 Elsevier Inc. All rights reserved.

Pimelic acid, the first precursor of the Bacillus subtilis biotin synthesis pathway, exists as the free acid and is assembled by fatty acid synthesis.

PubMed

Manandhar, Miglena; Cronan, John E

2017-05-01

Biotin synthetic pathways are readily separated into two stages, synthesis of the seven carbon α, ω-dicarboxylic acid pimelate moiety and assembly of the fused heterocyclic rings. The biotin pathway genes responsible for pimelate moiety synthesis vary widely among bacteria whereas the ring synthesis genes are highly conserved. Bacillus subtilis seems to have redundant genes, bioI and bioW, for generation of the pimelate intermediate. Largely consistent with previous genetic studies it was found that deletion of bioW caused a biotin auxotrophic phenotype whereas deletion of bioI did not. BioW is a pimeloyl-CoA synthetase that converts pimelic acid to pimeloyl-CoA. The essentiality of BioW for biotin synthesis indicates that the free form of pimelic acid is an intermediate in biotin synthesis although this is not the case in E. coli. Since the origin of pimelic acid in Bacillus subtilis is unknown, 13 C-NMR studies were carried out to decipher the pathway for its generation. The data provided evidence for the role of free pimelate in biotin synthesis and the involvement of fatty acid synthesis in pimelate production. Cerulenin, an inhibitor of the key fatty acid elongation enzyme, FabF, markedly decreased biotin production by B. subtilis resting cells whereas a strain having a cerulenin-resistant FabF mutant produced more biotin. In addition, supplementation with pimelic acid fully restored biotin production in cerulenin-treated cells. These results indicate that pimelic acid originating from fatty acid synthesis pathway is a bona fide precursor of biotin in B. subtilis. © 2017 John Wiley & Sons Ltd.

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

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

Kang, Aram; George, Kevin W.; Wang, George

Branched C 5 alcohols are promising biofuels with excellent combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C 5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C 5 alcohols and initial precursor to longermore » chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C 5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.« less

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

DOE PAGES

Kang, Aram; George, Kevin W.; Wang, George; ...

2015-12-17

Branched C 5 alcohols are promising biofuels with excellent combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C 5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C 5 alcohols and initial precursor to longermore » chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C 5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.« less

Understanding nanoparticle-mediated nucleation pathways of anisotropic nanoparticles

NASA Astrophysics Data System (ADS)

Laramy, Christine R.; Fong, Lam-Kiu; Jones, Matthew R.; O'Brien, Matthew N.; Schatz, George C.; Mirkin, Chad A.

2017-09-01

Several seed-mediated syntheses of low symmetry anisotropic nanoparticles yield broad product distributions with multiple defect structures. This observation challenges the role of the nanoparticle precursor as a seed for certain syntheses and suggests the possibility of alternate nucleation pathways. Herein, we report a method to probe the role of the nanoparticle precursor in anisotropic nanoparticle nucleation with compositional and structural 'labels' to track their fate. We use the synthesis of gold triangular nanoprisms (Au TPs) as a model system. We propose a mechanism in which, rather than acting as a template, the nanoparticle precursor catalyzes homogenous nucleation of Au TPs.

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid.

PubMed

Farré, Gemma; Perez-Fons, Laura; Decourcelle, Mathilde; Breitenbach, Jürgen; Hem, Sonia; Zhu, Changfu; Capell, Teresa; Christou, Paul; Fraser, Paul D; Sandmann, Gerhard

2016-08-01

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a β-carotene hydroxylase and a β-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the β-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.

On the structural intricacies of a metabolic precursor: Direct spectroscopic detection of water-induced conformational reshaping of mevalonolactone

NASA Astrophysics Data System (ADS)

Domingos, Sérgio R.; Pérez, Cristóbal; Schnell, Melanie

2017-09-01

We use high-resolution rotational spectroscopy to investigate the structural intricacies of the lactone form of mevalonic acid, precursor of the mevalonate pathway. By combining microwave spectroscopy with supersonic expansions and quantum-chemical calculations, we determine the two most stable conformations of the precursor. Complementary micro-solvation studies reveal that aggregation of the first water molecule induces a substantial structural rearrangement comprising a hydroxy rotation and an endocyclic core torsion to create a favourable geometry to accommodate the water molecule. We discuss the conformational aspects of the precursor in isolation and under micro-hydrated conditions.

Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.

PubMed

Zawadzke, Laura E; Norcia, Michael; Desbonnet, Charlene R; Wang, Hong; Freeman-Cook, Kevin; Dougherty, Thomas J

2008-02-01

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.

The Significance of Different Diacylgycerol Synthesis Pathways on Plant Oil Composition and Bioengineering

PubMed Central

Bates, Philip D.; Browse, John

2012-01-01

The unique properties of vegetable oils from different plants utilized for food, industrial feedstocks, and fuel is dependent on the fatty acid (FA) composition of triacylglycerol (TAG). Plants can use two main pathways to produce diacylglycerol (DAG), the immediate precursor molecule to TAG synthesis: (1) De novo DAG synthesis, and (2) conversion of the membrane lipid phosphatidylcholine (PC) to DAG. The FA esterified to PC are also the substrate for FA modification (e.g., desaturation, hydroxylation, etc.), such that the FA composition of PC-derived DAG can be substantially different than that of de novo DAG. Since DAG provides two of the three FA in TAG, the relative flux of TAG synthesis from de novo DAG or PC-derived DAG can greatly affect the final oil FA composition. Here we review how the fluxes through these two alternate pathways of DAG/TAG synthesis are determined and present evidence that suggests which pathway is utilized in different plants. Additionally, we present examples of how the endogenous DAG synthesis pathway in a transgenic host plant can produce bottlenecks for engineering of plant oil FA composition, and discuss alternative strategies to overcome these bottlenecks to produce crop plants with designer vegetable oil compositions. PMID:22783267

An Evaluation of Active Learning Causal Discovery Methods for Reverse-Engineering Local Causal Pathways of Gene Regulation

PubMed Central

Ma, Sisi; Kemmeren, Patrick; Aliferis, Constantin F.; Statnikov, Alexander

2016-01-01

Reverse-engineering of causal pathways that implicate diseases and vital cellular functions is a fundamental problem in biomedicine. Discovery of the local causal pathway of a target variable (that consists of its direct causes and direct effects) is essential for effective intervention and can facilitate accurate diagnosis and prognosis. Recent research has provided several active learning methods that can leverage passively observed high-throughput data to draft causal pathways and then refine the inferred relations with a limited number of experiments. The current study provides a comprehensive evaluation of the performance of active learning methods for local causal pathway discovery in real biological data. Specifically, 54 active learning methods/variants from 3 families of algorithms were applied for local causal pathways reconstruction of gene regulation for 5 transcription factors in S. cerevisiae. Four aspects of the methods’ performance were assessed, including adjacency discovery quality, edge orientation accuracy, complete pathway discovery quality, and experimental cost. The results of this study show that some methods provide significant performance benefits over others and therefore should be routinely used for local causal pathway discovery tasks. This study also demonstrates the feasibility of local causal pathway reconstruction in real biological systems with significant quality and low experimental cost. PMID:26939894

Dissolved Organic Carbon and Disinfection By-Product Precursor Release from Managed Peat Soils

USGS Publications Warehouse

Fleck, J.A.; Bossio, D.A.; Fujii, R.

2004-01-01

A wetland restoration demonstration project examined the effects of a permanently flooded wetland on subsidence of peat soils. The project, started in 1997, was done on Twitchell Island, in the Sacramento-San Joaquin Delta of California. Conversion of agricultural land to a wetland has changed many of the biogeochemical processes controlling dissolved organic carbon (DOC) release from the peat soils, relative to the previous land use. Dissolved organic C in delta waters is a concern because it reacts with chlorine, added as a disinfectant in municipal drinking waters, to form carcinogenic disinfection byproducts (DBPs), including trihalomethanes (THMs) and haloacetic acids (HAAs). This study explores the effects of peat soil biogeochemistry on DOC and DBP release under agricultural and wetland management. Results indicate that organic matter source, extent of soil organic matter decomposition, and decomposition pathways all are factors in THM formation. The results show that historical management practices dominate the release of DOC and THM precursors. However, within-site differences indicate that recent management decisions can contribute to changes in DOC quality and THM precursor formation. Not all aromatic forms of carbon are highly reactive and certain environmental conditions produce the specific carbon structures that form THMs. Both HAA and THM precursors are elevated in the DOC released under wetland conditions. The findings of this study emphasize the need to further investigate the roles of organic matter sources, microbial decomposition pathways, and decomposition status of soil organic matter in the release of DOC and DBP precursors from delta soils under varying land-use practices.

Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol.

PubMed

May, Bianca; Lange, B Markus; Wüst, Matthias

2013-11-01

The participation of the mevalonic acid (MVA) and 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-(2)H2]-1-deoxy-d-xylulose and [5,5-(2)H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol

PubMed Central

May, Bianca; Lange, B. Markus; Wüst, Matthias

2013-01-01

The participation of the mevalonic acid (MVA) and 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-2H2]-1-deoxy-D-xylulose and [5,5-2H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries. PMID:23954075

Self-Assembly of Mesoscale Isomers: The Role of Pathways and Degrees of Freedom

PubMed Central

Pandey, Shivendra; Johnson, Daniel; Kaplan, Ryan; Klobusicky, Joseph; Menon, Govind; Gracias, David H.

2014-01-01

The spontaneous self-organization of conformational isomers from identical precursors is of fundamental importance in chemistry. Since the precursors are identical, it is the multi-unit interactions, characteristics of the intermediates, and assembly pathways that determine the final conformation. Here, we use geometric path sampling and a mesoscale experimental model to investigate the self-assembly of a model polyhedral system, an octahedron, that forms two isomers. We compute the set of all possible assembly pathways and analyze the degrees of freedom or rigidity of intermediates. Consequently, by manipulating the degrees of freedom of a precursor, we were able to experimentally enrich the formation of one isomer over the other. Our results suggest a new approach to direct pathways in both natural and synthetic self-assembly using simple geometric criteria. We also compare the process of folding and unfolding in this model with a geometric model for cyclohexane, a well-known molecule with chair and boat conformations. PMID:25299051

Pathway engineering to improve ethanol production by thermophilic bacteria

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

Lynd, L.R.

1998-12-31

Continuation of a research project jointly funded by the NSF and DOE is proposed. The primary project goal is to develop and characterize strains of C. thermocellum and C. thermosaccharolyticum having ethanol selectivity similar to more convenient ethanol-producing organisms. An additional goal is to document the maximum concentration of ethanol that can be produced by thermophiles. These goals build on results from the previous project, including development of most of the genetic tools required for pathway engineering in the target organisms. As well, we demonstrated that the tolerance of C. thermosaccharolyticum to added ethanol is sufficiently high to allow practicalmore » utilization should similar tolerance to produced ethanol be demonstrated, and that inhibition by neutralizing agents may explain the limited concentrations of ethanol produced in studies to date. Task 1 involves optimization of electrotransformation, using either modified conditions or alternative plasmids to improve upon the low but reproducible transformation, frequencies we have obtained thus far.« less

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

PubMed Central

Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

2011-01-01

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule. PMID:21504897

Molecular variation of the nonribosomal peptide-polyketide siderophore yersiniabactin through biosynthetic and metabolic engineering.

PubMed

Ahmadi, Mahmoud Kamal; Fawaz, Samar; Fang, Lei; Yu, Zhipeng; Pfeifer, Blaine A

2016-05-01

The production of the mixed nonribosomal peptide-polyketide natural product yersiniabactin (Ybt) has been established using E. coli as a heterologous host. In this study, precursor-directed biosynthesis was used to generate five new analogs of Ybt, demonstrating the flexibility of the heterologous system and the biosynthetic process in allowing compound diversity. A combination of biosynthetic and cellular engineering was then used to influence the production metrics of the resulting analogs. First, the cellular levels and activity of FadL, a hydrocarbon transport protein, were tested for subsequent influence upon exogenous precursor uptake and Ybt analog production with a positive correlation observed between FadL over-production and analog formation. Next, a Ybt biosynthetic editing enzyme was removed from the heterologous system which decreased native compound production but increased analog formation. A final series of experiments enhanced endogenous anthranilate towards complete pathway formation of the associated analog which showed a selective ability to bind gold. © 2015 Wiley Periodicals, Inc.

The Exosome Secretory Pathway Transports Amyloid Precursor Protein Carboxyl-terminal Fragments from the Cell into the Brain Extracellular Space*

PubMed Central

Perez-Gonzalez, Rocio; Gauthier, Sebastien A.; Kumar, Asok; Levy, Efrat

2012-01-01

In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-β precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-β (Aβ). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphology were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were observed in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the number of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs associated with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and Aβ, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted Aβ, is harmful to the brain. PMID:23129776

Exogenous feeding of immediate precursors reveals synergistic effect on picroside-I biosynthesis in shoot cultures of Picrorhiza kurroa Royle ex Benth

NASA Astrophysics Data System (ADS)

Kumar, Varun; Sharma, Neha; Sood, Hemant; Chauhan, Rajinder Singh

2016-07-01

In the current study, we asked how the supply of immediate biosynthetic precursors i.e. cinnamic acid (CA) and catalpol (CAT) influences the synthesis of picroside-I (P-I) in shoot cultures of P. kurroa. Our results revealed that only CA and CA+CAT stimulated P-I production with 1.6-fold and 4.2-fold, respectively at 2.5 mg/100 mL concentration treatment. Interestingly, feeding CA+CAT not only directed flux towards p-Coumaric acid (p-CA) production but also appeared to trigger the metabolic flux through both shikimate/phenylpropanoid and iridoid pathways by utilizing more of CA and CAT for P-I biosynthesis. However, a deficiency in the supply of either the iridoid or the phenylpropanoid precursor limits flux through the respective pathways as reflected by feedback inhibition effect on PAL and decreased transcripts expressions of rate limiting enzymes (DAHPS, CM, PAL, GS and G10H). It also appears that addition of CA alone directed flux towards both p-CA and P-I production. Based on precursor feeding and metabolic fluxes, a current hypothesis is that precursors from both the iridoid and shikimate/phenylpropanoid pathways are a flux limitation for P-I production in shoot cultures of P. kurroa plants. This work thus sets a stage for future endeavour to elevate production of P-I in cultured plant cells.

Microbial engineering for the production of fatty acids and fatty acid derivatives

DOEpatents

Stephanopoulos, Gregory; Abidi, Syed Hussain Imam

2014-07-01

Some aspects of this invention relate to methods useful for the conversion of a carbon source to a biofuel or biofuel precursor using engineered microbes. Some aspects of this invention relate to the discovery of a key regulator of lipid metabolism in microbes. Some aspects of this invention relate to engineered microbes for biofuel or biofuel precursor production.

Investigating Diesel Engines as an Atmospheric Source of Isocyanic Acid in Urban Areas

NASA Astrophysics Data System (ADS)

Farmer, D.; Jathar, S.; Heppding, C.; Link, M.; Akherati, A.; Kleeman, M.; De Gouw, J. A.; Veres, P. R.; Roberts, J. M.

2017-12-01

Isocyanic acid (HNCO), an acidic gas found in tobacco smoke, urban environments and biomass burning-affected regions, has been linked to adverse health outcomes. Gasoline- and diesel-powered engines and biomass burning are known to emit HNCO and hypothesized to emit precursors such as amides that can photochemically react to produce HNCO in the atmosphere. Increasingly, diesel engines in developed countries like the United States are required to use Selective Catalytic Reduction (SCR) systems to reduce tailpipe emissions of oxides of nitrogen. SCR chemistry is known to produce HNCO as an intermediate product, and SCR systems have been implicated as an atmospheric source of HNCO. In this work, we measure HNCO emissions from an SCR system-equipped diesel engine and, in combination with earlier data, use a three-dimensional chemical transport model (CTM) to simulate the ambient concentrations and source/pathway contributions to HNCO in an urban environment. Engine tests were conducted at three different engine loads, using two different fuels and at multiple operating points. HNCO was measured using an acetate chemical ionization mass spectrometer. The diesel engine was found to emit primary HNCO (3-90 mg kg-fuel-1) but we did not find any evidence that the SCR system or other aftertreatment devices (i.e., oxidation catalyst and particle filter) produced or enhanced HNCO emissions. The CTM predictions compared well with the only available observational data sets for HNCO in urban areas but under-predicted the contribution from secondary processes. The comparison implied that diesel-powered engines were the largest source of HNCO in urban areas. The CTM also predicted that daily-averaged concentrations of HNCO reached a maximum of 110 pptv but were an order of magnitude lower than the 1 ppbv level that could be associated with physiological effects in humans. Precursor contributions from other combustion sources (gasoline and biomass burning) and wintertime conditions

Investigating diesel engines as an atmospheric source of isocyanic acid in urban areas

NASA Astrophysics Data System (ADS)

Jathar, Shantanu H.; Heppding, Christopher; Link, Michael F.; Farmer, Delphine K.; Akherati, Ali; Kleeman, Michael J.; de Gouw, Joost A.; Veres, Patrick R.; Roberts, James M.

2017-07-01

Isocyanic acid (HNCO), an acidic gas found in tobacco smoke, urban environments, and biomass-burning-affected regions, has been linked to adverse health outcomes. Gasoline- and diesel-powered engines and biomass burning are known to emit HNCO and hypothesized to emit precursors such as amides that can photochemically react to produce HNCO in the atmosphere. Increasingly, diesel engines in developed countries like the United States are required to use selective catalytic reduction (SCR) systems to reduce tailpipe emissions of oxides of nitrogen. SCR chemistry is known to produce HNCO as an intermediate product, and SCR systems have been implicated as an atmospheric source of HNCO. In this work, we measure HNCO emissions from an SCR system-equipped diesel engine and, in combination with earlier data, use a three-dimensional chemical transport model (CTM) to simulate the ambient concentrations and source/pathway contributions to HNCO in an urban environment. Engine tests were conducted at three different engine loads, using two different fuels and at multiple operating points. HNCO was measured using an acetate chemical ionization mass spectrometer. The diesel engine was found to emit primary HNCO (3-90 mg kg fuel-1) but we did not find any evidence that the SCR system or other aftertreatment devices (i.e., oxidation catalyst and particle filter) produced or enhanced HNCO emissions. The CTM predictions compared well with the only available observational datasets for HNCO in urban areas but underpredicted the contribution from secondary processes. The comparison implied that diesel-powered engines were the largest source of HNCO in urban areas. The CTM also predicted that daily-averaged concentrations of HNCO reached a maximum of ˜ 110 pptv but were an order of magnitude lower than the 1 ppbv level that could be associated with physiological effects in humans. Precursor contributions from other combustion sources (gasoline and biomass burning) and wintertime

Space Shuttle main engine product improvement

NASA Technical Reports Server (NTRS)

Lucci, A. D.; Klatt, F. P.

1985-01-01

The current design of the Space Shuttle Main Engine has passed 11 certification cycles, amassed approximately a quarter million seconds of engine test time in 1200 tests and successfully launched the Space Shuttle 17 times of 51 engine launches through May 1985. Building on this extensive background, two development programs are underway at Rocketdyne to improve the flow of hot gas through the powerhead and evaluate the changes to increase the performance margins in the engine. These two programs, called Phase II+ and Technology Test Bed Precursor program are described. Phase II+ develops a two-tube hot-gas manifold that improves the component environment. The Precursor program will evaluate a larger throat main combustion chamber, conduct combustion stability testing of a baffleless main injector, fabricate an experimental weld-free heat exchanger tube, fabricate and test a high pressure oxidizer turbopump with an improved inlet, and develop and test methods for reducing temperature transients at start and shutdown.

Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

PubMed Central

Fresquet-Corrales, Sandra; Roque, Edelín; Sarrión-Perdigones, Alejandro; Rochina, Maricruz; López-Gresa, María P.; Díaz-Mula, Huertas M.; Bellés, José M.; Tomás-Barberán, Francisco; Beltrán, José P.

2017-01-01

Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce “bloat-safe” plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed. PMID:28902886

A mobile precursor determines protein resistance on nanostructured surfaces.

PubMed

Wang, Kang; Chen, Ye; Gong, Xiangjun; Xia, Jianlong; Zhao, Junpeng; Shen, Lei

2018-05-09

Biomaterials are often engineered with nanostructured surfaces to control interactions with proteins and thus regulate their biofunctions. However, the mechanism of how nanostructured surfaces resist or attract proteins together with the underlying design rules remains poorly understood at a molecular level, greatly limiting attempts to develop high-performance biomaterials and devices through the rational design of nanostructures. Here, we study the dynamics of nonspecific protein adsorption on block copolymer nanostructures of varying adhesive domain areas in a resistant matrix. Using surface plasmon resonance and single molecule tracking techniques, we show that weakly adsorbed proteins with two-dimensional diffusivity are critical precursors to protein resistance on nanostructured surfaces. The adhesive domain areas must be more than tens or hundreds of times those of the protein footprints to slow down the 2D-mobility of the precursor proteins for their irreversible adsorption. This precursor model can be used to quantitatively analyze the kinetics of nonspecific protein adsorption on nanostructured surfaces. Our method is applicable to precisely manipulate protein adsorption and resistance on various nanostructured surfaces, e.g., amphiphilic, low-surface-energy, and charged nanostructures, for the design of protein-compatible materials.

Production of the forskolin precursor 11β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities.

PubMed

Ignea, Codruta; Ioannou, Efstathia; Georgantea, Panagiota; Trikka, Fotini A; Athanasakoglou, Anastasia; Loupassaki, Sofia; Roussis, Vassilios; Makris, Antonios M; Kampranis, Sotirios C

2016-02-26

Several plant diterpenes have important biological properties. Among them, forskolin is a complex labdane-type diterpene whose biological activity stems from its ability to activate adenylyl cyclase and to elevate intracellular cAMP levels. As such, it is used in the control of blood pressure, in the protection from congestive heart failure, and in weight-loss supplements. Chemical synthesis of forskolin is challenging, and production of forskolin in engineered microbes could provide a sustainable source. To this end, we set out to establish a platform for the production of forskolin and related epoxy-labdanes in yeast. Since the forskolin biosynthetic pathway has only been partially elucidated, and enzymes involved in terpene biosynthesis frequently exhibit relaxed substrate specificity, we explored the possibility of reconstructing missing steps of this pathway employing surrogate enzymes. Using CYP76AH24, a Salvia pomifera cytochrome P450 responsible for the oxidation of C-12 and C-11 of the abietane skeleton en route to carnosic acid, we were able to produce the forskolin precursor 11β-hydroxy-manoyl oxide in yeast. To improve 11β-hydroxy-manoyl oxide production, we undertook a chassis engineering effort involving the combination of three heterozygous yeast gene deletions (mct1/MCT1, whi2/WHI2, gdh1/GDH1) and obtained a 9.5-fold increase in 11β-hydroxy-manoyl oxide titers, reaching 21.2 mg L(-1). In this study, we identify a surrogate enzyme for the specific and efficient hydroxylation of manoyl oxide at position C-11β and establish a platform that will facilitate the synthesis of a broad range of tricyclic (8,13)-epoxy-labdanes in yeast. This platform forms a basis for the heterologous production of forskolin and will facilitate the elucidation of subsequent steps of forskolin biosynthesis. In addition, this study highlights the usefulness of using surrogate enzymes for the production of intermediates of complex biosynthetic pathways. The combination of

A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents

PubMed Central

Johnson, L. Jeffrey

2012-01-01

Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition. PMID:22777049

An Enzymatic Platform for the Synthesis of Isoprenoid Precursors

PubMed Central

Rodriguez, Sofia B.; Leyh, Thomas S.

2014-01-01

The isoprenoid family of compounds is estimated to contain ∼65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway (which produces mevalonate) to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ∼85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation. PMID:25153179

Application of isotope labeling experiments and (13)C flux analysis to enable rational pathway engineering.

PubMed

McAtee, Allison G; Jazmin, Lara J; Young, Jamey D

2015-12-01

Isotope labeling experiments (ILEs) and (13)C flux analysis provide actionable information for metabolic engineers to identify knockout, overexpression, and/or media optimization targets. ILEs have been used in both academic and industrial labs to increase product formation, discover novel metabolic functions in previously uncharacterized organisms, and enhance the metabolic efficiency of host cell factories. This review highlights specific examples of how ILEs have been used in conjunction with enzyme or metabolic engineering to elucidate host cell metabolism and improve product titer, rate, or yield in a directed manner. We discuss recent progress and future opportunities involving the use of ILEs and (13)C flux analysis to characterize non-model host organisms and to identify and subsequently eliminate wasteful byproduct pathways or metabolic bottlenecks. Copyright © 2015 Elsevier Ltd. All rights reserved.

Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light.

PubMed

Kusakabe, Tamami; Tatsuke, Tsuneyuki; Tsuruno, Keigo; Hirokawa, Yasutaka; Atsumi, Shota; Liao, James C; Hanai, Taizo

2013-11-01

Production of alternate fuels or chemicals directly from solar energy and carbon dioxide using engineered cyanobacteria is an attractive method to reduce petroleum dependency and minimize carbon emissions. Here, we constructed a synthetic pathway composed of acetyl-CoA acetyl transferase (encoded by thl), acetoacetyl-CoA transferase (encoded by atoAD), acetoacetate decarboxylase (encoded by adc) and secondary alcohol dehydrogenase (encoded by adh) in Synechococcus elongatus strain PCC 7942 to produce isopropanol. The enzyme-coding genes, heterogeneously originating from Clostridium acetobutylicum ATCC 824 (thl and adc), Escherichia coli K-12 MG1655 (atoAD) and Clostridium beijerinckii (adh), were integrated into the S. elongatus genome. Under the optimized production conditions, the engineered cyanobacteria produced 26.5 mg/L of isopropanol after 9 days. © 2013 Published by Elsevier Inc.

Engineering yeast with bifunctional minicellulosome and cellodextrin pathway for co-utilization of cellulose-mixed sugars.

PubMed

Fan, Li-Hai; Zhang, Zi-Jian; Mei, Sen; Lu, Yang-Yang; Li, Mei; Wang, Zai-Yu; Yang, Jian-Guo; Yang, Shang-Tian; Tan, Tian-Wei

2016-01-01

Consolidated bioprocessing (CBP), integrating cellulase production, cellulose saccharification, and fermentation into one step has been widely considered as the ultimate low-cost configuration for producing second-generation fuel ethanol. However, the requirement of a microbial strain able to hydrolyze cellulosic biomass and convert the resulting sugars into high-titer ethanol limits CBP application. In this work, cellulolytic yeasts were developed by engineering Saccharomyces cerevisiae with a heterologous cellodextrin utilization pathway and bifunctional minicellulosomes. The cell-displayed minicellulosome was two-scaffoldin derived, and contained an endoglucanase and an exoglucanase, while the intracellular cellodextrin pathway consisted of a cellodextrin transporter and a β-glucosidase, which mimicked the unique cellulose-utilization system in Clostridium thermocellum and allowed S. cerevisiae to degrade and use cellulose without glucose inhibition/repression on cellulases and mixed-sugar uptake. Consequently, only a small inoculation of the non-induced yeast cells was required to efficiently co-convert both cellulose and galactose to ethanol in a single-step co-fermentation process, achieving a high specific productivity of ~62.61 mg cellulosic ethanol/g cell·h from carboxymethyl cellulose and ~56.37 mg cellulosic ethanol/g cell·h from phosphoric acid-swollen cellulose. Our work provides a versatile engineering strategy for co-conversion of cellulose-mixed sugars to ethanol by S. cerevisiae, and the achievements in this work may further promote cellulosic biofuel production.

EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors

PubMed Central

Perrotta, Silverio; Cucciolla, Valeria; Ferraro, Marcella; Ronzoni, Luisa; Tramontano, Annunziata; Rossi, Francesca; Scudieri, Anna Chiara; Borriello, Adriana; Roberti, Domenico; Nobili, Bruno; Cappellini, Maria Domenica; Oliva, Adriana; Amendola, Giovanni; Migliaccio, Anna Rita; Mancuso, Patrizia; Martin-Padura, Ines; Bertolini, Francesco; Yoon, Donghoon; Prchal, Josef T.; Della Ragione, Fulvio

2010-01-01

Background Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. Methodology/Principal Findings We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. Conclusions/Significance Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis. PMID:20700488

Product Distribution from Precursor Bite Angle Variation in Multitopic Alkyne Metathesis: Evidence for a Putative Kinetic Bottleneck.

PubMed

Moneypenny, Timothy P; Yang, Anna; Walter, Nathan P; Woods, Toby J; Gray, Danielle L; Zhang, Yang; Moore, Jeffrey S

2018-05-02

In the dynamic synthesis of covalent organic frameworks and molecular cages, the typical synthetic approach involves heuristic methods of discovery. While this approach has yielded many remarkable products, the ability to predict the structural outcome of subjecting a multitopic precursor to dynamic covalent chemistry (DCC) remains a challenge in the field. The synthesis of covalent organic cages is a prime example of this phenomenon, where precursors designed with the intention of affording a specific product may deviate dramatically when the DCC synthesis is attempted. As such, rational design principles are needed to accelerate discovery in cage synthesis using DCC. Herein, we test the hypothesis that precursor bite angle contributes significantly to the energy landscape and product distribution in multitopic alkyne metathesis (AM). By subjecting a series of precursors with varying bite angles to AM, we experimentally demonstrate that the product distribution, and convergence toward product formation, is strongly dependent on this geometric attribute. Surprisingly, we discovered that precursors with the ideal bite angle (60°) do not afford the most efficient pathway to the product. The systematic study reported here illustrates how seemingly minor adjustments in precursor geometry greatly affect the outcome of DCC systems. This research illustrates the importance of fine-tuning precursor geometric parameters in order to successfully realize desirable targets.

Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition.

PubMed

Chwa, Jun-Won; Kim, Wook Jin; Sim, Sang Jun; Um, Youngsoon; Woo, Han Min

2016-08-01

Capture and conversion of CO2 to valuable chemicals is intended to answer global challenges on environmental issues, climate change and energy security. Engineered cyanobacteria have been enabled to produce industry-relevant chemicals from CO2 . However, the final products from cyanobacteria have often been mixed with fermented metabolites during dark fermentation. In this study, our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO2 to volatile acetone as sole product. This process occurred during lighted, aerobic culture via both ATP-driven malonyl-CoA synthesis pathway and heterologous phosphoketolase (PHK)-phosphotransacetylase (Pta) pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl-CoA directly from CO2 in the engineered strain, led to sole production of acetone (22.48 mg/L ± 1.00) without changing nutritional constraints, and without an anaerobic shift. Our engineered S. elongatus strains, designed for acetone production, could be modified to create biosolar cell factories for sustainable photosynthetic production of acetyl-CoA-derived biochemicals. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Engineering The Unicellular Alga Phaeodactylum tricornutum For High-Value Plant Triterpenoid Production.

PubMed

D'Adamo, Sarah; Schiano di Visconte, Gino; Lowe, Gavin; Szaub-Newton, Joanna; Beacham, Tracey; Landels, Andrew; Allen, Michael J; Spicer, Andrew; Matthijs, Michiel

2018-05-13

Plant triterpenoids constitute a diverse class of organic compounds that play a major role in development, plant defense and environmental interaction. Several triterpenes have demonstrated potential as pharmaceuticals. One example is betulin, which has shown promise as a pharmaceutical precursor for the treatment of certain cancers and HIV. Major challenges for triterpenoid commercialization include their low production levels and their cost-effective purification from the complex mixtures present in their natural hosts. Therefore, attempts to produce these compounds in industrially relevant microbial systems such as bacteria and yeasts have attracted great interest. Here we report the production of the triterpenes betulin and its precursor lupeol in the photosynthetic diatom Phaeodactylum tricornutum, a unicellular eukaryotic alga. This was achieved by introducing three plant enzymes in the microalga: a Lotus japonicus oxidosqualene cyclase and a Medicago truncatula cytochrome P450 along with its native reductase. The introduction of the L. japonicus oxidosqualene cyclase perturbed the mRNA expression levels of the native mevalonate and sterol biosynthesis pathway. The best performing strains were selected and grown in a 550L pilot scale photobioreactor facility. To our knowledge, this is the most extensive pathway engineering undertaken in a diatom and the first time that a sapogenin has been artificially produced in a microalga, demonstrating the feasibility of the photo-bio-production of more complex high-value, metabolites in microalgae. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Heterologous expression of the mevalonic acid pathway in cyanobacteria enhances endogenous carbon partitioning to isoprene.

PubMed

Bentley, Fiona K; Zurbriggen, Andreas; Melis, Anastasios

2014-01-01

Heterologous expression of the isoprene synthase gene in the cyanobacterium Synechocystis PCC 6803 conferred upon these microorganisms the property of photosynthetic isoprene (C₅H₈) hydrocarbons production. Continuous production of isoprene from CO₂ and H₂O was achieved in the light, occurring via the endogenous methylerythritol-phosphate (MEP) pathway, in tandem with the growth of Synechocystis. This work addressed the issue of photosynthetic carbon partitioning between isoprene and biomass in Synechocystis. Evidence is presented to show heterologous genomic integration and cellular expression of the mevalonic acid (MVA) pathway genes in Synechocystis endowing a non-native pathway for carbon flux amplification to isopentenyl-diphosphate (IPP) and dimethylallyl-diphosphate (DMAPP) precursors of isoprene. Heterologous expression of the isoprene synthase in combination with the MVA pathway enzymes resulted in photosynthetic isoprene yield improvement by approximately 2.5-fold, compared with that measured in cyanobacteria transformed with the isoprene synthase gene only. These results suggest that the MVA pathway introduces a bypass in the flux of endogenous cellular substrate in Synechocystis to IPP and DMAPP, overcoming flux limitations of the native MEP pathway. The work employed a novel chromosomal integration and expression of synthetic gene operons in Synechocystis, comprising up to four genes under the control of a single promoter, and expressing three operons simultaneously. This is the first time an entire biosynthetic pathway with seven recombinant enzymes has been heterologously expressed in a photosynthetic microorganism. It constitutes contribution to the genetic engineering toolkit of photosynthetic microorganisms and a paradigm in the pursuit of photosynthetic approaches for the renewable generation of high-impact products.

Precursors to potential severe core damage accidents: 1994, a status report. Volume 22: Appendix I

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

Belles, R.J.; Cletcher, J.W.; Copinger, D.A.

Nine operational events that affected eleven commercial light-water reactors (LWRs) during 1994 and that are considered to be precursors to potential severe core damage are described. All these events had conditional probabilities of subsequent severe core damage greater than or equal to 1.0 {times} 10{sup {minus}6}. These events were identified by computer-screening the 1994 licensee event reports from commercial LWRs to identify those that could be potential precursors. Candidate precursors were then selected and evaluated in a process similar to that used in previous assessments. Selected events underwent engineering evaluation that identified, analyzed, and documented the precursors. Other events designatedmore » by the Nuclear Regulatory Commission (NRC) also underwent a similar evaluation. Finally, documented precursors were submitted for review by licensees and NRC headquarters and regional offices to ensure that the plant design and its response to the precursor were correctly characterized. This study is a continuation of earlier work, which evaluated 1969--1981 and 1984--1993 events. The report discusses the general rationale for this study, the selection and documentation of events as precursors, and the estimation of conditional probabilities of subsequent severe core damage for events. This document is bound in two volumes: Vol. 21 contains the main report and Appendices A--H; Vol. 22 contains Appendix 1.« less

Heading in the right direction: thermodynamics-based network analysis and pathway engineering.

PubMed

Ataman, Meric; Hatzimanikatis, Vassily

2015-12-01

Thermodynamics-based network analysis through the introduction of thermodynamic constraints in metabolic models allows a deeper analysis of metabolism and guides pathway engineering. The number and the areas of applications of thermodynamics-based network analysis methods have been increasing in the last ten years. We review recent applications of these methods and we identify the areas that such analysis can contribute significantly, and the needs for future developments. We find that organisms with multiple compartments and extremophiles present challenges for modeling and thermodynamics-based flux analysis. The evolution of current and new methods must also address the issues of the multiple alternatives in flux directionalities and the uncertainties and partial information from analytical methods. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome*

PubMed Central

Trazzi, Stefania; Fuchs, Claudia; Valli, Emanuele; Perini, Giovanni; Bartesaghi, Renata; Ciani, Elisabetta

2013-01-01

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS. PMID:23740250

Production of C2-C4 diols from renewable bioresources: new metabolic pathways and metabolic engineering strategies.

PubMed

Zhang, Ye; Liu, Dehua; Chen, Zhen

2017-01-01

C2-C4 diols classically derived from fossil resource are very important bulk chemicals which have been used in a wide range of areas, including solvents, fuels, polymers, cosmetics, and pharmaceuticals. Production of C2-C4 diols from renewable resources has received significant interest in consideration of the reducing fossil resource and the increasing environmental issues. While bioproduction of certain diols like 1,3-propanediol has been commercialized in recent years, biosynthesis of many other important C2-C4 diol isomers is highly challenging due to the lack of natural synthesis pathways. Recent advances in synthetic biology have enabled the de novo design of completely new pathways to non-natural molecules from renewable feedstocks. In this study, we review recent advances in bioproduction of C2-C4 diols, focusing on new metabolic pathways and metabolic engineering strategies being developed. We also discuss the challenges and future trends toward the development of economically competitive processes for bio-based diol production.

Mitotic position and morphology of committed precursor cells in the zebrafish retina adapt to architectural changes upon tissue maturation.

PubMed

Weber, Isabell P; Ramos, Ana P; Strzyz, Paulina J; Leung, Louis C; Young, Stephen; Norden, Caren

2014-04-24

The development of complex neuronal tissues like the vertebrate retina requires the tight orchestration of cell proliferation and differentiation. Although the complexity of transcription factors and signaling pathways involved in retinogenesis has been studied extensively, the influence of tissue maturation itself has not yet been systematically explored. Here, we present a quantitative analysis of mitotic events during zebrafish retinogenesis that reveals three types of committed neuronal precursors in addition to the previously known apical progenitors. The identified precursor types present at distinct developmental stages and exhibit different mitotic location (apical versus nonapical), cleavage plane orientation, and morphology. Interestingly, the emergence of nonapically dividing committed bipolar cell precursors can be linked to an increase in apical crowding caused by the developing photoreceptor cell layer. Furthermore, genetic interference with neuronal subset specification induces ectopic divisions of committed precursors, underlining the finding that progressing morphogenesis can effect precursor division position. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Pathways into Science for High-School Girls

NASA Astrophysics Data System (ADS)

Simmons, Elizabeth

2004-03-01

This talk discusses the Pathways program ( www.bu.edu/lernet/pathways ) which I founded to provide encouragement and role models for young women interested in pursuing studies and careers in science, mathematics or engineering. I will describe the observations which led me to found Pathways, explain how the program has operated for the past 10 years, and then discuss its effect on the young women who attend... and the science and engineering professionals who volunteer to run the program.

Leveraging microbial biosynthetic pathways for the generation of 'drop-in' biofuels.

PubMed

Zargar, Amin; Bailey, Constance B; Haushalter, Robert W; Eiben, Christopher B; Katz, Leonard; Keasling, Jay D

2017-06-01

Advances in retooling microorganisms have enabled bioproduction of 'drop-in' biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gas-turbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), 'drop-in' biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical properties (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel. Copyright © 2017 Elsevier Ltd. All rights reserved.

Vitamins and aging: pathways to NAD+ synthesis.

PubMed

Denu, John M

2007-05-04

Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction.

RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

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

Dabney-Smith, Carole

Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembledmore » proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cp

How does the serrated polyp pathway alter CRC screening and surveillance?

PubMed

Kahi, Charles J

2015-03-01

Screening and surveillance for colorectal cancer (CRC) reduces mortality through the detection of early-stage adenocarcinoma, and more importantly the detection and removal of premalignant polyps. While adenomas have historically been considered the most common and screening-relevant precursor lesions, there is accumulating evidence showing that the serrated pathway is an important contributor to CRC, and a disproportionate contributor to interval or postcolonoscopy CRC, particularly in the proximal colon. The serrated pathway is characterized by mutations in the BRAF gene, high levels of methylation of promoter CpG islands (CIMP-high), and the sessile serrated adenoma/polyp (SSA/P) is the most important precursor lesion. The study of serrated polyps has been complicated by evolving nomenclature, substantial variation among pathologists in the identification of SSA/Ps, high variability in endoscopic detection rates, and uncertainty regarding the relation to synchronous and metachronous colonic neoplasia. This paper presents an overview of the serrated polyp pathway and discusses its clinical implications including its impact on CRC screening.

Evaluation of Ethanol Production Activity by Engineered Saccharomyces cerevisiae Fermenting Cellobiose through the Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation of Cellulose.

PubMed

Lee, Won-Heong; Jin, Yong-Su

2017-09-28

In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered Saccharomyces cerevisiae capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type S. cerevisiae fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular β-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting S. cerevisiae strains performed similarly under the anoxic SSF conditions, the hydrolytic S. cerevisiae performed slightly better than the phosphorolytic S. cerevisiae under the microaerobic SSF conditions. Nonetheless, the phosphorolytic S. cerevisiae expressing the mutant CDT-1 showed better ethanol production than the glucose-fermenting S. cerevisiae with an extracellular β-glucosidase, regardless of SSF conditions. These results clearly prove that introduction of the intracellular cellobiose metabolic pathway into yeast can be effective on cellulosic ethanol production in SSF. They also demonstrate that enhancement of cellobiose transport activity in engineered yeast is the most important factor affecting the efficiency of SSF of cellulose.

Precursor engineering and controlled conversion for the synthesis of monodisperse thiolate-protected metal nanoclusters

NASA Astrophysics Data System (ADS)

Yu, Yong; Yao, Qiaofeng; Luo, Zhentao; Yuan, Xun; Lee, Jim Yang; Xie, Jianping

2013-05-01

In very recent years, thiolate-protected metal nanoclusters (or thiolated MNCs) with core sizes smaller than 2 nm have emerged as a new direction in nanoparticle research due to their discrete and size dependent electronic structures and molecular-like properties, such as HOMO-LUMO transitions in optical absorptions, quantized charging, and strong luminescence. Synthesis of monodisperse thiolated MNCs in sufficiently large quantities (up to several hundred micrograms) is necessary for establishing reliable size-property relationships and exploring potential applications. This Feature Article reviews recent progress in the development of synthetic strategies for the production of monodisperse thiolated MNCs. The preparation of monodisperse thiolated MNCs is viewed as an engineerable process where both the precursors (input) and their conversion chemistry (processing) may be rationally designed to achieve the desired outcome - monodisperse thiolated MNCs (output). Several strategies for tailoring the precursor and the conversion process are analyzed to arrive at a unifying understanding of the processes involved.

Arabidopsis chlorophyll biosynthesis: an essential balance between the methylerythritol phosphate and tetrapyrrole pathways.

PubMed

Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

2013-12-01

Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)-derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis.

Engine Test and Measurements

NASA Technical Reports Server (NTRS)

Wey, Chown Chou

1999-01-01

Although the importance of aerosols and their precursors are now well recognized, the characterization of current subsonic engines for these emissions is far from complete. Furthermore, since the relationship of engine operating parameters to aerosol emissions is not known, extrapolation to untested and unbuilt engines necessarily remains highly uncertain. 1997 NASA LaRC engine test, as well as the parallel 1997 NASA LaRC flight measurement, attempts to address both issues by expanding measurements of aerosols and aerosol precursors with fuels containing different levels of fuel sulfur content. The specific objective of the 1997 engine test is to obtain a database of sulfur oxides emissions as well as the non-volatile particulate emission properties as a function of fuel sulfur and engine operating conditions. Four diagnostic systems, extractive and non-intrusive (optical), will be assembled for the gaseous and particulate emissions characterization measurements study. NASA is responsible for the extractive gaseous emissions measurement system which contains an array of analyzers dedicated to examining the concentrations of specific gases (NO, NO(x), CO, CO2, O2, THC, SO2) and the smoke number. University of Missouri-Rolla uses the Mobile Aerosol Sampling System to measure aerosol/particulate total concentration, size distribution, volatility and hydration property. Air Force Research Laboratory uses the Chemical Ionization Mass Spectrometer to measure SO2, SO3/H2SO4, and HN03 Aerodyne Research, Inc. uses Infrared Tunable Diode Laser system to measure SO2, SO3, NO, H2O, and CO2.

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

Single-step production of the simvastatin precursor monacolin J by engineering of an industrial strain of Aspergillus terreus.

PubMed

Huang, Xuenian; Liang, Yajing; Yang, Yong; Lu, Xuefeng

2017-07-01

Monacolin J is a key precursor for the synthesis of simvastatin (Zocor), an important drug for treating hypercholesterolemia. Industrially, monacolin J is manufactured through alkaline hydrolysis of lovastatin, a fungal polyketide produced by Aspergillus terreus. Multistep chemical processes for the conversion of lovastatin to simvastatin are laborious, cost expensive and environmentally unfriendly. A biocatalysis process for monacolin J conversion to simvastatin has been developed. However, direct bioproduction of monacolin J has not yet been achieved. Here, we identified a lovastatin hydrolase from Penicillium chrysogenum, which displays a 232-fold higher catalytic efficiency for the in vitro hydrolysis of lovastatin compared to a previously patented hydrolase, but no activity for simvastatin. Furthermore, we showed that an industrial A. terreus strain heterologously expressing this lovastatin hydrolase can produce monacolin J through single-step fermentation with high efficiency, approximately 95% of the biosynthesized lovastatin was hydrolyzed to monacolin J. Our results demonstrate a simple and green technical route for the production of monacolin J, which makes complete bioproduction of the cholesterol-lowering drug simvastatin feasible and promising. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Improved heterologous production of the nonribosomal peptide-polyketide siderophore yersiniabactin through metabolic engineering and induction optimization.

PubMed

Ahmadi, Mahmoud Kamal; Pfeifer, Blaine A

2016-11-01

Biosynthesis of complex natural products like polyketides and nonribosomal peptides using Escherichia coli as a heterologous host provides an opportunity to access these molecules. The value in doing so stems from the fact that many compounds hold some therapeutic or other beneficial property and their original production hosts are intractable for a variety of reasons. In this work, metabolic engineering and induction variable optimization were used to increase production of the polyketide-nonribosomal peptide compound yersiniabactin, a siderophore that has been utilized to selectively remove metals from various solid and aqueous samples. Specifically, several precursor substrate support pathways were altered through gene expression and exogenous supplementation in order to boost production of the final compound. The gene expression induction process was also analyzed to identify the temperatures and inducer concentrations resulting in highest final production levels. When combined, yersiniabactin production was extended to ∼175 mg L -1 . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1412-1417, 2016. © 2016 American Institute of Chemical Engineers.

Serrated Colon Polyps as Precursors to Colorectal Cancer

PubMed Central

Sweetser, Seth; Smyrk, Thomas C.; Sinicrope, Frank A.

2013-01-01

Identification of the serrated neoplasia pathway has improved our understanding of the pathogenesis of colorectal cancer (CRC). Insights have included an increased recognition of the malignant potential of different types of serrated polyps, such as sessile and traditional serrated adenomas. Sessile serrated adenomas share molecular features with colon tumors, such as microsatellite instability and a methylator phenotype, indicating that these lesions are precursors that progress via the serrated neoplasia pathway. There is evidence that the serrated pathway contributes to interval or missed cancers. These data have important implications for clinical practice and CRC prevention, since hyperplastic polyps were previously regarded as having no malignant potential. Endoscopic detection of serrated polyps is a challenge because they are often inconspicuous with indistinct margins, and are frequently covered by adherent mucus. It is important for gastroenterologists to recognize the subtle endoscopic features of serrated polyps, which would facilitate their detection and removal, to ensure a high-quality colonoscopy examination. Recognition of the role of serrated polyps in colon carcinogenesis has led to the inclusion of these lesions in post-polypectomy surveillance guidelines. However, an enhanced effort is needed to identify and completely remove serrated adenomas, with the goal of increasing the effectiveness of colonoscopy to reduce CRC incidence. PMID:23267866

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

PubMed Central

John, Maliyakal E.; Keller, Greg

1996-01-01

Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry. PMID:11038522

Leveraging microbial biosynthetic pathways for the generation of ‘drop-in’ biofuels

DOE PAGES

Zargar, Amin; Bailey, Constance B.; Haushalter, Robert W.; ...

2017-04-17

Advances in retooling microorganisms have enabled bioproduction of ‘drop-in’ biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gasturbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), ‘drop-in’ biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical propertiesmore » (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel.« less

Flavonoids from Engineered Tomatoes Inhibit Gut Barrier Pro-inflammatory Cytokines and Chemokines, via SAPK/JNK and p38 MAPK Pathways

PubMed Central

Tomlinson, Matthew L.; Butelli, Eugenio; Martin, Cathie; Carding, Simon R.

2017-01-01

Flavonoids are a diverse group of plant secondary metabolites, known to reduce inflammatory bowel disease symptoms. How they achieve this is largely unknown. Our study focuses on the gut epithelium as it receives high topological doses of dietary constituents, maintains gut homeostasis, and orchestrates gut immunity. Dysregulation leads to chronic gut inflammation, via dendritic cell (DC)-driven immune responses. Tomatoes engineered for enriched sets of flavonoids (anthocyanins or flavonols) provided a unique and complex naturally consumed food matrix to study the effect of diet on chronic inflammation. Primary murine colonic epithelial cell-based inflammation assays consist of chemokine induction, apoptosis and proliferation, and effects on kinase pathways. Primary murine leukocytes and DCs were used to assay effects on transmigration. A murine intestinal cell line was used to assay wound healing. Engineered tomato extracts (enriched in anthocyanins or flavonols) showed strong and specific inhibitory effects on a set of key epithelial pro-inflammatory cytokines and chemokines. Chemotaxis assays showed a resulting reduction in the migration of primary leukocytes and DCs. Activation of epithelial cell SAPK/JNK and p38 MAPK signaling pathways were specifically inhibited. The epithelial wound healing-associated STAT3 pathway was unaffected. Cellular migration, proliferation, and apoptosis assays confirmed that wound healing processes were not affected by flavonoids. We show flavonoids target epithelial pro-inflammatory kinase pathways, inhibiting chemotactic signals resulting in reduced leukocyte and DC chemotaxis. Thus, both anthocyanins and flavonols modulate epithelial cells to become hyporesponsive to bacterial stimulation. Our results identify a viable mechanism to explain the in vivo anti-inflammatory effects of flavonoids. PMID:29326940

Flavonoids from Engineered Tomatoes Inhibit Gut Barrier Pro-inflammatory Cytokines and Chemokines, via SAPK/JNK and p38 MAPK Pathways.

PubMed

Tomlinson, Matthew L; Butelli, Eugenio; Martin, Cathie; Carding, Simon R

2017-01-01

Flavonoids are a diverse group of plant secondary metabolites, known to reduce inflammatory bowel disease symptoms. How they achieve this is largely unknown. Our study focuses on the gut epithelium as it receives high topological doses of dietary constituents, maintains gut homeostasis, and orchestrates gut immunity. Dysregulation leads to chronic gut inflammation, via dendritic cell (DC)-driven immune responses. Tomatoes engineered for enriched sets of flavonoids (anthocyanins or flavonols) provided a unique and complex naturally consumed food matrix to study the effect of diet on chronic inflammation. Primary murine colonic epithelial cell-based inflammation assays consist of chemokine induction, apoptosis and proliferation, and effects on kinase pathways. Primary murine leukocytes and DCs were used to assay effects on transmigration. A murine intestinal cell line was used to assay wound healing. Engineered tomato extracts (enriched in anthocyanins or flavonols) showed strong and specific inhibitory effects on a set of key epithelial pro-inflammatory cytokines and chemokines. Chemotaxis assays showed a resulting reduction in the migration of primary leukocytes and DCs. Activation of epithelial cell SAPK/JNK and p38 MAPK signaling pathways were specifically inhibited. The epithelial wound healing-associated STAT3 pathway was unaffected. Cellular migration, proliferation, and apoptosis assays confirmed that wound healing processes were not affected by flavonoids. We show flavonoids target epithelial pro-inflammatory kinase pathways, inhibiting chemotactic signals resulting in reduced leukocyte and DC chemotaxis. Thus, both anthocyanins and flavonols modulate epithelial cells to become hyporesponsive to bacterial stimulation. Our results identify a viable mechanism to explain the in vivo anti-inflammatory effects of flavonoids.

Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

PubMed Central

Jarchow-Choy, Sarah K; Koppisch, Andrew T; Fox, David T

2014-01-01

Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined. PMID:25009443

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

PubMed Central

2012-01-01

Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co

High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

DOE PAGES

Kang, Aram; Meadows, Corey W.; Canu, Nicolas; ...

2017-04-05

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonatemore » monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either k cat or K i of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.« less

High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

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

Kang, Aram; Meadows, Corey W.; Canu, Nicolas

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonatemore » monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either k cat or K i of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.« less

Microglia modulate hippocampal neural precursor activity in response to exercise and aging.

PubMed

Vukovic, Jana; Colditz, Michael J; Blackmore, Daniel G; Ruitenberg, Marc J; Bartlett, Perry F

2012-05-09

Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor, CX(3)CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process.

From Solute, Fluidic and Particulate Precursors to Complex Organizations of Matter.

PubMed

Rao, Ashit; Cölfen, Helmut

2018-03-24

The organization of matter from its constitutive units recruits intermediate states with distinctive degrees of self-association and molecular order. Existing as clusters, droplets, gels as well as amorphous and crystalline nanoparticles, these precursor forms have fundamental contributions towards the composition and structure of inorganic and organic architectures. In this personal account, we show that the transitions from atoms, molecules or ionic species to superstructures of higher order are intertwined with the interfaces and interactions of precursor and intermediate states. Structural organizations distributed across different length scales are explained by the multistep nature of nucleation and crystallization, which can be guided towards functional hybrid materials by the strategic application of additives, templates and reaction environments. Thus, the non-classical pathways for material formation and growth offer conceptual frameworks for elucidating, inducing and directing fascinating material organizations of biogenic and synthetic origins. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic biology and metabolic engineering.

PubMed

Stephanopoulos, Gregory

2012-11-16

Metabolic engineering emerged 20 years ago as the discipline occupied with the directed modification of metabolic pathways for the microbial synthesis of various products. As such, it deals with the engineering (design, construction, and optimization) of native as well as non-natural routes of product synthesis, aided in this task by the availability of synthetic DNA, the core enabling technology of synthetic biology. The two fields, however, only partially overlap in their interest in pathway engineering. While fabrication of biobricks, synthetic cells, genetic circuits, and nonlinear cell dynamics, along with pathway engineering, have occupied researchers in the field of synthetic biology, the sum total of these areas does not constitute a coherent definition of synthetic biology with a distinct intellectual foundation and well-defined areas of application. This paper reviews the origins of the two fields and advances two distinct paradigms for each of them: that of unit operations for metabolic engineering and electronic circuits for synthetic biology. In this context, metabolic engineering is about engineering cell factories for the biological manufacturing of chemical and pharmaceutical products, whereas the main focus of synthetic biology is fundamental biological research facilitated by the use of synthetic DNA and genetic circuits.

Metaplasia in the Stomach—Precursor of Gastric Cancer?

PubMed Central

Kinoshita, Hiroto; Koike, Kazuhiko

2017-01-01

Despite a significant decrease in the incidence of gastric cancer in Western countries over the past century, gastric cancer is still one of the leading causes of cancer-related deaths worldwide. Most human gastric cancers develop after long-term Helicobacter pylori infection via the Correa pathway: the progression is from gastritis, atrophy, intestinal metaplasia, dysplasia, to cancer. However, it remains unclear whether metaplasia is a direct precursor of gastric cancer or merely a marker of high cancer risk. Here, we review human studies on the relationship between metaplasia and cancer in the stomach, data from mouse models of metaplasia regarding the mechanism of metaplasia development, and the cellular responses induced by H. pylori infection. PMID:28953255

Metaplasia in the Stomach-Precursor of Gastric Cancer?

PubMed

Kinoshita, Hiroto; Hayakawa, Yoku; Koike, Kazuhiko

2017-09-27

Despite a significant decrease in the incidence of gastric cancer in Western countries over the past century, gastric cancer is still one of the leading causes of cancer-related deaths worldwide. Most human gastric cancers develop after long-term Helicobacter pylori infection via the Correa pathway: the progression is from gastritis, atrophy, intestinal metaplasia, dysplasia, to cancer. However, it remains unclear whether metaplasia is a direct precursor of gastric cancer or merely a marker of high cancer risk. Here, we review human studies on the relationship between metaplasia and cancer in the stomach, data from mouse models of metaplasia regarding the mechanism of metaplasia development, and the cellular responses induced by H. pylori infection.

The development of Sustainability Graduate Community (SGC) as a learning pathway for sustainability education - a framework for engineering programmes in Malaysia Technical Universities Network (MTUN)

NASA Astrophysics Data System (ADS)

Johan, Kartina; Mohd Turan, Faiz

2016-11-01

‘Environmental and sustainability’ is one of the Program Outcome (PO) designated by the Board of Engineers Malaysia (BEM) as one of the accreditation program requirement. However, to-date the implementation of sustainability elements in engineering programme in the technical universities in Malaysia is within individual faculty's curriculum plan and lack of university-level structured learning pathway, which enable all students to have access to an education in sustainability across all disciplines. Sustainability Graduate Community (SGC) is a framework designed to provide a learning pathway in the curriculum of engineering programs to inculcate sustainability education among engineering graduates. This paper aims to study the required attributes in Sustainability Graduate Community (SGC) framework to produce graduates who are not just engineers but also skilful in sustainability competencies using Global Project Management (GPM) P5 Standard for Sustainability. The development of the conceptual framework is to provide a constructive teaching and learning plan for educators and policy makers to work on together in developing the Sustainability Graduates (SG), the new kind of graduates from Malaysia Technical Universities Network (MTUN) in Malaysia who are literate in sustainability practices. The framework also support the call for developing holistic students based on Malaysian Education Blueprint (Higher Education) and address the gap between the statuses of engineering qualification to the expected competencies from industries in Malaysia in particular by achieving the SG attributes outlined in the framework

Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells

PubMed Central

Tai, Ta-Wei; Chen, Ching-Yu; Su, Fong-Chin; Tu, Yuan-Kun; Tsai, Tsung-Ting; Lin, Chiou-Feng; Jou, I.-Ming

2017-01-01

Inhibiting osteoclasts and osteoclast precursors to reduce bone resorption is an important strategy to treat osteoclast-related diseases, such as osteoporosis, inflammatory bone loss, and malignant bone metastasis. However, the mechanism by which apoptosis is induced in the osteoclasts and their precursors are not completely understood. Here, we used nitrogen-containing bisphosphonate zoledronic acid (ZA) to induce cell apoptosis in human and murine osteoclast precursors and mature osteoclast-like cells. Caspase-3-mediated cell apoptosis occurred following the ZA (100 μM) treatment. Reactive oxygen species (ROS) were also generated in a time-dependent manner. Following knock-down of the p47phox expression, which is required for ROS activation, or co-treatment with the ROS inhibitor, N-acetyl-L-cysteine, ZA-induced apoptosis was significantly suppressed in both osteoclast precursors and mature osteoclast-like cells. The ROS-activated mitogen-activated protein kinases pathways did not trigger cell apoptosis. However, a ROS-regulated Mcl-1 decrease simultaneously with glycogen synthase kinase (GSK)-3β promoted cell apoptosis. These findings show that ZA induces apoptosis in osteoclast precursors and mature osteoclast-like cells by triggering ROS- and GSK-3β-mediated Mcl-1 down-regulation. PMID:28281643

Metabolic Engineering of Microalgal Based Biofuel Production: Prospects and Challenges.

PubMed

Banerjee, Chiranjib; Dubey, Kashyap K; Shukla, Pratyoosh

2016-01-01

The current scenario in renewable energy is focused on development of alternate and sustainable energy sources, amongst which microalgae stands as one of the promising feedstock for biofuel production. It is well known that microalgae generate much larger amounts of biofuels in a shorter time than other sources based on plant seeds. However, the greatest challenge in a transition to algae-based biofuel production is the various other complications involved in microalgal cultivation, its harvesting, concentration, drying and lipid extraction. Several green microalgae accumulate lipids, especially triacylglycerols (TAGs), which are main precursors in the production of lipid. The various aspects on metabolic pathway analysis of an oleaginous microalgae i.e., Chlamydomonas reinhardtii have elucidated some novel metabolically important genes and this enhances the lipid production in this microalgae. Adding to it, various other aspects in metabolic engineering using OptFlux and effectual bioprocess design also gives an interactive snapshot of enhancing lipid production which ultimately improvises the oil yield. This article reviews the current status of microalgal based technologies for biofuel production, bioreactor process design, flux analysis and it also provides various strategies to increase lipids accumulation via metabolic engineering.

Genome engineering for improved recombinant protein expression in Escherichia coli.

PubMed

Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

2014-12-19

A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

Arabidopsis Chlorophyll Biosynthesis: An Essential Balance between the Methylerythritol Phosphate and Tetrapyrrole Pathways[C][W

PubMed Central

Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

2013-01-01

Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)–derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis. PMID:24363312

Pathways to an Engineering Career

ERIC Educational Resources Information Center

Pearson, Willie, Jr.; Miller, Jon D.

2012-01-01

Utilizing data from the 20-year record of the Longitudinal Study of American Youth (LSAY), this analysis uses a set of variables to predict employment in engineering for a national sample of adults aged 34 to 37. The LSAY is one of the longest longitudinal studies of the impact of secondary education and postsecondary education conducted in the…

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.

Biosynthetic approaches to creating bioactive fungal metabolites: Pathway engineering and activation of secondary metabolism.

PubMed

Motoyama, Takayuki; Osada, Hiroyuki

2016-12-15

The diversity of natural products is greater than that of combinatorial chemistry compounds and is similar to that of drugs. Compounds rich in sp 3 carbons, such as natural products, typically exhibit high structural complexity and high specificity to molecular targets. Microorganisms can synthesize such sp 3 carbon-rich compounds and can be used as excellent factories for making bioactive compounds. Here, we mainly focus on pathway engineering of two sp 3 carbon-rich bioactive indole alkaloids, fumitremorgin C and terpendole E. We also demonstrate the importance of activation of secondary metabolism by focusing on tenuazonic acid, a bioactive tetramic acid compound, as an example. Copyright © 2016 Elsevier Ltd. All rights reserved.

Metabolic assessment of E. coli as a Biofactory for commercial products.

PubMed

Zhang, Xiaolin; Tervo, Christopher J; Reed, Jennifer L

2016-05-01

Metabolic engineering uses microorganisms to synthesize chemicals from renewable resources. Given the thousands of known metabolites, it is unclear what valuable chemicals could be produced by a microorganism and what native and heterologous reactions are needed for their synthesis. To answer these questions, a systematic computational assessment of Escherichia coli's potential ability to produce different chemicals was performed using an integrated metabolic model that included native E.coli reactions and known heterologous reactions. By adding heterologous reactions, a total of 1777 non-native products could theoretically be produced in E. coli under glucose minimal medium conditions, of which 279 non-native products have commercial applications. Synthesis pathways involving native and heterologous reactions were identified from eight central metabolic precursors to the 279 non-native commercial products. These pathways were used to evaluate the dependence on, and diversity of, native and heterologous reactions to produce each non-native commercial product, as well as to identify each product׳s closest central metabolic precursor. Analysis of the synthesis pathways (with 5 or fewer reaction steps) to non-native commercial products revealed that isopentenyl diphosphate, pyruvate, and oxaloacetate are the closest central metabolic precursors to the most non-native commercial products. Additionally, 4-hydroxybenzoate, tyrosine, and phenylalanine were found to be common precursors to a large number of non-native commercial products. Strains capable of producing high levels of these precursors could be further engineered to create strains capable of producing a variety of commercial non-native chemicals. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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

Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast

PubMed Central

Misra, Ashish; Conway, Matthew F.; Johnnie, Joseph; Qureshi, Tabish M.; Lige, Bao; Derrick, Anne M.; Agbo, Eddy C.; Sriram, Ganesh

2013-01-01

Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo 13C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. PMID:23898325

Synthesis of an A'B' Precursor to Angelmicin B: Product Diversification in the Suárez Lactol Fragmentation.

PubMed

Li, Jialiang; Todaro, Louis; Mootoo, David R

2011-11-01

We describe a synthetic strategy for the angelimicin family of anthraquinoid natural products that involves converting a central highly oxygenated decalin intermediate to the AB and A'B' subunits. Herein, we report the synthesis of the bicyclic A'B' subunit that complements our earlier route to the tricyclic AB framework. The differentiating tact in the two syntheses focused on controlling the Suárez radical fragmentation of lactol precursors by modulating the substrate's structural rigidity. A more flexible lactol gave the tricyclic AB framework, whereas a more rigid substrate led to the bicyclic A'B' precursor, presumably through divergent pathways from the radical produced in the initial fragmentation step. These results establish a versatile advanced synthetic precursor for the angelimicins, and on a more general note, illustrate strategies for applying the Suárez fragmentation to diverse and complex molecular frameworks.

Mechanical mixtures of metal oxides and phosphorus pentoxide as novel precursors for the synthesis of transition-metal phosphides.

PubMed

Guo, Lijuan; Zhao, Yu; Yao, Zhiwei

2016-01-21

This study presents a new type of precursor, mechanical mixtures of metal oxides (MOs) and phosphorus pentoxide (P2O5) are used to synthesize Ni2P, Co2P and MoP phosphides by the H2 reduction method. In addition, this is first report of common solid-state P2O5 being used as a P source for the synthesis of metal phosphides. The traditional precursors are usually prepared via a complicated preparation procedure involving dissolution, drying and calcination steps. However, these novel MOs/P2O5 precursors can be obtained only by simple mechanical mixing of the starting materials. Furthermore, unlike the direct transformation from amorphous phases to phosphides, various specific intermediates were involved in the transformation from MOs/P2O5 to phosphides. It is worthy to note that the dispersions of Ni2P, Co2P and MoP obtained from MOs/P2O5 precursors were superior to those of the corresponding phosphides prepared from the abovementioned traditional precursors. It is suggested that the morphology of the as-prepared metal phosphides might be inherited from the corresponding MOs. Based on the results of XRD, XPS, SEM and TEM, the formation pathway of phosphides can be defined as MOs/P2O5 precursors → complex intermediates (metals, metal phosphates and metal oxide-phosphates) → metal phosphides.

Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

PubMed

Zhu, Wen-Liang; Cui, Jin-Yu; Cui, Lan-Yu; Liang, Wei-Fan; Yang, Song; Zhang, Chong; Xing, Xin-Hui

2016-03-01

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae

PubMed Central

Kozak, Barbara U.; van Rossum, Harmen M.; Luttik, Marijke A. H.; Akeroyd, Michiel; Benjamin, Kirsten R.; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T.

2014-01-01

ABSTRACT The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. PMID:25336454

Engineered Plants Make Potential Precursor to Raw Material for Plastics

ScienceCinema

Shanklin, John

2018-06-12

In a first step toward achieving industrial-scale green production, scientists from BNL and collaborators at Dow AgroSciences report engineering a plant that produces industrially relevant levels of chemicals that could potentially be used to make plastics.

Ethanol production by engineered thermophiles.

PubMed

Olson, Daniel G; Sparling, Richard; Lynd, Lee R

2015-06-01

We compare a number of different strategies that have been pursued to engineer thermophilic microorganisms for increased ethanol production. Ethanol production from pyruvate can proceed via one of four pathways, which are named by the key pyruvate dissimilating enzyme: pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH), pyruvate formate lyase (PFL), and pyruvate ferredoxin oxidoreductase (PFOR). For each of these pathways except PFL, we see examples where ethanol production has been engineered with a yield of >90% of the theoretical maximum. In each of these cases, this engineering was achieved mainly by modulating expression of native genes. We have not found an example where a thermophilic ethanol production pathway has been transferred to a non-ethanol-producing organism to produce ethanol at high yield. A key reason for the lack of transferability of ethanol production pathways is the current lack of understanding of the enzymes involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

One step DNA assembly for combinatorial metabolic engineering.

PubMed

Coussement, Pieter; Maertens, Jo; Beauprez, Joeri; Van Bellegem, Wouter; De Mey, Marjan

2014-05-01

The rapid and efficient assembly of multi-step metabolic pathways for generating microbial strains with desirable phenotypes is a critical procedure for metabolic engineering, and remains a significant challenge in synthetic biology. Although several DNA assembly methods have been developed and applied for metabolic pathway engineering, many of them are limited by their suitability for combinatorial pathway assembly. The introduction of transcriptional (promoters), translational (ribosome binding site (RBS)) and enzyme (mutant genes) variability to modulate pathway expression levels is essential for generating balanced metabolic pathways and maximizing the productivity of a strain. We report a novel, highly reliable and rapid single strand assembly (SSA) method for pathway engineering. The method was successfully optimized and applied to create constructs containing promoter, RBS and/or mutant enzyme libraries. To demonstrate its efficiency and reliability, the method was applied to fine-tune multi-gene pathways. Two promoter libraries were simultaneously introduced in front of two target genes, enabling orthogonal expression as demonstrated by principal component analysis. This shows that SSA will increase our ability to tune multi-gene pathways at all control levels for the biotechnological production of complex metabolites, achievable through the combinatorial modulation of transcription, translation and enzyme activity. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Engineering microbial cell factories for the production of plant natural products: from design principles to industrial-scale production.

PubMed

Liu, Xiaonan; Ding, Wentao; Jiang, Huifeng

2017-07-19

Plant natural products (PNPs) are widely used as pharmaceuticals, nutraceuticals, seasonings, pigments, etc., with a huge commercial value on the global market. However, most of these PNPs are still being extracted from plants. A resource-conserving and environment-friendly synthesis route for PNPs that utilizes microbial cell factories has attracted increasing attention since the 1940s. However, at the present only a handful of PNPs are being produced by microbial cell factories at an industrial scale, and there are still many challenges in their large-scale application. One of the challenges is that most biosynthetic pathways of PNPs are still unknown, which largely limits the number of candidate PNPs for heterologous microbial production. Another challenge is that the metabolic fluxes toward the target products in microbial hosts are often hindered by poor precursor supply, low catalytic activity of enzymes and obstructed product transport. Consequently, despite intensive studies on the metabolic engineering of microbial hosts, the fermentation costs of most heterologously produced PNPs are still too high for industrial-scale production. In this paper, we review several aspects of PNP production in microbial cell factories, including important design principles and recent progress in pathway mining and metabolic engineering. In addition, implemented cases of industrial-scale production of PNPs in microbial cell factories are also highlighted.

Selective growth of chirality-enriched semiconducting carbon nanotubes by using bimetallic catalysts from salt precursors.

PubMed

Zhao, Xiulan; Yang, Feng; Chen, Junhan; Ding, Li; Liu, Xiyan; Yao, Fengrui; Li, Meihui; Zhang, Daqi; Zhang, Zeyao; Liu, Xu; Yang, Juan; Liu, Kaihui; Li, Yan

2018-04-19

Bimetallic catalysts play important roles in the selective growth of single-walled carbon nanotubes (SWNTs). Using the simple salts (NH4)6W7O24·6H2O and Co(CH3COO)2·4H2O as precursors, tungsten-cobalt catalysts were prepared. The catalysts were composed of W6Co7 intermetallic compounds and tungsten-dispersed cobalt. With the increase of the W/Co ratio in the precursors, the content of W6Co7 was increased. Because the W6Co7 intermetallic compound can enable the chirality specified growth of SWNTs, the selectivity of the resulting SWNTs is improved at a higher W/Co ratio. At a W/Co ratio of 6 : 4 and under optimized chemical vapor deposition conditions, we realized the direct growth of semiconducting SWNTs with the purity of ∼96%, in which ∼62% are (14, 4) tubes. Using salts as precursors to prepare tungsten-cobalt bimetallic catalysts is flexible and convenient. This offers an efficient pathway for the large-scale preparation of chirality enriched semiconducting SWNTs.

Becoming an Engineer: Toward a Three Dimensional View of Engineering Learning. Research Brief

ERIC Educational Resources Information Center

Stevens, Reed; O'Connor, Kevin; Garrison, Lari; Jocuns, Andrew; Amos, Daniel M.

2008-01-01

In this paper, the authors develop an analytical framework referred to as "Becoming an Engineer" that focuses upon changes occurring over time as students traverse their undergraduate educations in engineering. This paper discusses three conceptual dimensions used to follow the engineering students' educational pathways: the development of…

Meat flavor precursors and factors influencing flavor precursors--A systematic review.

PubMed

Khan, Muhammad Issa; Jo, Cheorun; Tariq, Muhammad Rizwan

2015-12-01

Flavor is the sensory impression sensed by taste and smell buds and is a leading factor determining the meat quality and purchasing decision of the consumer. Meat flavor is characteristic of volatiles produced as a result of reactions of non-volatile components that are induced thermally. The water soluble compounds having low molecular weight and meat lipids are important precursors of cooked meat flavor. The Maillard reaction, lipid oxidation, and vitamin degradation are leading reactions during cooking which develop meat flavor from uncooked meat with little aroma and bloody taste. The pre-slaughter and postmortem factors like animal breed, sex, age, feed, aging and cooking conditions contribute to flavor development of cooked meat. The objective of this review is to highlight the flavor chemistry, meat flavor precursors and factors affecting meat flavor precursors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Arginine decarboxylase (ADC) and agmatinase (AGMAT): an alternative pathway for synthesis of polyamines in pig conceptuses and uteri

USDA-ARS?s Scientific Manuscript database

Arginine, a precursor for the synthesis of nitric oxide (NO) and polyamines, is critical for implantation and development of the conceptus. We first reported that the arginine decarboxylase (ADC)/agmatinase(AGMAT) pathway as an alternative pathway for synthesis of polyamines in the ovine conceptuses...

A multi-pathway model for photosynthetic reaction center

NASA Astrophysics Data System (ADS)

Qin, M.; Shen, H. Z.; Yi, X. X.

2016-03-01

Charge separation occurs in a pair of tightly coupled chlorophylls at the heart of photosynthetic reaction centers of both plants and bacteria. Recently it has been shown that quantum coherence can, in principle, enhance the efficiency of a solar cell, working like a quantum heat engine. Here, we propose a biological quantum heat engine (BQHE) motivated by Photosystem II reaction center (PSII RC) to describe the charge separation. Our model mainly considers two charge-separation pathways which is more than that typically considered in the published literature. We explore how these cross-couplings increase the current and power of the charge separation and discuss the effects of multiple pathways in terms of current and power. The robustness of the BQHE against the charge recombination in natural PSII RC and dephasing induced by environments is also explored, and extension from two pathways to multiple pathways is made. These results suggest that noise-induced quantum coherence helps to suppress the influence of acceptor-to-donor charge recombination, and besides, nature-mimicking architectures with engineered multiple pathways for charge separations might be better for artificial solar energy devices considering the influence of environments.

Metabolic engineering of the pentose phosphate pathway for enhanced limonene production in the cyanobacterium Synechocysti s sp. PCC 6803.

PubMed

Lin, Po-Cheng; Saha, Rajib; Zhang, Fuzhong; Pakrasi, Himadri B

2017-12-13

Isoprenoids are diverse natural compounds, which have various applications as pharmaceuticals, fragrances, and solvents. The low yield of isoprenoids in plants makes them difficult for cost-effective production, and chemical synthesis of complex isoprenoids is impractical. Microbial production of isoprenoids has been considered as a promising approach to increase the yield. In this study, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 for sustainable production of a commercially valuable isoprenoid, limonene. Limonene synthases from the plants Mentha spicata and Citrus limon were expressed in cyanobacteria for limonene production. Production of limonene was two-fold higher with limonene synthase from M. spicata than that from C. limon. To enhance isoprenoid production, computational strain design was conducted by applying the OptForce strain design algorithm on Synechocystis 6803. Based on the metabolic interventions suggested by this algorithm, genes (ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase) in the pentose phosphate pathway were overexpressed, and a geranyl diphosphate synthase from the plant Abies grandis was expressed to optimize the limonene biosynthetic pathway. The optimized strain produced 6.7 mg/L of limonene, a 2.3-fold improvement in productivity. Thus, this study presents a feasible strategy to engineer cyanobacteria for photosynthetic production of isoprenoids.

Protein engineering approaches to chemical biotechnology.

PubMed

Chen, Zhen; Zeng, An-Ping

2016-12-01

Protein engineering for the improvement of properties of biocatalysts and for the generation of novel metabolic pathways plays more and more important roles in chemical biotechnology aiming at the production of chemicals from biomass. Although widely used in single-enzyme catalysis process, protein engineering is only being increasingly explored in recent years to achieve more complex in vitro and in vivo biocatalytic processes. This review focuses on major contributions of protein engineering to chemical biotechnology in the field of multi-enzymatic cascade catalysis and metabolic engineering. Especially, we discuss and highlight recent strategies for combining pathway design and protein engineering for the production of novel products. Copyright © 2016. Published by Elsevier Ltd.

ACBC to Balcite: Bioinspired Synthesis of a Highly Substituted High-Temperature Phase from an Amorphous Precursor

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

Whittaker, Michael L.; Joester, Derk

2017-04-28

Energy-efficient synthesis of materials locked in compositional and structural states far from equilibrium remains a challenging goal, yet biomineralizing organisms routinely assemble such materials with sophisticated designs and advanced functional properties, often using amorphous precursors. However, incorporation of organics limits the useful temperature range of these materials. Herein, the bioinspired synthesis of a highly supersaturated calcite (Ca0.5Ba0.5CO3) called balcite is reported, at mild conditions and using an amorphous calcium–barium carbonate (ACBC) (Ca1- x Ba x CO3·1.2H2O) precursor. Balcite not only contains 50 times more barium than the solubility limit in calcite but also displays the rotational disorder on carbonate sitesmore » that is typical for high-temperature calcite. It is significantly harder (30%) and less stiff than calcite, and retains these properties after heating to elevated temperatures. Analysis of balcite local order suggests that it may require the formation of the ACBC precursor and could therefore be an example of nonclassical nucleation. These findings demonstrate that amorphous precursor pathways are powerfully enabling and provide unprecedented access to materials far from equilibrium, including high-temperature modifications by room-temperature synthesis.« less

Biofuel metabolic engineering with biosensors.

PubMed

Morgan, Stacy-Anne; Nadler, Dana C; Yokoo, Rayka; Savage, David F

2016-12-01

Metabolic engineering offers the potential to renewably produce important classes of chemicals, particularly biofuels, at an industrial scale. DNA synthesis and editing techniques can generate large pathway libraries, yet identifying the best variants is slow and cumbersome. Traditionally, analytical methods like chromatography and mass spectrometry have been used to evaluate pathway variants, but such techniques cannot be performed with high throughput. Biosensors - genetically encoded components that actuate a cellular output in response to a change in metabolite concentration - are therefore a promising tool for rapid and high-throughput evaluation of candidate pathway variants. Applying biosensors can also dynamically tune pathways in response to metabolic changes, improving balance and productivity. Here, we describe the major classes of biosensors and briefly highlight recent progress in applying them to biofuel-related metabolic pathway engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Polyimide Precursor Solid Residuum

NASA Technical Reports Server (NTRS)

Weiser, Erik S. (Inventor); St.Clair, Terry L. (Inventor); Echigo, Yoshiaki (Inventor); Kaneshiro, Hisayasu (Inventor)

2001-01-01

A polyimide precursor solid residuum is an admixture of an aromatic dianhydride or derivative thereof and an aromatic diamine or derivative thereof plus a complexing agent, which is complexed with the admixture by hydrogen bonding. The polyimide precursor solid residuum is effectively employed in the preparation of polyimide foam and the fabrication of polyimide foam structures.

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

Metabolic Engineering VII Conference

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

Kevin Korpics

The aims of this Metabolic Engineering conference are to provide a forum for academic and industrial researchers in the field; to bring together the different scientific disciplines that contribute to the design, analysis and optimization of metabolic pathways; and to explore the role of Metabolic Engineering in the areas of health and sustainability. Presentations, both written and oral, panel discussions, and workshops will focus on both applications and techniques used for pathway engineering. Various applications including bioenergy, industrial chemicals and materials, drug targets, health, agriculture, and nutrition will be discussed. Workshops focused on technology development for mathematical and experimental techniquesmore » important for metabolic engineering applications will be held for more in depth discussion. This 2008 meeting will celebrate our conference tradition of high quality and relevance to both industrial and academic participants, with topics ranging from the frontiers of fundamental science to the practical aspects of metabolic engineering.« less

Total N-nitrosamine Precursor Adsorption with Carbon Nanotubes: Elucidating Controlling Physiochemical Properties and Developing a Size-Resolved Precursor Surrogate

NASA Astrophysics Data System (ADS)

Needham, Erin Michelle

As drinking water sources become increasingly impaired with nutrients and wastewater treatment plant (WWTP) effluent, formation of disinfection byproducts (DBPs)--such as trihalomethanes (THMs), dihaloacetonitriles (DHANs), and N-nitrosamines--during water treatment may also increase. N-nitrosamines may comprise the bulk of the chronic toxicity in treated drinking waters despite forming at low ng/L levels. This research seeks to elucidate physicochemical properties of carbon nanotubes (CNTs) for removal of DBP precursors, with an emphasis on total N-nitrosamines (TONO). Batch experiments with CNTs were completed to assess adsorption of THM, DHAN, and TONO precursors; physiochemical properties of CNTs were quantified through gas adsorption isotherms and x-ray photoelectron spectroscopy. Numerical modeling was used to elucidate characteristics of CNTs controlling DBP precursor adsorption. Multivariate models developed with unmodified CNTs revealed that surface carboxyl groups and, for TONO precursors, cumulative pore volume (CPV), controlled DBP precursor adsorption. Models developed with modified CNTs revealed that specific surface area controlled adsorption of THM and DHAN precursors while CPV and surface oxygen content were significant for adsorption of TONO precursors. While surrogates of THM and DHAN precursors leverage metrics from UV absorbance and fluorescence spectroscopy, a TONO precursor surrogate has proved elusive. This is important as measurements of TONO formation potential (TONOFP) require large sample volumes and long processing times, which impairs development of treatment processes. TONO precursor surrogates were developed using samples that had undergone oxidative or sorption treatments. Precursors were analyzed with asymmetric flow field-flow fractionation (AF4) with inline fluorescence detection (FLD) and whole water fluorescence excitation-emission matrices (EEMs). TONO precursor surrogates were discovered, capable of predicting changes in

Engineering Robustness of Microbial Cell Factories.

PubMed

Gong, Zhiwei; Nielsen, Jens; Zhou, Yongjin J

2017-10-01

Metabolic engineering and synthetic biology offer great prospects in developing microbial cell factories capable of converting renewable feedstocks into fuels, chemicals, food ingredients, and pharmaceuticals. However, prohibitively low production rate and mass concentration remain the major hurdles in industrial processes even though the biosynthetic pathways are comprehensively optimized. These limitations are caused by a variety of factors unamenable for host cell survival, such as harsh industrial conditions, fermentation inhibitors from biomass hydrolysates, and toxic compounds including metabolic intermediates and valuable target products. Therefore, engineered microbes with robust phenotypes is essential for achieving higher yield and productivity. In this review, the recent advances in engineering robustness and tolerance of cell factories is described to cope with these issues and briefly introduce novel strategies with great potential to enhance the robustness of cell factories, including metabolic pathway balancing, transporter engineering, and adaptive laboratory evolution. This review also highlights the integration of advanced systems and synthetic biology principles toward engineering the harmony of overall cell function, more than the specific pathways or enzymes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An Engineering Degree Does Not (Necessarily) an Engineer Make: Career Decision Making among Undergraduate Engineering Majors. Research Brief

ERIC Educational Resources Information Center

Lichtenstein, Gary; Loshbaugh, Heidi G.; Claar, Brittany; Chen, Helen L.; Jackson, Kristyn; Sheppard, Sheri

2009-01-01

This paper explores the career-related decision making of seniors enrolled in undergraduate engineering programs at two nationally recognized institutions. This strand of the Academic Pathways Study (APS) research revealed that many engineering students were undecided about their career plans, even late into their senior years and that many were…

Kinesin molecular motors: Transport pathways, receptors, and human disease

NASA Astrophysics Data System (ADS)

Goldstein, Lawrence S. B.

2001-06-01

Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.

Brillouin precursors in Debye media

NASA Astrophysics Data System (ADS)

Macke, Bruno; Ségard, Bernard

2015-05-01

We theoretically study the formation of Brillouin precursors in Debye media. We point out that the precursors are visible only at propagation distances such that the impulse response of the medium is essentially determined by the frequency dependence of its absorption and is practically Gaussian. By simple convolution, we then obtain explicit analytical expressions of the transmitted waves generated by reference incident waves, distinguishing precursor and main signal by a simple examination of the long-time behavior of the overall signal. These expressions are in good agreement with the signals obtained in numerical or real experiments performed on water in the radio-frequency domain and explain in particular some observed shapes of the precursor. Results are obtained for other remarkable incident waves. In addition, we show quite generally that the shape of the Brillouin precursor appearing alone at sufficiently large propagation distance and the law giving its amplitude as a function of this distance do not depend on the precise form of the incident wave but only on its integral properties. The incidence of a static conductivity of the medium is also examined and explicit analytical results are again given in the limit of weak and strong conductivities.

Natural Modulators of Amyloid-Beta Precursor Protein Processing

PubMed Central

Zhang, Can; Tanzi, Rudolph E.

2013-01-01

Alzheimer’s disease (AD) is a devastating neurodegenerative disease and the primary cause of dementia, with no cure currently available. The pathogenesis of AD is believed to be primarily driven by Aβ, the principal component of senile plaques. Aβ is an ~4 kDa peptide generated from the amyloid-β precursor protein (APP) through proteolytic secretases. Natural products, particularly those utilized in traditional Chinese medicine (TCM), have a long history alleviating common clinical disorders, including dementia. However, the cell/molecular pathways mediated by these natural products are largely unknown until recently when the underlying molecular mechanisms of the disorders begin to be elucidated. Here, the mechanisms with which natural products modulate the pathogenesis of AD are discussed, in particular, by focusing on their roles in the processing of APP. PMID:22998566

Multi-Step Crystallization of Barium Carbonate: Rapid Interconversion of Amorphous and Crystalline Precursors.

PubMed

Whittaker, Michael L; Smeets, Paul J M; Asayesh-Ardakani, Hasti; Shahbazian-Yassar, Reza; Joester, Derk

2017-12-11

The direct observation of amorphous barium carbonate (ABC), which transforms into a previously unknown barium carbonate hydrate (herewith named gortatowskite) within a few hundred milliseconds of formation, is described. In situ X-ray scattering, cryo-, and low-dose electron microscopy were used to capture the transformation of nanoparticulate ABC into gortatowskite crystals, highly anisotropic sheets that are up to 1 μm in width, yet only about 10 nm in thickness. Recrystallization of gortatowskite to witherite starts within 30 seconds. We describe a bulk synthesis and report a first assessment of the composition, vibrational spectra, and structure of gortatowskite. Our findings indicate that transient amorphous and crystalline precursors can play a role in aqueous precipitation pathways that may often be overlooked owing to their extremely short lifetimes and small dimensions. However, such transient precursors may be integral to the formation of more stable phases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance.

PubMed

Liang, Keming; Shen, Claire R

2017-01-01

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k cat /K m ≈0.4mM -1 s -1 ) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V max ≈6U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme

Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803

PubMed Central

2014-01-01

the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH. PMID:24991233

Engineering Cellular Metabolism.

PubMed

Nielsen, Jens; Keasling, Jay D

2016-03-10

Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation. Copyright © 2016 Elsevier Inc. All rights reserved.

Combined Effects of Early Season Leaf Removal and Climatic Conditions on Aroma Precursors in Sauvignon Blanc Grapes.

PubMed

Sivilotti, Paolo; Falchi, Rachele; Herrera, Jose Carlos; Škvarč, Branka; Butinar, Lorena; Sternad Lemut, Melita; Bubola, Marijan; Sabbatini, Paolo; Lisjak, Klemen; Vanzo, Andreja

2017-09-27

Early leaf removal around the cluster zone is a common technique applied in cool climate viticulture, to regulate yield components and improve fruit quality. Despite the increasing amount of information on early leaf removal and its impact on total soluble solids, anthocyanins, and polyphenols, less is known regarding aroma compounds. In order to verify the hypothesis that defoliation, applied before or after flowering, could impact the biosynthesis of thiol precursors, we performed a two year (2013 and 2014) experiment on Sauvignon blanc. We provided evidence that differential accumulation of thiol precursors in berries is affected by the timing of defoliation, and this impact was related to modifications in the biosynthetic pathway. Furthermore, the possible interaction between leaf removal treatment and seasonal weather conditions, and its effect on the biosynthesis of volatile precursors are discussed. Our results suggested that in Sauvignon blanc the relative proportion of 4-S-glutathionyl-4-methylpentan-2-one (G-4MSP) and 3-S-glutathionylhexan-1-ol (G-3SH) precursors can be affected by defoliation, and this could be related to the induction of two specific genes encoding glutathione-S-transferases (VvGST3 and VvGST5), while no significant effects on basic fruit chemical parameters, polyphenols, and methoxypyrazines were ascertained under our experimental conditions.

Ezh2 phosphorylation state determines its capacity to maintain CD8+ T memory precursors for antitumor immunity.

PubMed

He, Shan; Liu, Yongnian; Meng, Lijun; Sun, Hongxing; Wang, Ying; Ji, Yun; Purushe, Janaki; Chen, Pan; Li, Changhong; Madzo, Jozef; Issa, Jean-Pierre; Soboloff, Jonathan; Reshef, Ran; Moore, Bethany; Gattinoni, Luca; Zhang, Yi

2017-12-14

Memory T cells sustain effector T-cell production while self-renewing in reaction to persistent antigen; yet, excessive expansion reduces memory potential and impairs antitumor immunity. Epigenetic mechanisms are thought to be important for balancing effector and memory differentiation; however, the epigenetic regulator(s) underpinning this process remains unknown. Herein, we show that the histone methyltransferase Ezh2 controls CD8 + T memory precursor formation and antitumor activity. Ezh2 activates Id3 while silencing Id2, Prdm1 and Eomes, promoting the expansion of memory precursor cells and their differentiation into functional memory cells. Akt activation phosphorylates Ezh2 and decreases its control of these transcriptional programs, causing enhanced effector differentiation at the expense of T memory precursors. Engineering T cells with an Akt-insensitive Ezh2 mutant markedly improves their memory potential and capability of controlling tumor growth compared to transiently inhibiting Akt. These findings establish Akt-mediated phosphorylation of Ezh2 as a critical target to potentiate antitumor immunotherapeutic strategies.

Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis.

PubMed

Pirie, Christopher M; De Mey, Marjan; Jones Prather, Kristala L; Ajikumar, Parayil Kumaran

2013-04-19

Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.

Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+.

PubMed

Belenky, Peter; Racette, Frances G; Bogan, Katrina L; McClure, Julie M; Smith, Jeffrey S; Brenner, Charles

2007-05-04

Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function.

Preparation of superconductor precursor powders

DOEpatents

Bhattacharya, R.

1998-08-04

A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic and/or reduced particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of all metals in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products. 7 figs.

Preparation of superconductor precursor powders

DOEpatents

Bhattacharya, Raghunath; Blaugher, Richard D.

1995-01-01

A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals, such as nitrate salts of thallium, barium, calcium, and copper, which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of thallium in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products.

Preparation of superconductor precursor powders

DOEpatents

Bhattacharya, Raghunath

1998-01-01

A process for the preparation of a precursor metallic powder composition for use in the subsequent formation of a superconductor. The process comprises the steps of providing an electrodeposition bath comprising an electrolyte medium and a cathode substrate electrode, and providing to the bath one or more soluble salts of one or more respective metals which are capable of exhibiting superconductor properties upon subsequent appropriate treatment. The bath is continually energized to cause the metallic and/or reduced particles formed at the electrode to drop as a powder from the electrode into the bath, and this powder, which is a precursor powder for superconductor production, is recovered from the bath for subsequent treatment. The process permits direct inclusion of all metals in the preparation of the precursor powder, and yields an amorphous product mixed on an atomic scale to thereby impart inherent high reactivity. Superconductors which can be formed from the precursor powder include pellet and powder-in-tube products.

1994 Accident sequence precursor program results

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

Belles, R.J.; Cletcher, J.W.; Copinger, D.A.

1996-01-01

The Accident Sequence Precursor (ASP) Program involves the systematic review and evaluation of operational events that have occurred at light-water reactors to identify and categorize precursors to potential severe core damage accident sequences. The results of the ASP Program are published in an annual report. The most recent report, which contains the analyses of the precursors for 1994, is NUREG/CR-4674, Vols. 21 and 22, Precursors to Potential Severe Core Damage Accidents: 1994, A Status Report, published in December 1995. This article provides an overview of the ASP review and evaluation process and a summary of the results for 1994. 12more » refs., 2 figs., 4 tabs.« less

The chemokine CXCL16 induces migration and invasion of glial precursor cells via its receptor CXCR6.

PubMed

Hattermann, Kirsten; Ludwig, Andreas; Gieselmann, Volkmar; Held-Feindt, Janka; Mentlein, Rolf

2008-09-01

Chemokines are implicated in developmental and inflammatory processes in the brain. The transmembrane chemokine CXCL16 is produced in brain endothelial and reactive astroglial cells and released by shedding. Its receptor CXCR6 is detected during brain development highest at postnatal day 6, found in glial precursor cells differentiated from neural stem cells and in an A2B5-positive glial precursor cell line. Their stimulation by soluble CXCL16 induces the PI3-kinase/Akt and Erk pathways resulting in the activation of the transcription factor AP-1. As biological responses, soluble CXCL16 upregulates its own receptor, increases cell proliferation, stimulates cell migration in wound-healing and in spheroid confrontation assays. Invasion of CXCR6-positive glial cells into CXCL16-expressing spheroids can be blocked by sheddase inhibitors and CXCL16-antibody. Since CXCL16 is induced by cytokines at sites of inflammation, neurodegeneration, ischemia and malignant transformation, it should attract CXCR6-positive glial precursor cells, enhance their invasion and proliferation and thus favor astrogliosis.

Accident Precursor Analysis and Management: Reducing Technological Risk Through Diligence

NASA Technical Reports Server (NTRS)

Phimister, James R. (Editor); Bier, Vicki M. (Editor); Kunreuther, Howard C. (Editor)

2004-01-01

Almost every year there is at least one technological disaster that highlights the challenge of managing technological risk. On February 1, 2003, the space shuttle Columbia and her crew were lost during reentry into the atmosphere. In the summer of 2003, there was a blackout that left millions of people in the northeast United States without electricity. Forensic analyses, congressional hearings, investigations by scientific boards and panels, and journalistic and academic research have yielded a wealth of information about the events that led up to each disaster, and questions have arisen. Why were the events that led to the accident not recognized as harbingers? Why were risk-reducing steps not taken? This line of questioning is based on the assumption that signals before an accident can and should be recognized. To examine the validity of this assumption, the National Academy of Engineering (NAE) undertook the Accident Precursors Project in February 2003. The project was overseen by a committee of experts from the safety and risk-sciences communities. Rather than examining a single accident or incident, the committee decided to investigate how different organizations anticipate and assess the likelihood of accidents from accident precursors. The project culminated in a workshop held in Washington, D.C., in July 2003. This report includes the papers presented at the workshop, as well as findings and recommendations based on the workshop results and committee discussions. The papers describe precursor strategies in aviation, the chemical industry, health care, nuclear power and security operations. In addition to current practices, they also address some areas for future research.

Patchoulol Production with Metabolically Engineered Corynebacterium glutamicum

PubMed Central

Wichmann, Julian; Baier, Thomas; Frohwitter, Jonas; Risse, Joe M.; Peters-Wendisch, Petra; Kruse, Olaf

2018-01-01

Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1. PMID:29673223

L-Arginine promotes protein synthesis and cell growth in brown adipocyte precursor cells via the mTOR signal pathway.

PubMed

Ma, Xi; Han, Meng; Li, Defa; Hu, Shengdi; Gilbreath, Kyler R; Bazer, Fuller W; Wu, Guoyao

2017-05-01

L-Arginine has been reported to enhance brown adipose tissue developments in fetal lambs of obese ewes, but the underlying mechanism is unknown. The present study tested the hypothesis that L-arginine stimulates growth and development of brown adipocyte precursor cells (BAPCs) through activation of mammalian target of rapamycin cell signaling. BAPCs isolated from fetal lambs at day 90 of gestation were incubated   for 6 h in arginine-free DMEM, and then cultured in DMEM with concentrations of 50, 100, 200, 500 or 1000 μmol L-arginine/L for 24-96 h. Cell proliferation, protein turnover, the mammalian target of rapamycin (mTOR) signaling pathway and pre-adipocyte differentiation markers were determined. L-arginine treatment enhanced (P < 0.05) BAPC growth and protein synthesis, while inhibiting proteolysis in a dose-dependent manner. Compared with 50 and 100 μmol/L (the concentrations of arginine in the maternal plasma of obese ewes), 200 μmol L-arginine/L (the concentrations of arginine in the maternal plasma of obese ewes receiving arginine supplementation) increased (P < 0.05) the abundances of phosphorylated mTOR, P70 S6K and 4EBP1, as well as the abundances of PGC1α, UCP1, BMP7 and PRDM16. These novel findings indicate that increasing extra-cellular arginine concentration from 50 to 200 µmol/L activates mTOR cell signaling in BAPCs and enhances their growth and development in a dose-dependent manner. Our results provide a mechanism for arginine supplementation to enhance the development of brown adipose tissue in fetal lambs.

Synthetic metabolism: metabolic engineering meets enzyme design.

PubMed

Erb, Tobias J; Jones, Patrik R; Bar-Even, Arren

2017-04-01

Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata

PubMed Central

Hussain, Tajammul; Plunkett, Blue; Ejaz, Mahwish; Espley, Richard V.; Kayser, Oliver

2018-01-01

The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for R. marginata. The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the Radulaceae family.

Kinetic analysis on precursors for iturin A production from Bacillus amyloliquefaciens BPD1.

PubMed

Wu, Jiun-Yan; Liao, Jen-Hung; Shieh, Chwen-Jen; Hsieh, Feng-Chia; Liu, Yung-Chuan

2018-06-12

In this study, the precursor effect for iturin A production was quantitatively analyzed. A strain identified as Bacillus amyloliquefaciens BPD1 (Ba-BPD1) was selected due to its ability to produce iturin A. The enhancement of iturin A production in a submerged culture was tested using various additives, including palmitic acid, oils, and complex amino acids. Among these, complex amino acids triggered the highest yield at 559 mg/L. The respective amino acids that contribute to the structure of iturin A were used as precursors. In fact, it was found that the addition of l-proline, l-glutamine, l-asparagine and l-serine could improve iturin A yield in the defined medium. However, during the kinetic analysis, all the amino acids exhibited a lower saturation level than l-serine, which exhibited a high saturation level at 1.2% resulting in an iturin A yield of 914 mg/L. In contrast, a negative effect was observed following the addition of l-tyrosine. To analyze the kinetic behavior of l-serine, three kinetic models were adopted: the kinetic order equation, the Langmuir kinetic equation, and a modified logistic equation. The regression results showed that the modified logistic model was the best fit for the kinetic behavior of l-serine as the major precursor, which could be further referred to the biosynthesis pathway of iturin A. Among the proposed processes for iturin A production, this study achieved the highest iturin A levels as a result of the addition of precursors. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Probabilistic pathway construction.

PubMed

Yousofshahi, Mona; Lee, Kyongbum; Hassoun, Soha

2011-07-01

Expression of novel synthesis pathways in host organisms amenable to genetic manipulations has emerged as an attractive metabolic engineering strategy to overproduce natural products, biofuels, biopolymers and other commercially useful metabolites. We present a pathway construction algorithm for identifying viable synthesis pathways compatible with balanced cell growth. Rather than exhaustive exploration, we investigate probabilistic selection of reactions to construct the pathways. Three different selection schemes are investigated for the selection of reactions: high metabolite connectivity, low connectivity and uniformly random. For all case studies, which involved a diverse set of target metabolites, the uniformly random selection scheme resulted in the highest average maximum yield. When compared to an exhaustive search enumerating all possible reaction routes, our probabilistic algorithm returned nearly identical distributions of yields, while requiring far less computing time (minutes vs. years). The pathways identified by our algorithm have previously been confirmed in the literature as viable, high-yield synthesis routes. Prospectively, our algorithm could facilitate the design of novel, non-native synthesis routes by efficiently exploring the diversity of biochemical transformations in nature. Copyright © 2011 Elsevier Inc. All rights reserved.

The NAD(+) precursor nicotinamide riboside decreases exercise performance in rats.

PubMed

Kourtzidis, Ioannis A; Stoupas, Andreas T; Gioris, Ioannis S; Veskoukis, Aristidis S; Margaritelis, Nikos V; Tsantarliotou, Maria; Taitzoglou, Ioannis; Vrabas, Ioannis S; Paschalis, Vassilis; Kyparos, Antonios; Nikolaidis, Michalis G

2016-01-01

Nicotinamide adenine dinucleotide (NAD(+)) and its phosphorylated form (NADP(+)) are key molecules in ubiquitous bioenergetic and cellular signaling pathways, regulating cellular metabolism and homeostasis. Thus, supplementation with NAD(+) and NADP(+) precursors emerged as a promising strategy to gain many and multifaceted health benefits. In this proof-of-concept study, we sought to investigate whether chronic nicotinamide riboside administration (an NAD(+) precursor) affects exercise performance. Eighteen Wistar rats were equally divided in two groups that received either saline vehicle or nicotinamide riboside at a dose of 300 mg/kg body weight/day for 21 days via gavage. At the end of the 21-day administration protocol, both groups performed an incremental swimming performance test. The nicotinamide riboside group showed a tendency towards worse physical performance by 35 % compared to the control group at the final 10 % load (94 ± 53 s for the nicotinamide riboside group and 145 ± 59 s for the control group; P = 0.071). Our results do not confirm the previously reported ergogenic effect of nicotinamide riboside. The potentially negative effect of nicotinamide riboside administration on physical performance may be attributed to the pleiotropic metabolic and redox properties of NAD(+) and NADP(+).

GAS6/Mer axis regulates the homing and survival of the E2A/PBX1 positive B-cell precursor acute lymphoblastic leukemia in the bone marrow niche

PubMed Central

Shiozawa, Yusuke; Pedersen, Elisabeth A.; Taichman, Russell S.

2009-01-01

Despite improvements in current combinational chemotherapy regimens, the prognosis of the (1;19)(q23;p13) translocation (E2A/PBX1) positive B-cell precursor acute lymphoblastic leukemia (ALL) is poor in pediatric leukemia patients. In this study, we examined the roles of GAS6/Mer axis in the interactions between E2A/PBX1 positive B-cell precursor ALL cells and the osteoblastic niche in the bone marrow. The data show that primary human osteoblasts secrete GAS6 in response to the Mer-over-expressed E2A/PBX1 positive ALL cells through MAPK signaling pathway and that leukemia cells migrate toward GAS6 using pathways activated by Mer. Importantly, GAS6 supports the survival and prevents apoptosis from chemotherapy of E2A/PBX1 positive ALL cells by inducing dormancy. Together, these data suggest that GAS6/Mer axis regulates the homing and survival of the E2A/PBX1 positive B-cell precursor ALL in the bone marrow niche. PMID:19922767

The seco-iridoid pathway from Catharanthus roseus

PubMed Central

Miettinen, Karel; Dong, Lemeng; Navrot, Nicolas; Schneider, Thomas; Burlat, Vincent; Pollier, Jacob; Woittiez, Lotte; van der Krol, Sander; Lugan, Raphaël; Ilc, Tina; Verpoorte, Robert; Oksman-Caldentey, Kirsi-Marja; Martinoia, Enrico; Bouwmeester, Harro; Goossens, Alain; Memelink, Johan; Werck-Reichhart, Danièle

2014-01-01

The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications. PMID:24710322

Serrated pathway: Alternative route to colorectal cancer

PubMed Central

Patai, Árpád V; Molnár, Béla; Tulassay, Zsolt; Sipos, Ferenc

2013-01-01

Serrated polyps have been an area of intense focus for gastroenterologists over the past several years. Contrary to what was thought before, a growing body of literature indicates that these polyps can be precursors of colorectal cancer (CRC). Most of these lesions, particularly those in the proximal colon, have so far been under-recognized and missed during colonoscopy, qualifying these lesions to be the main cause of interval cancers. It is estimated that 10%-20% of CRCs evolve through this alternative, serrated pathway, with a distinct genetic and epigenetic profile. Aberrant DNA methylation plays a central role in the development of this CRC subtype. This characteristic molecular background is reflected in a unique pathological and clinical manifestation different from cancers arising via the traditional pathway. In this review we would like to highlight morphological, molecular and clinical features of this emerging pathway that are essential for gastroenterologists and may influence their everyday practice. PMID:23431044

A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.

PubMed

Karim, Ashty S; Jewett, Michael C

2016-07-01

Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Modularization of genetic elements promotes synthetic metabolic engineering.

PubMed

Qi, Hao; Li, Bing-Zhi; Zhang, Wen-Qian; Liu, Duo; Yuan, Ying-Jin

2015-11-15

In the context of emerging synthetic biology, metabolic engineering is moving to the next stage powered by new technologies. Systematical modularization of genetic elements makes it more convenient to engineer biological systems for chemical production or other desired purposes. In the past few years, progresses were made in engineering metabolic pathway using synthetic biology tools. Here, we spotlighted the topic of implementation of modularized genetic elements in metabolic engineering. First, we overviewed the principle developed for modularizing genetic elements and then discussed how the genetic modules advanced metabolic engineering studies. Next, we picked up some milestones of engineered metabolic pathway achieved in the past few years. Last, we discussed the rapid raised synthetic biology field of "building a genome" and the potential in metabolic engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

Single Source Precursors for Thin Film Solar Cells

NASA Technical Reports Server (NTRS)

Banger, Kulbinder K.; Hollingsworth, Jennifer A.; Harris, Jerry D.; Cowen, Jonathan; Buhro, William E.; Hepp, Aloysius F.

2002-01-01

The development of thin film solar cells on flexible, lightweight, space-qualified substrates provides an attractive cost solution to fabricating solar arrays with high specific power, (W/kg). The use of a polycrystalline chalcopyrite absorber layer for thin film solar cells is considered as the next generation photovoltaic devices. At NASA GRC we have focused on the development of new single source precursors (SSP) and their utility to deposit the chalcopyrite semi-conducting layer (CIS) onto flexible substrates for solar cell fabrication. The syntheses and thermal modulation of SSPs via molecular engineering is described. Thin-film fabrication studies demonstrate the SSPs can be used in a spray CVD (chemical vapor deposition) process, for depositing CIS at reduced temperatures, which display good electrical properties, suitable for PV (photovoltaic) devices.

Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

PubMed

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2016-03-01

Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

The Interrelationships of Mathematical Precursors in Kindergarten

PubMed Central

Cirino, Paul T.

2011-01-01

This study evaluated the interrelations among cognitive precursors across quantitative, linguistic, and spatial attention domains that have been implicated for math achievement in young children. The dimensionality of the quantity precursors was evaluated in 286 Kindergarteners via latent variable techniques, and the contribution of precursors from each domain was established for small sums addition. Results showed a five factor structure for the quantity precursors with the major distinction between nonsymbolic and symbolic tasks. The overall model demonstrated good fit, and strong predictive power (R2 = 55%) for addition number combinations. Linguistic and spatial attention domains showed indirect relationships with outcomes, with their effects mediated by symbolic quantity measures. These results have implications for the measurement of mathematical precursors, and yield promise for predicting future math performance. PMID:21194711

Engineering Bacterial Thiosulfate and Tetrathionate Sensors for Detecting Gut Inflammation

DTIC Science & Technology

2017-04-03

Article Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation Kristina N-M Daeffler1 , Jeffrey D Galley2, Ravi U...interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we...understood. Genetically engineered sensor bacteria have untapped potential as tools for analyzing gut pathways. Bacteria have evolved sensors of a large

Rational synthetic pathway refactoring of natural products biosynthesis in actinobacteria.

PubMed

Tan, Gao-Yi; Liu, Tiangang

2017-01-01

Natural products (NPs) and their derivatives are widely used as frontline treatments for many diseases. Actinobacteria spp. are used to produce most of NP antibiotics and have also been intensively investigated for NP production, derivatization, and discovery. However, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in Actinobacteria, especially in the cases of genome mining and heterologous expression, it is often difficult to rationally and systematically engineer synthetic pathways to maximize biosynthetic efficiency. With the emergence of new tools and methods in metabolic engineering, the synthetic pathways of many chemicals, such as fatty acids and biofuels, in model organisms (e.g. Escherichia coli ), have been refactored to realize precise and flexible control of production. These studies also offer a promising approach for synthetic pathway refactoring in Actinobacteria. In this review, the great potential of Actinobacteria as a microbial cell factory for biosynthesis of NPs is discussed. To this end, recent progress in metabolic engineering of NP synthetic pathways in Actinobacteria are summarized and strategies and perspectives to rationally and systematically refactor synthetic pathways in Actinobacteria are highlighted. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of D-glucose and L-arabinose.

PubMed

Kawaguchi, Hideo; Yoshihara, Kumiko; Hara, Kiyotaka Y; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

2018-05-17

L-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to D-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as D-glucose, leading to a preferential utilization of D-glucose over pentoses. In order to simultaneously utilize both D-glucose and L-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis. Corynebacterium glutamicum ATCC 31831 utilized D-glucose and L-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized D-glucose over L-arabinose at a high concentration (15 g/L each), although L-arabinose and D-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for D-glucose and L-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of L-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of D-glucose and L-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized D-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of L-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of D-glucose and L-arabinose. Metabolome analysis

Functional Reconstitution of a Pyruvate Dehydrogenase in the Cytosol of Saccharomyces cerevisiae through Lipoylation Machinery Engineering.

PubMed

Lian, Jiazhang; Zhao, Huimin

2016-07-15

Acetyl-CoA is a key precursor for the biosynthesis of a wide range of fuels, chemicals, and value-added compounds, whose biosynthesis in Saccharomyces cerevisiae involves acetyl-CoA synthetase (ACS) and is energy intensive. Previous studies have demonstrated that functional expression of a pyruvate dehydrogenase (PDH) could fully replace the endogenous ACS-dependent pathway for cytosolic acetyl-CoA biosynthesis in an ATP-independent manner. However, the requirement for lipoic acid (LA) supplementation hinders its wide industrial applications. In the present study, we focus on the engineering of a de novo synthetic lipoylation machinery for reconstitution of a functional PDH in the cytosol of yeast. First, a LA auxotrophic yeast strain was constructed through the expression of the Escherichia coli PDH structural genes and a lipoate-protein ligase gene in an ACS deficient (acs1Δ acs2Δ) strain, based on which an in vivo acetyl-CoA reporter was developed for following studies. Then the de novo lipoylation pathway was reconstituted in the cytosol of yeast by coexpressing the yeast mitochondrial lipoylation machinery genes and the E. coli type II fatty acid synthase (FAS) genes. Alternatively, an unnatural de novo synthetic lipoylation pathway was constructed by combining the reversed β-oxidation pathway with an acyl-ACP synthetase gene. To the best of our knowledge, reconstitution of natural and unnatural de novo synthetic lipoylation pathways for functional expression of a PDH in the cytosol of yeast has never been reported. Our study has laid a solid foundation for the construction and further optimization of acetyl-CoA overproducing yeast strains.

Dual role of wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila.

PubMed

Sinenko, Sergey A; Mandal, Lolitika; Martinez-Agosto, Julian A; Banerjee, Utpal

2009-05-01

In Drosophila, blood development occurs in a specialized larval hematopoietic organ, the lymph gland (LG), within which stem-like hemocyte precursors or prohemocytes differentiate to multiple blood cell types. Here we show that components of the Wingless (Wg) signaling pathway are expressed in prohemocytes. Loss- and gain-of-function analysis indicates that canonical Wg signaling is required for maintenance of prohemocytes and negatively regulates their differentiation. Wg signals locally in a short-range fashion within different compartments of the LG. In addition, Wg signaling positively regulates the proliferation and maintenance of cells that function as a hematopoietic niche in Drosophila, the posterior signaling center (PSC), and in the proliferation of crystal cells. Our studies reveal a conserved function of Wg signaling in the maintenance of stem-like blood progenitors and reveal an involvement of this pathway in the regulation of hemocyte differentiation through its action in the hematopoietic niche.

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis.

PubMed

Cardenas, Javier; Da Silva, Nancy A

2016-07-01

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP(+) for acetyl-CoA production. After 24h of cultivation, a 3.7-fold increase in NADPH/NADP(+) ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2-3-fold over the base strain (up to 0.8g/L), and in combination to 1.4g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16g/g glucose), the highest reported to date. These biological driving

A Social Cognitive Approach to Understanding Engineering Career Interest and Expectations among Underrepresented Students in School-Based Clubs

ERIC Educational Resources Information Center

Dika, Sandra L.; Alvarez, Jaquelina; Santos, Jeannette; Suárez, Oscar Marcelo

2016-01-01

Interest in engineering at early stages of the educational career is one important precursor to choosing to study engineering in college, and engineering-related clubs are designed to foster such interest and diversify the engineering pipeline. In this study, the researchers employed a social cognitive career theory framework to examine level of…

Averufanin is an aflatoxin B1 precursor between averantin and averufin in the biosynthetic pathway.

PubMed Central

McCormick, S P; Bhatnagar, D; Lee, L S

1987-01-01

Wild-type Aspergillus parasiticus produces, in addition to the colorless aflatoxins, a number of pigmented secondary metabolites. Examination of these pigments demonstrated that a major component was an anthraquinone, averufanin. Radiolabeling studies with [14C]averufanin showed that 23% of the label was incorporated into aflatoxin B1 by the wild type and that 31% of the label was incorporated into O-methylsterigmatocystin by a non-aflatoxin-producing isolate. In similar studies with blocked mutants of A. parasiticus the 14C label from averufanin was accumulated in averufin (72%) and versicolorin A (54%) but not averantin. The results demonstrate that averufanin is a biosynthetic precursor of aflatoxin B1 between averantin and averufin. PMID:3103529

Career Pathways of Science, Engineering and Technology Research Postgraduates

ERIC Educational Resources Information Center

Giles, Marnie; Ski, Chantal; Vrdoljak, Davorin

2009-01-01

Suitably qualified scientists and engineers are essential for research and development, innovation and, in turn, the growth of the economy. Science, engineering and technology skills are therefore necessary for Australia to remain competitive in a global market. This article reports findings from a nationwide study investigating the career…

Serrated pathway in colorectal carcinogenesis

PubMed Central

Yamane, Letícia; Scapulatempo-Neto, Cristovam; Reis, Rui Manuel; Guimarães, Denise Peixoto

2014-01-01

Serrated adenocarcinoma is a recently described subset of colorectal cancer (CRC), which account for about 10% of all CRCs and follows an alternative pathway in which serrated polyps replace the traditional adenoma as the precursor lesion to CRC. Serrated polyps form a heterogeneous group of colorectal lesions that includes hyperplastic polyps (HPs), sessile serrated adenoma (SSA), traditional serrated adenoma (TSA) and mixed polyps. HPs are the most common serrated polyp followed by SSA and TSA. This distinct histogenesis is believed to have a major influence in prevention strategies, patient prognosis and therapeutic impact. Genetically, serrated polyps exhibited also a distinct pattern, with KRAS and BRAF having an important contribution to its development. Two other molecular changes that have been implicated in the serrated pathway include microsatellite instability and the CpG island methylator phenotype. In the present review we will address the current knowledge of serrated polyps, clinical pathological features and will update the most recent findings of its molecular pathways. The understanding of their biology and malignancy potential is imperative to implement a surveillance approach in order to prevent colorectal cancer development. PMID:24627599

On the difficulties of detecting PP precursors

NASA Astrophysics Data System (ADS)

Lessing, Stephan; Thomas, Christine; Saki, Morvarid; Schmerr, Nicholas; Vanacore, Elizabeth

2015-06-01

The PP precursors are seismic waves that form from underside reflections of P waves off discontinuities in the upper mantle transition zone (MTZ). These seismic phases are used to map discontinuity topography, sharpness, and impedance contrasts; the resulting structural variations are then often interpreted as evidence for temperature and/or mineralogy variations within the mantle. The PP precursors as well as other seismic phases have been used to establish the global presence of seismic discontinuities at 410 and 660 km depth. Intriguingly, in more than 80 per cent of PP precursor observations the seismic wave amplitudes are significantly weaker than the amplitudes predicted by seismic reference models. Even more perplexing is the observation that 1-5 per cent of all earthquakes (which are 20-25 per cent of earthquakes with clear PP waveforms) do not show any evidence for the PP precursors from the discontinuities even in the presence of well-developed PP waveforms. Non-detections are found in six different data sets consisting of tens to hundreds of events. We use synthetic modelling to examine a suite of factors that could be responsible for the absence of the PP precursors. The take-off angles for PP and the precursors differ by only 1.2-1.5°; thus source-related complexity would affect PP and the precursors. A PP wave attenuated in the upper mantle would increase the relative amplitude of the PP precursors. Attenuation within the transition zone could reduce precursor amplitudes, but this would be a regional phenomenon restricted to particular source receiver geometries. We also find little evidence for deviations from the theoretical travel path of seismic rays expected for scattered arrivals. Factors that have a strong influence include the stacking procedures used in seismic array techniques in the presence of large, interfering phases, the presence of topography on the discontinuities on the order of tens of kilometres, and 3-D lateral heterogeneity in

Metabolic Engineering of Plants to Produce Precursors (Phloroglucinol and 1,2,4-butanetriol) of Energetic Materials

DTIC Science & Technology

2015-01-02

phloroglucinol, which are precursors of energetic materials butanetriol trinitrate (BTTN) and l ,3,5-triamino-2,4,6 trinitrobenzene (TATB), respectively, in...of energetic materials butanetriol trinitrate (BTTN) and l ,3,5-triamino-2,4,6 trinitrobenzene (TATB), respectively, in plants. The strategy was to... phenylalanine , valine and hexose sugars. On the other hand the metabolites that are completely depleted in the chloroplastic lines and partially in

Career Pursuit Pathways among Emerging Adult Men and Women: Psychosocial Correlates and Precursors

ERIC Educational Resources Information Center

Shulman, Shmuel; Barr, Tamuz; Livneh, Yaara; Nurmi, Jari-Erik; Vasalampi, Kati; Pratt, Michael

2015-01-01

The present study examined career pursuit pathways in 100 Israeli emerging adults (54 men) who were followed from age 22 to 29. Employing a semi-structured interview at the age of 29, participants were asked about current work and educational status, work and educational goals and status changes in recent years, and to reflect on the meaning of…

Toward Engineering Synthetic Microbial Metabolism

PubMed Central

McArthur, George H.; Fong, Stephen S.

2010-01-01

The generation of well-characterized parts and the formulation of biological design principles in synthetic biology are laying the foundation for more complex and advanced microbial metabolic engineering. Improvements in de novo DNA synthesis and codon-optimization alone are already contributing to the manufacturing of pathway enzymes with improved or novel function. Further development of analytical and computer-aided design tools should accelerate the forward engineering of precisely regulated synthetic pathways by providing a standard framework for the predictable design of biological systems from well-characterized parts. In this review we discuss the current state of synthetic biology within a four-stage framework (design, modeling, synthesis, analysis) and highlight areas requiring further advancement to facilitate true engineering of synthetic microbial metabolism. PMID:20037734

Engineered Nanomaterials Elicit Cellular Stress Responses

EPA Science Inventory

Engineered nanomaterials are being developed continuously and incorporated into consumer products, resulting in increased human exposures. The study of engineered nanomaterials has focused largely on toxicity endpoints without further investigating potential mechanisms or pathway...

[Biosynthesis of indigo and indirubin by whole-cell catalyst designed by combination of protein engineering and metabolic engineering].

PubMed

Li, Yang; Zhu, Junge; Wang, Jianjun; Xia, Huanzhang; Wu, Sheng

2016-01-01

The phenylacetone monooxygenase, isolated from Thermobifida fusca, mainly catalyzes Baeyer-Villiger oxidation reaction towards aromatic compounds. Met446 plays a vital role in catalytic promiscuity, based on the structure and function of phenylacetone monooxygenase. Mutation in Met446 locus can offer enzyme new catalytic feature to activate C-H bond, oxidizing indole to finally generate indigo and indirubin, but the yield was only 1.89 mg/L. In order to further improve the biosynthesis efficiency of the whole-cell catalyst, metabolic engineering was applied to change glucose metabolism pathway of Escherichia coli. Blocking glucose isomerase gene pgi led to pentose phosphate pathway instead of the glycolytic pathway to become the major metabolic pathways of glucose, which provided more cofactor NADPH needed in enzymatic oxidation of indole. Engineering the host E. coli led to synthesis of indigo and indirubin efficiency further increased to 25 mg/L. Combination of protein and metabolic engineering to design efficient whole-cell catalysts not only improves the synthesis of indigo and indirubin, but also provides a novel strategy for whole-cell catalyst development.

The return of metabolism: biochemistry and physiology of the pentose phosphate pathway

PubMed Central

Stincone, Anna; Prigione, Alessandro; Cramer, Thorsten; Wamelink, Mirjam M. C.; Campbell, Kate; Cheung, Eric; Olin-Sandoval, Viridiana; Grüning, Nana-Maria; Krüger, Antje; Alam, Mohammad Tauqeer; Keller, Markus A.; Breitenbach, Michael; Brindle, Kevin M.; Rabinowitz, Joshua D.; Ralser, Markus

2015-01-01

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner–Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the ‘Warburg effect’ of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

PubMed Central

Summer, Elizabeth J.; Cline, Kenneth

1999-01-01

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways. PMID:9952453

Pathways to Engineering: The Validation Experiences of Transfer Students

ERIC Educational Resources Information Center

Zhang, Yi; Ozuna, Taryn

2015-01-01

Community college engineering transfer students are a critical student population of engineering degree recipients and technical workforce in the United States. Focusing on this group of students, we adopted Rendón's (1994) validation theory to explore the students' experiences in community colleges prior to transferring to a four-year…

Chaotic Time Series Analysis Method Developed for Stall Precursor Identification in High-Speed Compressors

NASA Technical Reports Server (NTRS)

1997-01-01

A new technique for rotating stall precursor identification in high-speed compressors has been developed at the NASA Lewis Research Center. This pseudo correlation integral method uses a mathematical algorithm based on chaos theory to identify nonlinear dynamic changes in the compressor. Through a study of four various configurations of a high-speed compressor stage, a multistage compressor rig, and an axi-centrifugal engine test, this algorithm, using only a single pressure sensor, has consistently predicted the onset of rotating stall.

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells*

PubMed Central

Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

2013-01-01

NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

Pyrolysis Pathways of Sulfonated Polyethylene, an Alternative Carbon Fiber Precursor

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

Younker, Jarod M; Saito, Tomonori; Hunt, Marcus A

2013-01-01

Sulfonated polyethylene is an emerging precursor for the production of carbon fibers. Pyrolysis of sulfonated polyethylene was characterized by thermogravimetric analysis (TGA). n-heptane-4-sulfonic acid (H4S) was selected as a model compound for the study of sulfonated polyethylene. Density functional theory and conventional transition state theory were used to determine the rate constants of pyrolysis for H4S from 300-1000 K. Multiple reaction channels from two different mechanisms were explored: 1) internal five-centered elimination (Ei 5) and 2) radical chain reaction. The pyrolysis of H4S was simulated with kinetic Monte Carlo (kMC) to obtain TGA plots that compared favorably to experiment. Wemore » observed that at tem- peratures < 550 K, the radical mechanism was dominant and yielded the trans-alkene, whereas cis-alkene was formed at higher temperatures from the internal elimination. The maximum rates of % mass loss became independent of initial OH radical concentration at 440-480 K. Experimentally, the maximum % mass loss occurred from 440-460 K (heating rate dependent). Activation energies derived from the kMC-simulated TGAs of H4S (26-29 kcal/mol) agreed with experiment for sulfonated polyethylene ( 31 kcal/mol). The simulations revealed that in this region, decomposition of radical HOSO2 became competitive to H abstraction by HOSO2, making OH the carrying radical for the reaction chain. The maximum rate of % mass loss for internal elimination was observed at temperatures > 600 K. Low-scale carbonization utilizes temperatures < 620 K; thus, internal elimination will not be competitive. Ei5 elimination has been studied for sulfoxides and sulfones, but this represents the first study of internal elimination in sulfonic acids. Nonlinear Arrhenius plots were found for all bimolecular reactions. The most significant nonlinear behavior was observed for reactions where the barrier was small. For reactions with low activation barriers, nonlinearity was traced

Synthesis and Characterization of the First Liquid Single Source Precursors for the Deposition of Ternary Chalcopyrite (CuInS2) Thin Film Materials

NASA Technical Reports Server (NTRS)

Banger, Kulbinder K.; Cowen, Jonathan; Hepp, Aloysius

2002-01-01

Molecular engineering of ternary single source precursors based on the [{PBu3}2Cu(SR')2In(SR')2] architecture have afforded the first liquid CIS ternary single source precursors (when R = Et, n-Pr), which are suitable for low temperature deposition (< 350 C). Thermogravimetric analyses (TGA) and modulated-differential scanning calorimetry (DSC) confirm their liquid phase and reduced stability. X-ray diffraction studies, energy dispersive analyzer (EDS), and scanning electron microscopy (SEM) support the formation of the single-phase chalcopyrite CuInS2 at low temperatures.

Functional Analyses and Treatment of Precursor Behavior

PubMed Central

Najdowski, Adel C; Wallace, Michele D; Ellsworth, Carrie L; MacAleese, Alicia N; Cleveland, Jackie M

2008-01-01

Functional analysis has been demonstrated to be an effective method to identify environmental variables that maintain problem behavior. However, there are cases when conducting functional analyses of severe problem behavior may be contraindicated. The current study applied functional analysis procedures to a class of behavior that preceded severe problem behavior (precursor behavior) and evaluated treatments based on the outcomes of the functional analyses of precursor behavior. Responding for all participants was differentiated during the functional analyses, and individualized treatments eliminated precursor behavior. These results suggest that functional analysis of precursor behavior may offer an alternative, indirect method to assess the operant function of severe problem behavior. PMID:18468282

Role of Elemental Sulfur in Forming Latent Precursors of H2S in Wine.

PubMed

Jastrzembski, Jillian A; Allison, Rachel B; Friedberg, Elle; Sacks, Gavin L

2017-12-06

The level of hydrogen sulfide (H 2 S) can increase during abiotic storage of wines, and potential latent sources of H 2 S are still under investigation. We demonstrate that elemental sulfur (S 0 ) residues on grapes not only can produce H 2 S during fermentation but also can form precursors capable of generating additional H 2 S after bottle storage for 3 months. H 2 S could be released from S 0 -derived precursors by addition of a reducing agent (TCEP), but not by addition of strong brine to induce release of H 2 S from metal sulfide complexes. The size of the TCEP-releasable pool varied among yeast strains. Using the TCEP assay, multiple polar S 0 -derived precursors were detected following normal-phase preparative chromatography. Using reversed-phase liquid chromatography and high-resolution mass spectrometry, we detected an increase in the levels of diglutathione trisulfane (GSSSG) and glutathione disulfide (GSSG) in S 0 -fermented red wine and an increase in the levels of glutathione S-sulfonate (GSSO 3 - ) and tetrathionate (S 4 O 6 2- ) in S 0 -fermented white wine as compared to controls. GSSSG, but not S 4 O 6 2- , was shown to evolve H 2 S in the presence of TCEP. Pathways for the formation of GSSSG, GSSG, GSSO 3 - , and S 4 O 6 2- from S 0 are proposed.

Engineering of the glycerol decomposition pathway and cofactor regulation in an industrial yeast improves ethanol production.

PubMed

Zhang, Liang; Tang, Yan; Guo, Zhongpeng; Shi, Guiyang

2013-10-01

Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD⁺-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.

Metabolic engineering of the shikimate pathway

DOEpatents

Juminaga, Darmawi; Keasling, Jay D.

2017-01-10

The present disclosure relates to engineered microorganisms that produce amino acids and amino acid intermediates. In particular, the disclosure relates to recombinant nucleic acids encoding operons that increase production of aromatic amino acids and the aromatic amino acid intermediate shikimate; microorganisms with increased production of aromatic amino acids and the aromatic amino acid intermediate shikimate; and methods related to the production of aromatic amino acids, the aromatic amino acid intermediate shikimate, and commodity chemicals derived therefrom.

The Drosophila T-box transcription factor Midline functions within the Notch–Delta signaling pathway to specify sensory organ precursor cell fates and regulates cell survival within the eye imaginal disc

PubMed Central

Das, Sudeshna; Chen, Q. Brent; Saucier, Joseph D.; Drescher, Brandon; Zong, Yan; Morgan, Sarah; Forstall, John; Meriwether, Andrew; Toranzo, Randy; Leal, Sandra M.

2014-01-01

We report that the T-box transcription factor Midline (Mid), an evolutionary conserved homolog of the vertebrate Tbx20 protein, functions within the Notch–Delta signaling pathway essential for specifying the fates of sensory organ precursor cells. This complements an established history of research showing that Mid regulates the cell-fate specification of diverse cell types within the developing heart, epidermis and central nervous system. Tbx20 has been detected in diverse neuronal and epithelial cells of embryonic eye tissues in both mice and humans. However, the mechanisms by which either Mid or Tbx20 function to regulate cell-fate specification or other critical aspects of eye development including cell survival have not yet been elucidated. We have also gathered preliminary evidence suggesting that Mid may play an indirect, but vital role in selecting SOP cells within the third-instar larval eye disc by regulating the expression of the proneural gene atonal. During subsequent pupal stages, Mid specifies SOP cell fates as a member of the Notch–Delta signaling hierarchy and is essential for maintaining cell viability within by inhibiting apoptotic pathways. We present several new hypotheses that seek to understand the role of Mid in regulating developmental processes downstream of the Notch receptor that are critical for specifying unique cell fates, patterning the adult eye and maintaining cellular homeostasis during eye disc morphogenesis. PMID:23962751

Advances in metabolic pathway and strain engineering paving the way for sustainable production of chemical building blocks.

PubMed

Chen, Yun; Nielsen, Jens

2013-12-01

Bio-based production of chemical building blocks from renewable resources is an attractive alternative to petroleum-based platform chemicals. Metabolic pathway and strain engineering is the key element in constructing robust microbial chemical factories within the constraints of cost effective production. Here we discuss how the development of computational algorithms, novel modules and methods, omics-based techniques combined with modeling refinement are enabling reduction in development time and thus advance the field of industrial biotechnology. We further discuss how recent technological developments contribute to the development of novel cell factories for the production of the building block chemicals: adipic acid, succinic acid and 3-hydroxypropionic acid. Copyright © 2013 Elsevier Ltd. All rights reserved.

Method of texturing a superconductive oxide precursor

DOEpatents

DeMoranville, Kenneth L.; Li, Qi; Antaya, Peter D.; Christopherson, Craig J.; Riley, Jr., Gilbert N.; Seuntjens, Jeffrey M.

1999-01-01

A method of forming a textured superconductor wire includes constraining an elongated superconductor precursor between two constraining elongated members placed in contact therewith on opposite sides of the superconductor precursor, and passing the superconductor precursor with the two constraining members through flat rolls to form the textured superconductor wire. The method includes selecting desired cross-sectional shape and size constraining members to control the width of the formed superconductor wire. A textured superconductor wire formed by the method of the invention has regular-shaped, curved sides and is free of flashing. A rolling assembly for single-pass rolling of the elongated precursor superconductor includes two rolls, two constraining members, and a fixture for feeding the precursor superconductor and the constraining members between the rolls. In alternate embodiments of the invention, the rolls can have machined regions which will contact only the elongated constraining members and affect the lateral deformation and movement of those members during the rolling process.

Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling

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

Pasoreck, Elise K.; Su, Jin; Silverman, Ian M.

The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were similar to 4300-fold higher in C and CN lines than in N, but all accumulated similar to 150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level ofmore » transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. In conclusion, the mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering.« less

Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling

DOE PAGES

Pasoreck, Elise K.; Su, Jin; Silverman, Ian M.; ...

2016-03-08

The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were similar to 4300-fold higher in C and CN lines than in N, but all accumulated similar to 150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level ofmore » transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. In conclusion, the mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering.« less

Fermentation and purification strategies for the production of betulinic acid and its lupane-type precursors in Saccharomyces cerevisiae.

PubMed

Czarnotta, Eik; Dianat, Mariam; Korf, Marcel; Granica, Fabian; Merz, Juliane; Maury, Jérôme; Baallal Jacobsen, Simo A; Förster, Jochen; Ebert, Birgitta E; Blank, Lars M

2017-11-01

Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L, and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering. © 2017 Wiley Periodicals, Inc.

Modular co-culture engineering, a new approach for metabolic engineering.

PubMed

Zhang, Haoran; Wang, Xiaonan

2016-09-01

With the development of metabolic engineering, employment of a selected microbial host for accommodation of a designed biosynthetic pathway to produce a target compound has achieved tremendous success in the past several decades. Yet, increasing requirements for sophisticated microbial biosynthesis call for establishment and application of more advanced metabolic engineering methodologies. Recently, important progress has been made towards employing more than one engineered microbial strains to constitute synthetic co-cultures and modularizing the biosynthetic labor between the co-culture members in order to improve bioproduction performance. This emerging approach, referred to as modular co-culture engineering in this review, presents a valuable opportunity for expanding the scope of the broad field of metabolic engineering. We highlight representative research accomplishments using this approach, especially those utilizing metabolic engineering tools for microbial co-culture manipulation. Key benefits and major challenges associated with modular co-culture engineering are also presented and discussed. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Fatty acid cosubstrates provide β-oxidation precursors for rhamnolipid biosynthesis in Pseudomonas aeruginosa, as evidenced by isotope tracing and gene expression assays.

PubMed

Zhang, Lin; Veres-Schalnat, Tracey A; Somogyi, Arpad; Pemberton, Jeanne E; Maier, Raina M

2012-12-01

Rhamnolipids have multiple potential applications as "green" surfactants for industry, remediation, and medicine. As a result, they have been intensively investigated to add to our understanding of their biosynthesis and improve yields. Several studies have noted that the addition of a fatty acid cosubstrate increases rhamnolipid yields, but a metabolic explanation has not been offered, partly because biosynthesis studies to date have used sugar or sugar derivatives as the carbon source. The objective of this study was to investigate the role of fatty acid cosubstrates in improving rhamnolipid biosynthesis. A combination of stable isotope tracing and gene expression assays was used to identify lipid precursors and potential lipid metabolic pathways used in rhamnolipid synthesis when fatty acid cosubstrates are present. To this end, we compared the rhamnolipids produced and their yields using either glucose alone or glucose and octadecanoic acid-d(35) as cosubstrates. Using a combination of sugar and fatty acids, the rhamnolipid yield was significantly higher (i.e., doubled) than when glucose was used alone. Two patterns of deuterium incorporation (either 1 or 15 deuterium atoms) in a single Rha-C(10) lipid chain were observed for octadecanoic acid-d(35) treatment, indicating that in the presence of a fatty acid cosubstrate, both de novo fatty acid synthesis and β-oxidation are used to provide lipid precursors for rhamnolipids. Gene expression assays showed a 200- to 600-fold increase in the expression of rhlA and rhlB rhamnolipid biosynthesis genes and a more modest increase of 3- to 4-fold of the fadA β-oxidation pathway gene when octadecanoic acid was present. Taken together, these results suggest that the simultaneous use of de novo fatty acid synthesis and β-oxidation pathways allows for higher production of lipid precursors, resulting in increased rhamnolipid yields.

Formation of Bi2Sr2CaCu2O x /Ag multifilamentary metallic precursor powder-in-tube wires

NASA Astrophysics Data System (ADS)

Zhang, Yun; Koch, Carl C.; Schwartz, Justin

2016-12-01

Previously, a metallic precursor (MP) approach to synthesizing Bi2Sr2CaCu2O x (Bi2212), with a homogeneous mixture of Bi, Sr, Ca, Cu and Ag was produced by mechanical alloying. Here, Bi2212/Ag round multifilamentary wire is manufactured using a metallic precursor powder-in-tube (MPIT) process. The MP powders were packed into a pure Ag tube in an Ar atmosphere and then sealed. After deformation, multifilamentary round wires and rolled tapes were heat treated in flowing oxygen through three stages: oxidation, conversion and partial-melt processing (PMP). Processing-microstructure-property relationships on 20-50 mm long multifilamentary round wires and rolled tapes were studied extensively. It is shown that conventional wire deformation processes, optimized for oxide-powder-in-tube wires, are not effective for deforming MPIT wires, and that as with prior studies of MPIT Bi2Sr2Ca2Cu3O y conductors, hot extrusion is required for obtaining a multifilamentary structure with fine filaments. As a result, the Bi2212 MPIT wires reported here have low engineering critical current density. Nonetheless, by focusing on sections of wires that remain intact after deformation, it is also shown that the first heat treatment stage, the oxidation stage, plays a crucial role in chemical homogeneity, phase transformation, and microstructural evolution and three reaction pathways for MP oxidation are presented. Furthermore, it is found the Bi2212 grain alignment within an MPIT filament is significantly different from that found in OPIT filaments after PMP, indicating the formation of highly dense filaments containing Bi2212 fine grains and Ag particles before PMP aids the formation of large, c-axis textured Bi2212 filaments during PMP. These results show that, with improved wire deformation, high critical current density may be obtained via a MPIT process.

The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

PubMed Central

Gao, Shouhong; Saechao, Saengking; Di, Peng; Chen, Junfeng; Chen, Wansheng

2011-01-01

Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors. PMID:22242141

Metabolite damage and repair in metabolic engineering design.

PubMed

Sun, Jiayi; Jeffryes, James G; Henry, Christopher S; Bruner, Steven D; Hanson, Andrew D

2017-11-01

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

Sol-gel precursors and products thereof

DOEpatents

Warren, Scott C.; DiSalvo, Jr., Francis J.; Weisner, Ulrich B.

2017-02-14

The present invention provides a generalizable single-source sol-gel precursor capable of introducing a wide range of functionalities to metal oxides such as silica. The sol-gel precursor facilitates a one-molecule, one-step approach to the synthesis of metal-silica hybrids with combinations of biological, catalytic, magnetic, and optical functionalities. The single-source precursor also provides a flexible route for simultaneously incorporating functional species of many different types. The ligands employed for functionalizing the metal oxides are derived from a library of amino acids, hydroxy acids, or peptides and a silicon alkoxide, allowing many biological functionalities to be built into silica hybrids. The ligands can coordinate with a wide range of metals via a carboxylic acid, thereby allowing direct incorporation of inorganic functionalities from across the periodic table. Using the single-source precursor a wide range of functionalized nanostructures such as monolith structures, mesostructures, multiple metal gradient mesostructures and Stober-type nanoparticles can be synthesized. ##STR00001##

Pathways to Science and Engineering Bachelor's Degrees for Men and Women.

PubMed

Legewie, Joscha; DiPrete, Thomas A

2014-02-18

Despite the striking reversal of the gender gap in educational attainment and the near-gender parity in math performance, women pursue science and engineering (S/E) degrees at much lower rates than their male peers do. Current efforts to increase the number of women in these fields focus on different life-course periods but lack a clear understanding of the importance of these periods and how orientations toward S/E fields develop over time. In this article, we examine the gendered pathways to a S/E bachelor's degree from middle school to high school and college based on a representative sample from the 1973 to 1974 birth cohort. Using a counterfactual decomposition analysis, we determine the relative importance of these different life-course periods and thereby inform the direction of future research and policy. Our findings confirm previous research that highlights the importance of early encouragement for gender differences in S/E degrees, but our findings also attest to the high school years as a decisive period for the gender gap, while challenging the focus on college in research and policy. Indeed, if female high school seniors had the same orientation toward and preparation for S/E fields as their male peers, the gender gap in S/E degrees would be closed by as much as 82 percent.

Detection of Chemical Precursors of Explosives

NASA Technical Reports Server (NTRS)

Li, Jing

2012-01-01

Certain selected chemicals associated with terrorist activities are too unstable to be prepared in final form. These chemicals are often prepared as precursor components, to be combined at a time immediately preceding the detonation. One example is a liquid explosive, which usually requires an oxidizer, an energy source, and a chemical or physical mechanism to combine the other components. Detection of the oxidizer (e.g. H2O2) or the energy source (e.g., nitromethane) is often possible, but must be performed in a short time interval (e.g., 5 15 seconds) and in an environment with a very small concentration (e.g.,1 100 ppm), because the target chemical(s) is carried in a sealed container. These needs are met by this invention, which provides a system and associated method for detecting one or more chemical precursors (components) of a multi-component explosive compound. Different carbon nanotubes (CNTs) are loaded (by doping, impregnation, coating, or other functionalization process) for detecting of different chemical substances that are the chemical precursors, respectively, if these precursors are present in a gas to which the CNTs are exposed. After exposure to the gas, a measured electrical parameter (e.g. voltage or current that correlate to impedance, conductivity, capacitance, inductance, etc.) changes with time and concentration in a predictable manner if a selected chemical precursor is present, and will approach an asymptotic value promptly after exposure to the precursor. The measured voltage or current are compared with one or more sequences of their reference values for one or more known target precursor molecules, and a most probable concentration value is estimated for each one, two, or more target molecules. An error value is computed, based on differences of voltage or current for the measured and reference values, using the most probable concentration values. Where the error value is less than a threshold, the system concludes that the target

Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

PubMed

van Deenen, Nicole; Bachmann, Anne-Lena; Schmidt, Thomas; Schaller, Hubert; Sand, Jennifer; Prüfer, Dirk; Schulze Gronover, Christian

2012-04-01

Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.

Precursor Mediated Synthesis of Nanostructured Silicas: From Precursor-Surfactant Ion Pairs to Structured Materials.

PubMed

Hesemann, Peter; Nguyen, Thy Phung; Hankari, Samir El

2014-04-11

The synthesis of nanostructured anionic-surfactant-templated mesoporous silica (AMS) recently appeared as a new strategy for the formation of nanostructured silica based materials. This method is based on the use of anionic surfactants together with a co-structure-directing agent (CSDA), mostly a silylated ammonium precursor. The presence of this CSDA is necessary in order to create ionic interactions between template and silica forming phases and to ensure sufficient affinity between the two phases. This synthetic strategy was for the first time applied in view of the synthesis of surface functionalized silica bearing ammonium groups and was then extended on the formation of materials functionalized with anionic carboxylate and bifunctional amine-carboxylate groups. In the field of silica hybrid materials, the "anionic templating" strategy has recently been applied for the synthesis of silica hybrid materials from cationic precursors. Starting from di- or oligosilylated imidazolium and ammonium precursors, only template directed hydrolysis-polycondensation reactions involving complementary anionic surfactants allowed accessing structured ionosilica hybrid materials. The mechanistic particularity of this approach resides in the formation of precursor-surfactant ion pairs in the hydrolysis-polycondensation mixture. This review gives a systematic overview over the various types of materials accessed from this cooperative ionic templating approach and highlights the high potential of this original strategy for the formation of nanostructured silica based materials which appears as a complementary strategy to conventional soft templating approaches.

Potato plants with genetically engineered tropane alkaloid precursors.

PubMed

Küster, Nadine; Rosahl, Sabine; Dräger, Birgit

2017-02-01

Solanum tuberosum tropinone reductase I reduced tropinone in vivo. Suppression of tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescine N -methyltransferase did not alter calystegine accumulation. Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato (Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N-methyltransferase (PMT) and tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N-methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants.

Shikimic Acid Production in Escherichia coli: From Classical Metabolic Engineering Strategies to Omics Applied to Improve Its Production.

PubMed

Martínez, Juan Andrés; Bolívar, Francisco; Escalante, Adelfo

2015-01-01

Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several "intuitive" metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the

Shikimic Acid Production in Escherichia coli: From Classical Metabolic Engineering Strategies to Omics Applied to Improve Its Production

PubMed Central

Martínez, Juan Andrés; Bolívar, Francisco; Escalante, Adelfo

2015-01-01

Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several “intuitive” metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for

Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A.

PubMed

Zhou, Liang; Ding, Qi; Jiang, Guo-Zhen; Liu, Zhen-Ning; Wang, Hai-Yan; Zhao, Guang-Rong

2017-05-16

Salvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases. Recently, production of SAA in engineered Escherichia coli was established via the artificial biosynthetic pathway of SAA on the multiple plasmids in our previous work. However, the plasmid-mediated system required to supplement expensive inducers and antibiotics during the fermentation process, restricting scale-up production of SAA. Microbial cell factory would be an attractive approach for constitutive production of SAA by chromosome engineering. The limited enzymatic reactions in SAA biosynthetic pathway from glucose were grouped into three modules, which were sequentially integrated into chromosome of engineered E. coli by λ Red homologous recombination method. With starting strain E. coli BAK5, in which the ptsG, pykF, pykA, pheA and tyrR genes were previously deleted, chassis strain BAK11 was constructed for constitutive production of precursor L-tyrosine by replacing the 17.7-kb mao-paa cluster with module 1 (P lacUV5 -aroG fbr -tyrA fbr -aroE) and the lacI gene with module 2 (P trc -glk-tktA-ppsA). The synthetic 5tacs promoter demonstrated the optimal strength to drive the expression of hpaBC-d-ldh Y52A in module 3, which then was inserted at the position between nupG and speC on the chromosome of strain BAK11. The final strain BKD13 produced 5.6 g/L of SAA by fed-batch fermentation in 60 h from glucose without any antibiotics and inducers supplemented. The plasmid-free and inducer-free strain for SAA production was developed by targeted integration of the constitutive expression of SAA biosynthetic genes into E. coli chromosome. Our work provides the industrial potential for constitutive production of SAA by the indel microbial cell factory and also sets an example of further producing other valuable natural and unnatural products.

Compartmentalization of metabolic pathways in yeast mitochondria improves production of branched chain alcohols

PubMed Central

Avalos, José L.; Fink, Gerald R.; Stephanopoulos, Gregory

2013-01-01

Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeted expression of metabolic pathways to mitochondria can increase production levels compared with expression of the same pathways in the cytoplasm. Compartmentalisation of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overexpressing only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains reveals that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves higher local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion, and reducing the loss of intermediates to competing pathways. PMID:23417095

2-Keto acids based biosynthesis pathways for renewable fuels and chemicals.

PubMed

Tashiro, Yohei; Rodriguez, Gabriel M; Atsumi, Shota

2015-03-01

Global energy and environmental concerns have driven the development of biological chemical production from renewable sources. Biological processes using microorganisms are efficient and have been traditionally utilized to convert biomass (i.e., glucose) to useful chemicals such as amino acids. To produce desired fuels and chemicals with high yield and rate, metabolic pathways have been enhanced and expanded with metabolic engineering and synthetic biology approaches. 2-Keto acids, which are key intermediates in amino acid biosynthesis, can be converted to a wide range of chemicals. 2-Keto acid pathways were engineered in previous research efforts and these studies demonstrated that 2-keto acid pathways have high potential for novel metabolic routes with high productivity. In this review, we discuss recently developed 2-keto acid-based pathways.

Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

PubMed

Chin, Young-Wook; Seo, Nari; Kim, Jae-Han; Seo, Jin-Ho

2016-11-01

2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A review of parameters and heuristics for guiding metabolic pathfinding.

PubMed

Kim, Sarah M; Peña, Matthew I; Moll, Mark; Bennett, George N; Kavraki, Lydia E

2017-09-15

Recent developments in metabolic engineering have led to the successful biosynthesis of valuable products, such as the precursor of the antimalarial compound, artemisinin, and opioid precursor, thebaine. Synthesizing these traditionally plant-derived compounds in genetically modified yeast cells introduces the possibility of significantly reducing the total time and resources required for their production, and in turn, allows these valuable compounds to become cheaper and more readily available. Most biosynthesis pathways used in metabolic engineering applications have been discovered manually, requiring a tedious search of existing literature and metabolic databases. However, the recent rapid development of available metabolic information has enabled the development of automated approaches for identifying novel pathways. Computer-assisted pathfinding has the potential to save biochemists time in the initial discovery steps of metabolic engineering. In this paper, we review the parameters and heuristics used to guide the search in recent pathfinding algorithms. These parameters and heuristics capture information on the metabolic network structure, compound structures, reaction features, and organism-specificity of pathways. No one metabolic pathfinding algorithm or search parameter stands out as the best to use broadly for solving the pathfinding problem, as each method and parameter has its own strengths and shortcomings. As assisted pathfinding approaches continue to become more sophisticated, the development of better methods for visualizing pathway results and integrating these results into existing metabolic engineering practices is also important for encouraging wider use of these pathfinding methods.

Earthquakes: hydrogeochemical precursors

USGS Publications Warehouse

Ingebritsen, Steven E.; Manga, Michael

2014-01-01

Earthquake prediction is a long-sought goal. Changes in groundwater chemistry before earthquakes in Iceland highlight a potential hydrogeochemical precursor, but such signals must be evaluated in the context of long-term, multiparametric data sets.

Enhanced wound healing of tissue-engineered human corneas through altered phosphorylation of the CREB and AKT signal transduction pathways.

PubMed

Couture, Camille; Desjardins, Pascale; Zaniolo, Karine; Germain, Lucie; Guérin, Sylvain L

2018-06-01

The cornea is a transparent organ, highly specialized and unique that is continually subjected to abrasive forces and occasional mechanical or chemical trauma because of its anatomical localization. Upon injury, the extracellular matrix (ECM) rapidly changes to promote wound healing through integrin-dependent activation of specific signal transduction mediators whose contribution is to favor faster closure of the wound by altering the adhesive and migratory properties of the cells surrounding the damaged area. In this study, we exploited the human tissue-engineered cornea (hTECs) as a model to study the signal transduction pathways that participate to corneal wound healing. By exploiting both gene profiling and activated kinases arrays, we could demonstrate the occurrence of important alterations in the level of expression and activation of a few mediators from the PI3K/Akt and CREB pathways in response to the ECM remodeling taking place during wound healing of damaged hTECs. Pharmacological inhibition of CREB with C646 considerably accelerated wound closure compared to controls. This process was considerably accelerated further when both C646 and SC79, an Akt agonist, were added together to wounded hTECs. Therefore, our study demonstrate that proper corneal wound healing requires the activation of Akt together with the inhibition of CREB and that wound healing in vitro can be altered by the use of pharmacological inhibitors (such as C646) or agonists (such as SC79) of these mediators. Corneal wounds account for a large proportion of all visual disabilities in North America. To our knowledge, this is the first time that a tissue-engineered human cornea (hTEC) entirely produced using normal untransformed human cells is used as a biomaterial to study the signal transduction pathways that are critical to corneal wound healing. Through the use of this biomaterial, we demonstrated that human corneal epithelial cells engaged in wound healing reduce phosphorylation of the

The logics of metabolic regulation in bacteria challenges biosensor-based metabolic engineering.

PubMed

Jules, Matthieu

2017-12-11

Synthetic Biology (SB) aims at the rational design and engineering of novel biological functions and systems. By facilitating the engineering of living organisms, SB promises to facilitate the development of many new applications for health, biomanufacturing, and the environment. Over the last decade, SB promoted the construction of libraries of components enabling the fine-tuning of genetic circuits expression and the development of novel genome engineering methodologies for many organisms of interest. SB thus opened new perspectives in the field of metabolic engineering, which was until then mainly limited to (over)producing naturally synthesized metabolic compounds. To engineer efficient cell factories, it is key to precisely reroute cellular resources from the central carbon metabolism (CCM) to the synthetic circuitry. This task is however difficult as there is still significant lack of knowledge regarding both the function of several metabolic components and the regulation of the CCM fluxes for many industrially important bacteria. Pyruvate is a pivotal metabolite at the heart of the CCM and a key precursor for the synthesis of several commodity compounds and fine chemicals. Numerous bacterial species can also use it as a carbon source when present in the environment but bacterial, pyruvate-specific uptake systems were to be discovered. This is an issue for metabolic engineering as one can imagine to make use of pyruvate transport systems to replenish synthetic metabolic pathways towards the synthesis of chemicals of interest. Here we describe a recent study (MBio 8(5): e00976-17), which identified and characterized a pyruvate transport system in the Gram-positive (G +ve ) bacterium Bacillus subtilis , a well-established biotechnological workhorse for the production of enzymes, fine chemicals and antibiotics. This study also revealed that the activity of the two-component system (TCS) responsible for its induction is retro-inhibited by the level of pyruvate

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity

DOE PAGES

Guo, Chun -Jun; Sun, Wei -Wen; Bruno, Kenneth S.; ...

2015-07-13

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPSlike genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas inmore » conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.« less

The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells

PubMed Central

Trötzmüller, Martin; Hinteregger, Barbara; Leko, Petra; Wieser, Beatrix I.; Grasmann, Gabriele; Bertsch, Alexandra L.; Züllig, Thomas; Stacher, Elvira; Valli, Alessandro; Prassl, Ruth; Olschewski, Andrea; Harris, Adrian L.; Köfeler, Harald C.; Olschewski, Horst; Hrzenjak, Andelko

2018-01-01

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M–dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation. PMID:29844165

Expanding the metabolic engineering toolbox with directed evolution.

PubMed

Abatemarco, Joseph; Hill, Andrew; Alper, Hal S

2013-12-01

Cellular systems can be engineered into factories that produce high-value chemicals from renewable feedstock. Such an approach requires an expanded toolbox for metabolic engineering. Recently, protein engineering and directed evolution strategies have started to play a growing and critical role within metabolic engineering. This review focuses on the various ways in which directed evolution can be applied in conjunction with metabolic engineering to improve product yields. Specifically, we discuss the application of directed evolution on both catalytic and non-catalytic traits of enzymes, on regulatory elements, and on whole genomes in a metabolic engineering context. We demonstrate how the goals of metabolic pathway engineering can be achieved in part through evolving cellular parts as opposed to traditional approaches that rely on gene overexpression and deletion. Finally, we discuss the current limitations in screening technology that hinder the full implementation of a metabolic pathway-directed evolution approach. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways.

PubMed

Caveney, Nathanael A; Li, Franco Kk; Strynadka, Natalie Cj

2018-06-06

The bacterial cell wall is a complex polymeric structure with essential roles in defence, survival and pathogenesis. Common to both Gram-positive and Gram-negative bacteria is the mesh-like peptidoglycan sacculus that surrounds the outer leaflet of the cytoplasmic membrane. Recent crystallographic studies of enzymes that comprise the peptidoglycan biosynthetic pathway have led to significant new understanding of all stages. These include initial multi-step cytosolic formation of sugar-pentapeptide precursors, transfer of the precursors to activated polyprenyl lipids at the membrane inner leaflet and flippase mediated relocalization of the resulting lipid II precursors to the outer leaflet where glycopolymerization and subsequent peptide crosslinking are finalized. Additional, species-specific enzymes allow customized peptidoglycan modifications and biosynthetic regulation that are important to bacterial virulence and survival. These studies have reinforced the unique and specific catalytic mechanisms at play in cell wall biogenesis and expanded the atomic foundation to develop novel, structure guided, antibacterial agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

Primary Emission and the Potential of Secondary Aerosol Formation from Chinese Gasoline Engine Exhaust

NASA Astrophysics Data System (ADS)

Hu, Min; Peng, Jianfei; Qin, Yanhong; Du, Zhuofei; Li, Mengjin; Zheng, Rong; Zheng, Jing; Shang, Dongjie; Lu, Sihua; Wu, Yusheng; Zeng, Limin; Guo, Song; Shao, Min; Wang, Yinhui; Shuai, Shijin

2017-04-01

Along with the urbanization and economic growth, vehicle population in China reached 269 million, ranked the second in the world in 2015. Gasoline vehicle is identified to be the main source for urban PM2.5 in China, accounting for 15%-31%. In this study the impact of fuel components on PM2.5 and volatile organic compounds (VOCs) emissions from a gasoline port fuel injection (PFI) engine and a gasoline direct injection (GDI) engine are discussed. Results show that, higher proportion of aromatics, alkenes or sulfur in gasoline fuel will lead to higher PM emissions. The PM from the PFI engine mainly consists of OC and a small amount of EC and inorganic ions, while the PM discharge from the GDI engine mainly consists of EC, OM and a small amount of inorganic ions. Since the GDI engines can reduce fuel consumption and CO2 emissions, and it would become more and more popular in the near future. The characteristics of POM component, emission factors and source profile were investigated from GDI engine, particularly focused on the effect of engine speed, load and the catalyst, which will be very much helpful for source identification as source indicators. Chamber experiments were conducted to quantify the potential of secondary aerosol formation from exhaust of a PFI gasoline engine and China V gasoline fuel. During 4-5 h simulation, equivalent to10 days of atmospheric photo-oxidation in Beijing, the extreme SOA production was 426 ± 85 mg/kg fuel, with high precursors and OH exposure. 14% of SOA measured in the chamber experiments could be explained through the oxidation of speciated single-ring aromatics. Unspeciated precursors, such as intermediate-volatility organic compounds and semi-volatility organic compounds, might be significant for SOA formation from gasoline VOCs. We concluded that reduction of emissions of aerosol precursor gases from vehicles is essential to mediate pollution in China.

Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo

PubMed Central

Young, Barry P.; Craven, Rachel A.; Reid, Peter J.; Willer, Martin; Stirling, Colin J.

2001-01-01

The translocation of secretory polypeptides into the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. In yeast, targeting of secretory precursors to the translocon can occur by two distinct pathways that are distinguished by their dependence upon the signal recognition particle (SRP). The SRP-dependent pathway requires SRP and its membrane-bound receptor, whereas the SRP-independent pathway requires a separate receptor complex consisting of Sec62p, Sec63p, Sec71p, Sec72p plus lumenal Kar2p/BiP. Here we demonstrate that Sec63p and Kar2p are also required for the SRP-dependent targeting pathway in vivo. Furthermore, we demonstrate multiple roles for Sec63p, at least one of which is exclusive to the SRP-independent pathway. PMID:11226176

Effect of elicitors and precursors on azadirachtin production in hairy root culture of Azadirachta indica.

PubMed

Srivastava, Smita; Srivastava, A K

2014-02-01

The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag(+), Hg(+2), Co(+2), Cu(+2)) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l(-1) of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g(-1)). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 % v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g(-1)) with respect to the control (3.3 mg g(-1)). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l(-1) in 25 days) was found to be the highest with cholesterol (50 mg l(-1)) addition as an indirect precursor in the medium.

Vaticaffinol, a resveratrol tetramer, exerts more preferable immunosuppressive activity than its precursor in vitro and in vivo through multiple aspects against activated T lymphocytes

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

Feng, Li-Li; Wu, Xue-Feng; Liu, Hai-Liang

2013-03-01

In the present study, we aimed to investigate the immunosuppressive activity of vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, on T lymphocytes both in vitro and in vivo, and further explored its potential molecular mechanism. Resveratrol had a wide spectrum of healthy beneficial effects with multiple targets. Interestingly, its tetramer, vaticaffinol, exerted more intensive immunosuppressive activity than resveratrol. Vaticaffinol significantly inhibited T cells proliferation activated by concanavalin A (Con A) or anti-CD3 plus anti-CD28 in a dose- and time-dependent manner. It also induced Con A-activated T cells undergoing apoptosis through mitochondrial pathway. Moreover, this compound prevented cells from enteringmore » S phase and G2/M phase during T cells activation. In addition, vaticaffinol inhibited ERK and AKT signaling pathways in Con A-activated T cells. Furthermore, vaticaffinol significantly ameliorated ear swelling in a mouse model of picryl chloride-induced ear contact dermatitis in vivo. In most of the aforementioned experiments, however, resveratrol had only slight effects on the inhibition of T lymphocytes compared with vaticaffinol. Taken together, our findings suggest that vaticaffinol exerts more preferable immunosuppressive activity than its precursor resveratrol both in vitro and in vivo by affecting multiple targets against activated T cells. - Graphical abstract: Vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, exerts more intensive immunosuppressive activity than its precursor resveratrol does in vitro and in vivo. Its mechanism may involve multiple effects against activated T cells: regulation of signalings involved in cell proliferation, G0/G1 arrest of T cells, as well as an apoptosis induction in activated effector T cells. Highlights: ► Vaticaffinol, a resveratrol tetramer, exerts more potent activity than its precursor. ► It inhibited T cells proliferation and prevented them from

Distinct polymer architecture mediates switching of complement activation pathways at the nanosphere-serum interface: implications for stealth nanoparticle engineering.

PubMed

Hamad, Islam; Al-Hanbali, Othman; Hunter, A Christy; Rutt, Kenneth J; Andresen, Thomas L; Moghimi, S Moein

2010-11-23

Nanoparticles with surface projected polyethyleneoxide (PEO) chains in "mushroom-brush" and "brush" configurations display stealth properties in systemic circulation and have numerous applications in site-specific targeting for controlled drug delivery and release as well as diagnostic imaging. We report on the "structure-activity" relationship pertaining to surface-immobilized PEO of various configurations on model nanoparticles, and the initiation of complement cascade, which is the most ancient component of innate human immunity, and its activation may induce clinically significant adverse reactions in some individuals. Conformational states of surface-projected PEO chains, arising from the block copolymer poloxamine 908 adsorption, on polystyrene nanoparticles trigger complement activation differently. Alteration of copolymer architecture on nanospheres from mushroom to brush configuration not only switches complement activation from C1q-dependent classical to lectin pathway but also reduces the level of generated complement activation products C4d, Bb, C5a, and SC5b-9. Also, changes in adsorbed polymer configuration trigger alternative pathway activation differently and through different initiators. Notably, the role for properdin-mediated activation of alternative pathway was only restricted to particles displaying PEO chains in a transition mushroom-brush configuration. Since nanoparticle-mediated complement activation is of clinical concern, our findings provide a rational basis for improved surface engineering and design of immunologically safer stealth and targetable nanosystems with polymers for use in clinical medicine.

A modular metabolic engineering approach for the production of 1,2-propanediol from glycerol by Saccharomyces cerevisiae.

PubMed

Islam, Zia-Ul; Klein, Mathias; Aßkamp, Maximilian R; Ødum, Anders S R; Nevoigt, Elke

2017-11-01

Compared to sugars, a major advantage of using glycerol as a feedstock for industrial bioprocesses is the fact that this molecule is more reduced than sugars. A compound whose biotechnological production might greatly profit from the substrate's higher reducing power is 1,2-propanediol (1,2-PDO). Here we present a novel metabolic engineering approach to produce 1,2-PDO from glycerol in S. cerevisiae. Apart from implementing the heterologous methylglyoxal (MG) pathway for 1,2-PDO formation from dihydroxyacetone phosphate (DHAP) and expressing a heterologous glycerol facilitator, the employed genetic modifications included the replacement of the native FAD-dependent glycerol catabolic pathway by the 'DHA pathway' for delivery of cytosolic NADH and the reduction of triosephosphate isomerase (TPI) activity for increased precursor (DHAP) supply. The choice of the medium had a crucial impact on both the strength of the metabolic switch towards fermentation in general (as indicated by the production of ethanol and 1,2-PDO) and on the ratio at which these two fermentation products were formed. For example, virtually no 1,2-PDO but only ethanol was formed in synthetic glycerol medium with urea as the nitrogen source. When nutrient-limited complex YG medium was used, significant amounts of 1,2-PDO were formed and it became obvious that the concerted supply of NADH and DHAP are essential for boosting 1,2-PDO production. Additionally, optimizing the flux into the MG pathway improved 1,2-PDO formation at the expense of ethanol. Cultivation of the best-performing strain in YG medium and a controlled bioreactor set-up resulted in a maximum titer of > 4gL -1 1,2-PDO which, to the best of our knowledge, has been the highest titer of 1,2-PDO obtained in yeast so far. Surprisingly, significant 1,2-PDO production was also obtained in synthetic glycerol medium after changing the nitrogen source towards ammonium sulfate and adding a buffer. Copyright © 2017 International Metabolic

Descriptive and Experimental Analyses of Potential Precursors to Problem Behavior

PubMed Central

Borrero, Carrie S.W; Borrero, John C

2008-01-01

We conducted descriptive observations of severe problem behavior for 2 individuals with autism to identify precursors to problem behavior. Several comparative probability analyses were conducted in addition to lag-sequential analyses using the descriptive data. Results of the descriptive analyses showed that the probability of the potential precursor was greater given problem behavior compared to the unconditional probability of the potential precursor. Results of the lag-sequential analyses showed a marked increase in the probability of a potential precursor in the 1-s intervals immediately preceding an instance of problem behavior, and that the probability of problem behavior was highest in the 1-s intervals immediately following an instance of the precursor. We then conducted separate functional analyses of problem behavior and the precursor to identify respective operant functions. Results of the functional analyses showed that both problem behavior and the precursor served the same operant functions. These results replicate prior experimental analyses on the relation between problem behavior and precursors and extend prior research by illustrating a quantitative method to identify precursors to more severe problem behavior. PMID:18468281

Precursor Mediated Synthesis of Nanostructured Silicas: From Precursor-Surfactant Ion Pairs to Structured Materials

PubMed Central

Hesemann, Peter; Nguyen, Thy Phung; Hankari, Samir El

2014-01-01

The synthesis of nanostructured anionic-surfactant-templated mesoporous silica (AMS) recently appeared as a new strategy for the formation of nanostructured silica based materials. This method is based on the use of anionic surfactants together with a co-structure-directing agent (CSDA), mostly a silylated ammonium precursor. The presence of this CSDA is necessary in order to create ionic interactions between template and silica forming phases and to ensure sufficient affinity between the two phases. This synthetic strategy was for the first time applied in view of the synthesis of surface functionalized silica bearing ammonium groups and was then extended on the formation of materials functionalized with anionic carboxylate and bifunctional amine-carboxylate groups. In the field of silica hybrid materials, the “anionic templating” strategy has recently been applied for the synthesis of silica hybrid materials from cationic precursors. Starting from di- or oligosilylated imidazolium and ammonium precursors, only template directed hydrolysis-polycondensation reactions involving complementary anionic surfactants allowed accessing structured ionosilica hybrid materials. The mechanistic particularity of this approach resides in the formation of precursor-surfactant ion pairs in the hydrolysis-polycondensation mixture. This review gives a systematic overview over the various types of materials accessed from this cooperative ionic templating approach and highlights the high potential of this original strategy for the formation of nanostructured silica based materials which appears as a complementary strategy to conventional soft templating approaches. PMID:28788602

Alternative Pathways to Engineering Success--Using Academic and Social Integration to Understand Two-Year Engineering Student Success

ERIC Educational Resources Information Center

Marra, Rose M.; Tsai, Chia-Lin; Bogue, Barbara; Pytel, Jean Landa

2015-01-01

The need for educating engineers in the United States continues as the projected demand is rising the number of high school seniors planning to enter engineering careers has remained relatively stable (Sargent, 2014). Additionally, figures show that attrition rates in undergraduate engineering continue to be an area of concern, (Sargent, 2014;…

Condensation of the isoprenoid and amino precursors in the biosynthesis of domoic acid.

PubMed

Savage, Thomas J; Smith, G Jason; Clark, Amy T; Saucedo, Portia N

2012-01-01

Understanding how environmental signals regulate production of domoic acid in blooms of Pseudo-nitzschia spp. at a molecular level requires description of the biochemical pathway to this kainoid neurotoxin. Precursor feeding studies have suggested domoic acid arises from the condensation of the C(10) isoprenoid geranyl diphosphate with glutamate, but the specific reactions leading to domoic acid from these precursors remain undescribed. Here, we develop a method to derivatize domoic acid with propyl chloroformate that enables gas chromatography-mass spectrometry (GC-MS) analysis to measure incorporation of stable isotopes into domoic acid generated in cultures incubated with isotopically-labeled substrates. We apply this method to demonstrate that both (2)H from [1-(2)H(2)]geraniol are incorporated into domoic acid, suggesting that the condensation of geranyl diphosphate with an amino group occurs by nucleophilic substitution of the diphosphate rather than by oxidation of geraniol to the aldehyde before reaction with an amino group to form an imine. Ultimately, these and similar studies will facilitate the identification of DA biosynthetic enzymes and genes which will enable the study of how environmental factors regulate DA biosynthesis at the molecular level. Copyright © 2011 Elsevier Ltd. All rights reserved.

Arginine pathways and the inflammatory response: interregulation of nitric oxide and polyamines: review article.

PubMed

Satriano, J

2004-07-01

An early response to an acute inflammatory insult, such as wound healing or experimental glomerulonephritis, is the conversion of arginine to the cytostatic molecule nitric oxide (NO). This 'anti-bacterial' phase is followed by the conversion of arginine to ornithine, which is the precursor for the pro-proliferative polyamines as well as proline for the production of extracellular matrix. This latter, pro-growth phase constitutes a 'repair' phase response. The temporal switch of arginine as a substrate for the cytostatic iNOS/NO axis to the pro-growth arginase/ ornithine/polyamine and proline axis is subject to regulation by inflammatory cytokines as well as interregulation by the arginine metabolites themselves. Arginine is also the precursor for another biogenic amine, agmatine. Here we describe the capacity of these three arginine pathways to interregulate, and propose a model whereby agmatine has the potential to serve in the coordination of the early and repair phase pathways of arginine in the inflammatory response by acting as a gating mechanism at the transition from the iNOS/NO axis to the arginase/ODC/polyamine axis. Due to the pathophysiologic and therapeutic potential, we will further examine the antiproliferative effects of agmatine on the polyamine pathway.

Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2.

PubMed

Wang, Bo; Pugh, Shawn; Nielsen, David R; Zhang, Weiwen; Meldrum, Deirdre R

2013-03-01

(S)- and (R)-3-hydroxybutyrate (3HB) are precursors to synthesize the biodegradable plastics polyhydroxyalkanoates (PHAs) and many fine chemicals. To date, however, their production has been restricted to petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economical feasibility. With the ability to fix CO2 photosynthetically, cyanobacteria have attracted increasing interest as a biosynthesis platform to produce fuels and chemicals from alternative renewable resources. To this end, synthesis metabolic pathways have been constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803 to photosynthetically produce (S)- and (R)-3HB directly from CO2. Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing pathway by deleting slr1829 and slr1830 (encoding PHB polymerase) from the Synechocystis genome further promoted the 3HB production. Up to 533.4mg/L 3HB has been produced after photosynthetic cultivation of the engineered cyanobacterium Synechocystis TABd for 21 days. Further analysis indicated that the phosphate consumption during the photoautrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. For the first time, the study has demonstrated the feasibility of photosynthetic production of (S)- and (R)-3HB directly from sunlight and CO2. Copyright © 2013 Elsevier Inc. All rights reserved.

Preparation of boron nitride fiber by organic precursor method

NASA Astrophysics Data System (ADS)

Zhou, Yingying; Sun, Runjun; Zhang, Zhaohuan; Fan, Wei; Zhou, Dan; Sheng, Cuihong

In this paper, boron nitride polymer precursor was made by boric acid, melamine, twelve sodium alkyl sulfate as raw materials and pure water as medium which is heated to 70 °C. Boron nitride precursor polymer was soluble in formic acid solution. The boron nitride precursor can be electrostatically spun at the voltage in 23 kV and the distance between the positive and negative poles is 15 cm. The formed fiber is very uniform. The properties of the precursors were analyzed through electron microscope, infrared spectrum, X-ray and ultraviolet spectrum. The aim of the job is to got the precursor of BN and spun it.

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

PubMed Central

2012-01-01

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts.

PubMed

Angaman, Djédoux Maxime; Petrizzo, Rocco; Hernández-Gras, Francesc; Romero-Segura, Carmen; Pateraki, Irene; Busquets, Montserrat; Boronat, Albert

2012-01-13

Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. We have set up a

Wnt and BMP Signaling Crosstalk in Regulating Dental Stem Cells: Implications in Dental Tissue Engineering

PubMed Central

Zhang, Fugui; Song, Jinglin; Zhang, Hongmei; Huang, Enyi; Song, Dongzhe; Tollemar, Viktor; Wang, Jing; Wang, Jinhua; Mohammed, Maryam; Wei, Qiang; Fan, Jiaming; Liao, Junyi; Zou, Yulong; Liu, Feng; Hu, Xue; Qu, Xiangyang; Chen, Liqun; Yu, Xinyi; Luu, Hue H.; Lee, Michael J.; He, Tong-Chuan; Ji, Ping

2016-01-01

Tooth is a complex hard tissue organ and consists of multiple cell types that are regulated by important signaling pathways such as Wnt and BMP signaling. Serious injuries and/or loss of tooth or periodontal tissues may significantly impact aesthetic appearance, essential oral functions and the quality of life. Regenerative dentistry holds great promise in treating oral/dental disorders. The past decade has witnessed a rapid expansion of our understanding of the biological features of dental stem cells, along with the signaling mechanisms governing stem cell self-renewal and differentiation. In this review, we first summarize the biological characteristics of seven types of dental stem cells, including dental pulp stem cells, stem cells from apical papilla, stem cells from human exfoliated deciduous teeth, dental follicle precursor cells, periodontal ligament stem cells, alveolar bone-derived mesenchymal stem cells (MSCs), and MSCs from gingiva. We then focus on how these stem cells are regulated by bone morphogenetic protein (BMP) and/or Wnt signaling by examining the interplays between these pathways. Lastly, we analyze the current status of dental tissue engineering strategies that utilize oral/dental stem cells by harnessing the interplays between BMP and Wnt pathways. We also highlight the challenges that must be addressed before the dental stem cells may reach any clinical applications. Thus, we can expect to witness significant progresses to be made in regenerative dentistry in the coming decade. PMID:28491933

SEARCH FOR PRECURSOR ERUPTIONS AMONG TYPE IIB SUPERNOVAE

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

Strotjohann, Nora L.; Ofek, Eran O.; Gal-Yam, Avishay

2015-10-01

The progenitor stars of several Type IIb supernovae (SNe) show indications of extended hydrogen envelopes. These envelopes might be the outcome of luminous energetic pre-explosion events, so-called precursor eruptions. We use the Palomar Transient Factory (PTF) pre-explosion observations of a sample of 27 nearby SNe IIb to look for such precursors during the final years prior to the SN explosion. No precursors are found when combining the observations in 15-day bins, and we calculate the absolute-magnitude-dependent upper limit on the precursor rate. At the 90% confidence level, SNe IIb have on average <0.86 precursors as bright as an absolute R-bandmore » magnitude of −14 in the final 3.5 years before the explosion and <0.56 events over the final year. In contrast, precursors among SNe IIn have a ≳5 times higher rate. The kinetic energy required to unbind a low-mass stellar envelope is comparable to the radiated energy of a few-weeks-long precursor that would be detectable for the closest SNe in our sample. Therefore, mass ejections, if they are common in such SNe, are radiatively inefficient or have durations longer than months. Indeed, when using 60-day bins, a faint precursor candidate is detected prior to SN 2012cs (∼2% false-alarm probability). We also report the detection of the progenitor of SN 2011dh that does not show detectable variability over the final two years before the explosion. The suggested progenitor of SN 2012P is still present, and hence is likely a compact star cluster or an unrelated object.« less

The Drosophila T-box transcription factor Midline functions within the Notch-Delta signaling pathway to specify sensory organ precursor cell fates and regulates cell survival within the eye imaginal disc.

PubMed

Das, Sudeshna; Chen, Q Brent; Saucier, Joseph D; Drescher, Brandon; Zong, Yan; Morgan, Sarah; Forstall, John; Meriwether, Andrew; Toranzo, Randy; Leal, Sandra M

2013-01-01

We report that the T-box transcription factor Midline (Mid), an evolutionary conserved homolog of the vertebrate Tbx20 protein, functions within the Notch-Delta signaling pathway essential for specifying the fates of sensory organ precursor (SOP) cells. These findings complement an established history of research showing that Mid regulates the cell-fate specification of diverse cell types within the developing heart, epidermis and central nervous system. Tbx20 has been detected in unique neuronal and epithelial cells of embryonic eye tissues in both mice and humans. However, the mechanisms by which either Mid or Tbx20 function to regulate cell-fate specification or other critical aspects of eye development including cell survival have not yet been elucidated. We have also gathered preliminary evidence suggesting that Mid may play an indirect, but vital role in selecting SOP cells within the third-instar larval eye disc by regulating the expression of the proneural gene atonal. During subsequent pupal stages, Mid specifies SOP cell fates as a member of the Notch-Delta signaling hierarchy and is essential for maintaining cell viability by inhibiting apoptotic pathways. We present several new hypotheses that seek to understand the role of Mid in regulating developmental processes downstream of the Notch receptor that are critical for specifying unique cell fates, patterning the adult eye and maintaining cellular homeostasis during eye disc morphogenesis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

40 CFR 80.1352 - What are the pre-compliance reporting requirements for the gasoline benzene program?

Code of Federal Regulations, 2010 CFR

2010-07-01

...) Benzene concentration. An estimate of the average gasoline benzene concentration corresponding to the time... engineering and permitting, Procurement and Construction, and Commissioning and startup. (7) Basic information regarding the selected technology pathway for compliance (e.g., precursor re-routing or other technologies...

Inhibitors targeting on cell wall biosynthesis pathway of MRSA.

PubMed

Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui

2012-11-01

Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA.

Precursor-Surface Reactions in Plasma Deposition of Silicon Thin Films

NASA Astrophysics Data System (ADS)

Bakos, Tamas

2005-03-01

Device-quality hydrogenated amorphous silicon (a-Si:H) thin films are usually grown by plasma deposition under conditions where the SiH3 radical is the dominant deposition precursor. In this presentation, we report results of first-principles density functional theory calculations on the interactions of the SiH3 radical with the crystalline Si(100)-(2x1):H surface in conjunction with molecular-dynamics simulations of a-Si:H thin film growth by SiH3 radicals, which elucidate the pathways and energetics of surface reactions that govern important film properties. In particular, we show that an SiH3 radical can insert into strained surface Si-Si dimer bonds, abstract surface H through an Eley-Rideal mechanism, and passivate surface dangling bonds; these reactions follow exothermic and barrierless pathways that lead to a temperature-independent growth rate in agreement with experimental measurements. We also identify a thermally activated surface H abstraction process, in which the SiH3 radical diffuses through overcoordinated surface Si atoms until it encounters a favorable site for H abstraction; the diffusion and H-abstraction steps have commensurate activation barriers. This mechanism explains partly the reduction of the film H content at elevated substrate temperatures.

Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system.

PubMed

Jang, Hui-Jeong; Yoon, Sang-Hwal; Ryu, Hee-Kyung; Kim, Jung-Hun; Wang, Chong-Long; Kim, Jae-Yean; Oh, Deok-Kun; Kim, Seon-Won

2011-07-29

Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay. Among the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which

40 CFR 766.38 - Reporting on precursor chemical substances.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Reporting on precursor chemical... SUBSTANCES CONTROL ACT DIBENZO-PARA-DIOXINS/DIBENZOFURANS Specific Chemical Testing/Reporting Requirements § 766.38 Reporting on precursor chemical substances. (a) Identification of precursor chemical substances...

40 CFR 766.38 - Reporting on precursor chemical substances.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Reporting on precursor chemical... SUBSTANCES CONTROL ACT DIBENZO-PARA-DIOXINS/DIBENZOFURANS Specific Chemical Testing/Reporting Requirements § 766.38 Reporting on precursor chemical substances. (a) Identification of precursor chemical substances...

40 CFR 766.38 - Reporting on precursor chemical substances.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Reporting on precursor chemical... SUBSTANCES CONTROL ACT DIBENZO-PARA-DIOXINS/DIBENZOFURANS Specific Chemical Testing/Reporting Requirements § 766.38 Reporting on precursor chemical substances. (a) Identification of precursor chemical substances...

40 CFR 766.38 - Reporting on precursor chemical substances.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Reporting on precursor chemical... SUBSTANCES CONTROL ACT DIBENZO-PARA-DIOXINS/DIBENZOFURANS Specific Chemical Testing/Reporting Requirements § 766.38 Reporting on precursor chemical substances. (a) Identification of precursor chemical substances...

40 CFR 766.38 - Reporting on precursor chemical substances.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Reporting on precursor chemical... SUBSTANCES CONTROL ACT DIBENZO-PARA-DIOXINS/DIBENZOFURANS Specific Chemical Testing/Reporting Requirements § 766.38 Reporting on precursor chemical substances. (a) Identification of precursor chemical substances...

Shape-programmed nanofabrication: understanding the reactivity of dichalcogenide precursors.

PubMed

Guo, Yijun; Alvarado, Samuel R; Barclay, Joshua D; Vela, Javier

2013-04-23

Dialkyl and diaryl dichalcogenides are highly versatile and modular precursors for the synthesis of colloidal chalcogenide nanocrystals. We have used a series of commercially available dichalcogenide precursors to unveil the molecular basis for the outcome of nanocrystal preparations, more specifically, how precursor molecular structure and reactivity affect the final shape and size of II-VI semiconductor nanocrystals. Dichalcogenide precursors used were diallyl, dibenzyl, di-tert-butyl, diisopropyl, diethyl, dimethyl, and diphenyl disulfides and diethyl, dimethyl, and diphenyl diselenides. We find that the presence of two distinctively reactive C-E and E-E bonds makes the chemistry of these precursors much richer and interesting than that of other conventional precursors such as the more common phosphine chalcogenides. Computational studies (DFT) reveal that the dissociation energy of carbon-chalcogen (C-E) bonds in dichalcogenide precursors (R-E-E-R, E=S or Se) increases in the order (R): diallylprecursor reactivity, leading to progressively slower nucleation and higher selectivity for anisotropic growth, all the way from dots to pods to tetrapods. Under identical experimental conditions, we obtain CdS and CdSe nanocrystals with spherical, elongated, or tetrapodal morphology by simply varying the identity and reactivity of the dichalcogenide precursor. Interestingly, we find that precursors with strong C-E and weak E-E bond dissociation energies such as Ph-S-S-Ph serve as a ready source of thiol radicals that appear to stabilize small CdE nuclei, facilitating anisotropic growth. These CdS and CdSe nanocrystals have been

New Polymeric Precursors of Silicon Carbide

NASA Technical Reports Server (NTRS)

Litt, M.; Kumar, K.

1987-01-01

Silicon carbide made by pyrolizing polymers. Method conceived for preparation of poly(decamethylcyclohexasilanes) as precursors for preparation of silicon carbide at high yield. Technical potential of polysilanes as precursors of SiC ceramics being explored. Potential limited by intractability of some polysilanes; formation of small, cyclic polycarbosilane fragments during pyrolysis; and overall low char yield and large shrinkage in conversion to ceramics.

Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

PubMed Central

Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; Hou, Guichuan; Zhang, Xuebin; Yang, Huijun; Feng, Huan; Miller, Lisa; Ralph, John; Liu, Chang-Jun

2016-01-01

Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications. PMID:27349324

Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N-methyltransferase

PubMed Central

McNeil, Scott D.; Nuccio, Michael L.; Ziemak, Michael J.; Hanson, Andrew D.

2001-01-01

Choline (Cho) is the precursor of the osmoprotectant glycine betaine and is itself an essential nutrient for humans. Metabolic engineering of Cho biosynthesis in plants could therefore enhance both their resistance to osmotic stresses (drought and salinity) and their nutritional value. The key enzyme of the plant Cho-synthesis pathway is phosphoethanolamine N-methyltransferase, which catalyzes all three of the methylations required to convert phosphoethanolamine to phosphocholine. We show here that overexpressing this enzyme in transgenic tobacco increased the levels of phosphocholine by 5-fold and free Cho by 50-fold without affecting phosphatidylcholine content or growth. Moreover, the expanded Cho pool led to a 30-fold increase in synthesis of glycine betaine via an engineered glycine betaine pathway. Supplying the transgenics with the Cho precursor ethanolamine (EA) further enhanced Cho levels even though the supplied EA was extensively catabolized. These latter results establish that there is further scope for improving Cho synthesis by engineering an increased endogenous supply of EA and suggest that this could be achieved by enhancing EA synthesis and/or by suppressing its degradation. PMID:11481443

Formation and specification of a Drosophila dopaminergic precursor cell.

PubMed

Watson, Joseph D; Crews, Stephen T

2012-09-01

Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb(+)/gsb-n(+) MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons.

Formation and specification of a Drosophila dopaminergic precursor cell

PubMed Central

Watson, Joseph D.; Crews, Stephen T.

2012-01-01

Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb+/gsb-n+ MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons. PMID:22874915

The Psen1-L166P-knock-in mutation leads to amyloid deposition in human wild-type amyloid precursor protein YAC transgenic mice

PubMed Central

Vidal, Ruben; Sammeta, Neeraja; Garringer, Holly J.; Sambamurti, Kumar; Miravalle, Leticia; Lamb, Bruce T.; Ghetti, Bernardino

2012-01-01

Genetically engineered mice have been generated to model cerebral β-amyloidosis, one of the hallmarks of Alzheimer disease (AD) pathology, based on the overexpression of a mutated cDNA of the amyloid-β precursor protein (AβPP) or by knock-in of the murine Aβpp gene alone or with presenilin1 mutations. Here we describe the generation and initial characterization of a new mouse line based on the presence of 2 copies of the human genomic region encoding the wild-type AβPP and the L166P presenilin 1 mutation. At ∼6 mo of age, double-mutant mice develop amyloid pathology, with signs of neuritic dystrophy, intracellular Aβ accumulation, and glial inflammation, an increase in AβPP C-terminal fragments, and an 8 times increase in Aβ42 levels with a 40% decrease in Aβ40 levels, leading to a significant increase (14 times) of Aβ42/Aβ40 ratios, with minimal effects on presenilin or the Notch1 pathway in the brain. We conclude that in mice, neither mutations in AβPP nor overexpression of an AβPP isoform are a prerequisite for Aβ pathology. This model will allow the study of AD pathogenesis and testing of therapeutic strategies in a more relevant environment without experimental artifacts due to the overexpression of a single-mutant AβPP isoform using exogenous promoters.—Vidal, R., Sammeta, N., Garringer, H. J., Sambamurti, K., Miravalle, L., Lamb B. T., Ghetti, B. The Psen1-L166P-knock-in mutation leads to amyloid deposition in human wild-type amyloid precursor protein YAC transgenic mice. PMID:22459153

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

PubMed

Body, A; Brownstein, B H

1978-01-01

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

PubMed Central

Body, Barbara A.; Brownstein, Bernard H.

1978-01-01

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction. PMID:412833

Elementary Mode Analysis: A Useful Metabolic Pathway Analysis Tool for Characterizing Cellular Metabolism

PubMed Central

Trinh, Cong T.; Wlaschin, Aaron; Srienc, Friedrich

2010-01-01

Elementary Mode Analysis is a useful Metabolic Pathway Analysis tool to identify the structure of a metabolic network that links the cellular phenotype to the corresponding genotype. The analysis can decompose the intricate metabolic network comprised of highly interconnected reactions into uniquely organized pathways. These pathways consisting of a minimal set of enzymes that can support steady state operation of cellular metabolism represent independent cellular physiological states. Such pathway definition provides a rigorous basis to systematically characterize cellular phenotypes, metabolic network regulation, robustness, and fragility that facilitate understanding of cell physiology and implementation of metabolic engineering strategies. This mini-review aims to overview the development and application of elementary mode analysis as a metabolic pathway analysis tool in studying cell physiology and as a basis of metabolic engineering. PMID:19015845

A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

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

Fan, J.; Xu, C.; Andre, C.

2011-06-23

Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to producemore » TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.« less

Instellar Exploration: Propulsion Options for Precursors and Beyond

NASA Technical Reports Server (NTRS)

Johnson, Charles Les; Leifer, Stephanie

1999-01-01

NASA is considering a mission to explore near-interstellar space early in the next decade as the first step toward a vigorous interstellar exploration program. A key enabling technology for such an ambitious science and exploration effort is the development of propulsion systems capable of providing fast trip times; mission duration should not exceed the professional lifetime of the investigative team. Advanced propulsion technologies that might support an interstellar precursor mission early in the next century include some combination of solar sails, nuclear electric propulsion systems, and aerogravity assists. Follow-on missions to far beyond the heliopause will require the development of propulsion technologies that are only at the conceptual stage today. These include 1) matter-antimatter annihilation, 2) beamed-energy sails, and 3) fusion systems. For years, the scientific community has been interested in the development of solar sail technology to support exploration of the inner and outer planets. Progress in thin-film technology and the development of technologies that may enable the remote assembly of large sails in space are only now maturing to the point where ambitious interstellar precursor missions can be considered. Electric propulsion is now being demonstrated for planetary exploration by the Deep Space 1 mission. The primary issues for it's adaptation to interstellar precursor applications include the nuclear reactor that would be required and the engine lifetime. For further term interstellar missions, matter-antimatter annihilation propulsion system concepts have the highest energy density of any propulsion systems using onboard propellants. However, there are numerous challenges to production and storage of antimatter that must be overcome before it can be seriously considered for interstellar flight. Off-board energy systems (laser sails) are candidates for long-distance interstellar flight but development of component technologies and

Engineering of carboligase activity reaction in Candida glabrata for acetoin production.

PubMed

Li, Shubo; Xu, Nan; Liu, Liming; Chen, Jian

2014-03-01

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction. Copyright © 2013. Published by Elsevier Inc.

A systematic review of methamphetamine precursor regulations.

PubMed

McKetin, Rebecca; Sutherland, Rachel; Bright, David A; Norberg, Melissa M

2011-11-01

To assess the effectiveness of methamphetamine precursor regulations in reducing illicit methamphetamine supply and use. A systematic review of 12 databases was used to identify studies that had evaluated the impact of methamphetamine precursor regulations on methamphetamine supply and/or use. The guidelines of the Effective Practice and Organization of Care Group (EPOC) of The Cochrane Collaboration were used to determine which study designs were included and assess their quality. Ten studies met the inclusion criteria. These studies evaluated 15 interventions (13 regulations and two related interdiction efforts), all of which were located in North America. Interventions had consistent impacts across various indicators of methamphetamine supply and use. Seven of the 15 interventions produced reductions in methamphetamine indicators (ranging from 12% to 77%). Two of the largest impacts were seen following interdiction efforts, involving the closure of rogue pharmaceutical companies. There was no evidence of a shift into other types of drug use, or injecting use, although the impact on the synthetic drug market was not examined. Null effects were related largely to the existence of alternative sources of precursor chemicals or the availability of imported methamphetamine. Methamphetamine precursor regulations can reduce indicators of methamphetamine supply and use. Further research is needed to determine whether regulations can be effective outside North America, particularly in developing countries, and what impact they have on the broader synthetic drug market. Improved data on precursor diversion are needed to facilitate the evaluation of precursor regulations. © 2011 The Authors, Addiction © 2011 Society for the Study of Addiction.

Intra-Engine Trace Species Chemistry

NASA Technical Reports Server (NTRS)

Waitz, Ian A.; Lukachko, S. P.; Chobot, A.; Miake-Lye, R. C.; Brown, R.

2002-01-01

Prompted by the needs of downstream plume-wake models, the Massachusetts Institute of Technology (MIT) and Aerodyne Research Incorporated (ART) initiated a collaborative effort, with funding from the NASA AEAP, to develop tools that would assist in understanding the fundamental drivers of chemical change within the intra-engine exhaust flow path. Efforts have been focused on the development of a modeling methodology that can adequately investigate the complex intra-engine environment. Over the history of this project, our research has increasingly pointed to the intra-engine environment as a possible site for important trace chemical activity. Modeling studies we initiated for the turbine and exhaust nozzle have contributed several important capabilities to the atmospheric effects of aviation assessment. These include a more complete understanding of aerosol precursor production, improved initial conditions for plume-wake modeling studies, and a more comprehensive analysis of ground-based test cell and in-flight exhaust measurement data. In addition, establishing a physical understanding of important flow and chemical processes through computational investigations may eventually assist in the design of engines to reduce undesirable species.

Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation.

PubMed

Su, Liqiu; Shen, Yanbing; Zhang, Wenkai; Gao, Tian; Shang, Zhihua; Wang, Min

2017-10-30

Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD + ) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD + -dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism. Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD + /NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD + /NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD + /NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D

3D Bioprinting of Developmentally Inspired Templates for Whole Bone Organ Engineering.

PubMed

Daly, Andrew C; Cunniffe, Gráinne M; Sathy, Binulal N; Jeon, Oju; Alsberg, Eben; Kelly, Daniel J

2016-09-01

The ability to print defined patterns of cells and extracellular-matrix components in three dimensions has enabled the engineering of simple biological tissues; however, bioprinting functional solid organs is beyond the capabilities of current biofabrication technologies. An alternative approach would be to bioprint the developmental precursor to an adult organ, using this engineered rudiment as a template for subsequent organogenesis in vivo. This study demonstrates that developmentally inspired hypertrophic cartilage templates can be engineered in vitro using stem cells within a supporting gamma-irradiated alginate bioink incorporating Arg-Gly-Asp adhesion peptides. Furthermore, these soft tissue templates can be reinforced with a network of printed polycaprolactone fibers, resulting in a ≈350 fold increase in construct compressive modulus providing the necessary stiffness to implant such immature cartilaginous rudiments into load bearing locations. As a proof-of-principal, multiple-tool biofabrication is used to engineer a mechanically reinforced cartilaginous template mimicking the geometry of a vertebral body, which in vivo supported the development of a vascularized bone organ containing trabecular-like endochondral bone with a supporting marrow structure. Such developmental engineering approaches could be applied to the biofabrication of other solid organs by bioprinting precursors that have the capacity to mature into their adult counterparts over time in vivo. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing

PubMed Central

Le Thomas, Adrien; Stuwe, Evelyn; Li, Sisi; Marinov, Georgi; Rozhkov, Nikolay; Chen, Yung-Chia Ariel; Luo, Yicheng; Sachidanandam, Ravi; Toth, Katalin Fejes; Patel, Dinshaw; Aravin, Alexei A.

2014-01-01

Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. PMID:25085419

The Interrelationships of Mathematical Precursors in Kindergarten

ERIC Educational Resources Information Center

Cirino, Paul T.

2011-01-01

This study evaluated the interrelations among cognitive precursors across quantitative, linguistic, and spatial attention domains that have been implicated for math achievement in young children. The dimensionality of the quantity precursors was evaluated in 286 kindergarteners via latent variable techniques, and the contribution of precursors…

The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells.

PubMed

Leithner, Katharina; Triebl, Alexander; Trötzmüller, Martin; Hinteregger, Barbara; Leko, Petra; Wieser, Beatrix I; Grasmann, Gabriele; Bertsch, Alexandra L; Züllig, Thomas; Stacher, Elvira; Valli, Alessandro; Prassl, Ruth; Olschewski, Andrea; Harris, Adrian L; Köfeler, Harald C; Olschewski, Horst; Hrzenjak, Andelko

2018-06-12

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M ( PCK2 ), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation. Copyright © 2018 the Author(s). Published by PNAS.

The Chemistry of Inorganic Precursors during the Chemical Deposition of Films on Solid Surfaces.

PubMed

Barry, Seán T; Teplyakov, Andrew V; Zaera, Francisco

2018-03-20

The deposition of thin solid films is central to many industrial applications, and chemical vapor deposition (CVD) methods are particularly useful for this task. For one, the isotropic nature of the adsorption of chemical species affords even coverages on surfaces with rough topographies, an increasingly common requirement in microelectronics. Furthermore, by splitting the overall film-depositing reactions into two or more complementary and self-limiting steps, as it is done in atomic layer depositions (ALD), film thicknesses can be controlled down to the sub-monolayer level. Thanks to the availability of a vast array of inorganic and metalorganic precursors, CVD and ALD are quite versatile and can be engineered to deposit virtually any type of solid material. On the negative side, the surface chemistry that takes place in these processes is often complex, and can include undesirable side reactions leading to the incorporation of impurities in the growing films. Appropriate precursors and deposition conditions need to be chosen to minimize these problems, and that requires a proper understanding of the underlying surface chemistry. The precursors for CVD and ALD are often designed and chosen based on their known thermal chemistry from inorganic chemistry studies, taking advantage of the vast knowledge developed in that field over the years. Although a good first approximation, however, this approach can lead to wrong choices, because the reactions of these precursors at gas-solid interfaces can be quite different from what is seen in solution. For one, solvents often aid in the displacement of ligands in metalorganic compounds, providing the right dielectric environment, temporarily coordinating to the metal, or facilitating multiple ligand-complex interactions to increase reaction probabilities; these options are not available in the gas-solid reactions associated with CVD and ALD. Moreover, solid surfaces act as unique "ligands", if these reactions are to be

Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene

Treesearch

Yi Lasanajak; Rakesh Minocha; Subhash C. Minocha; Ravinder Goyal; Tahira Fatima; Avtar K. Handa; Autar K. Mattoo

2014-01-01

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in...

Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells

PubMed Central

Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

2016-01-01

Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event

High-Solids Polyimide Precursor Solutions

NASA Technical Reports Server (NTRS)

Chuang, Chun-Hua (Inventor)

2004-01-01

The invention is a highly concentrated stable solution of polymide precursors (monometers) having a solids content ranging from about 80 to 98 percent by weight in lower aliphatic alcohols i.e. methyl and/or ethylalcohol. the concentrated polyimide precursos solution comparisons effective amounts of at least one aromatic diamine, at least one aromatic dianhydride, and a monofunctional endcap including monoamines, monoanhydrides and lower alkyl esters of said monoanhydrides. These concentrated polyimide precursor solutions are particularly useful for the preparation of fibrous prepregs and composites for use in structural materials for military and civil applications.

Metabolite damage and repair in metabolic engineering design

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

Sun, Jiayi; Jeffryes, James G.; Henry, Christopher S.

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields,more » and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.« less

Triggering signaling pathways using F-actin self-organization.

PubMed

Colin, A; Bonnemay, L; Gayrard, C; Gautier, J; Gueroui, Z

2016-10-04

The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity.