Computational enzyme design: transitioning from catalytic proteins to enzymes.

PubMed

Mak, Wai Shun; Siegel, Justin B

2014-08-01

The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward. Published by Elsevier Ltd.

Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

PubMed

Yang, Mingfeng; Li, Xuefeng; Bu, Chunya; Wang, Hui; Shi, Guanglu; Yang, Xiushan; Hu, Yong; Wang, Xiaoqin

2014-11-01

Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

Computer-generated Model of Purine Nucleoside Phosphorylase (PNP)

NASA Technical Reports Server (NTRS)

1987-01-01

Purine Nucleoside Phosphorylase (PNP) is an important target enzyme for the design of anti-cancer and immunosuppressive drugs. Bacterial PNP, which is slightly different from the human enzyme, is used to synthesize chemotherapuautic agents. Knowledge of the three-dimensional structure of the bacterial PNP molecule is useful in efforts to engineer different types of PNP enzymes, that can be used to produce new chemotherapeutic agents. This picture shows a computer model of bacterial PNP, which looks a lot like a display of colorful ribbons. Principal Investigator was Charles Bugg.

Production of extremophilic bacterial cellulase enzymes in aspergillus niger.

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

Gladden, John Michael

2013-09-01

Enzymes can be used to catalyze a myriad of chemical reactions and are a cornerstone in the biotechnology industry. Enzymes have a wide range of uses, ranging from medicine with the production of pharmaceuticals to energy were they are applied to biofuel production. However, it is difficult to produce large quantities of enzymes, especially if they are non-native to the production host. Fortunately, filamentous fungi, such as Aspergillus niger, are broadly used in industry and show great potential for use a heterologous enzyme production hosts. Here, we present work outlining an effort to engineer A. niger to produce thermophilic bacterialmore » cellulases relevant to lignocellulosic biofuel production.« less

Virus scaffolds as enzyme nano-carriers.

PubMed

Cardinale, Daniela; Carette, Noëlle; Michon, Thierry

2012-07-01

The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.

PubMed

Whitaker, W Brian; Jones, J Andrew; Bennett, R Kyle; Gonzalez, Jacqueline E; Vernacchio, Victoria R; Collins, Shannon M; Palmer, Michael A; Schmidt, Samuel; Antoniewicz, Maciek R; Koffas, Mattheos A; Papoutsakis, Eleftherios T

2017-01-01

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13 C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Application of dextranases in sugarcane factory: overcoming practical problems

USDA-ARS?s Scientific Manuscript database

Dextranases only have a small market and low volume sales compared to many other industrial enzymes. Consequently, research and development efforts to engineer tailormade properties of destranases to specific conditions of industrial processes have not occurred and are not expected soon. This book...

Engineering Lipases: walking the fine line between activity and stability

NASA Astrophysics Data System (ADS)

Dasetty, Siva; Blenner, Mark A.; Sarupria, Sapna

2017-11-01

Lipases are enzymes that hydrolyze lipids and have several industrial applications. There is a tremendous effort in engineering the activity, specificity and stability of lipases to render them functional in a variety of environmental conditions. In this review, we discuss the recent experimental and simulation studies focused on engineering lipases. Experimentally, mutagenesis studies have demonstrated that the activity, stability, and specificity of lipases can be modulated by mutations. It has been particularly challenging however, to elucidate the underlying mechanisms through which these mutations affect the lipase properties. We summarize results from experiments and molecular simulations highlighting the emerging picture to this end. We end the review with suggestions for future research which underscores the delicate balance of various facets in the lipase that affect their activity and stability necessitating the consideration of the enzyme as a network of interactions.

Cell-free metabolic engineering: Biomanufacturing beyond the cell

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

Dudley, QM; Karim, AS; Jewett, MC

2014-10-15

Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedentedmore » level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L-1 h(-1), reaction scales of >100 L, and new directions in protein purification, spatial organization, and enzyme stability. In the coming years, CFME will offer exciting opportunities to: (i) debug and optimize biosynthetic pathways; (ii) carry out design-build-test iterations without re-engineering organisms; and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility.« less

Cell-Free Metabolic Engineering: Biomanufacturing beyond the cell

PubMed Central

Dudley, Quentin M.; Karim, Ashty S.; Jewett, Michael C.

2014-01-01

Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L−1 hr−1, reaction scales of >100L, and new directions in protein purification, spatial organization and enzyme stability. In the coming years, CFME will offer exciting opportunities to (i) debug and optimize biosynthetic pathways, (ii) carry out design-build-test iterations without re-engineering organisms, and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility. PMID:25319678

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

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli

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

Whitaker, W. Brian; Jones, J. Andrew; Bennett, R. Kyle

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolyticmore » intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.« less

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli

DOE PAGES

Whitaker, W. Brian; Jones, J. Andrew; Bennett, R. Kyle; ...

2016-11-01

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolyticmore » intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.« less

Structural and biophysical characterization of the α-carbonic anhydrase from the gammaproteobacterium Thiomicrospira crunogena XCL-2: insights into engineering thermostable enzymes for CO2 sequestration.

PubMed

Díaz-Torres, Natalia A; Mahon, Brian P; Boone, Christopher D; Pinard, Melissa A; Tu, Chingkuang; Ng, Robert; Agbandje-McKenna, Mavis; Silverman, David; Scott, Kathleen; McKenna, Robert

2015-08-01

Biocatalytic CO2 sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an α-CA recently discovered in the mesophilic hydrothermal vent-isolate extremophile Thiomicrospira crunogena XCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9°C versus 59.5°C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other α-CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer α-CAs for applications in industrial CO2 sequestration.

Designing industrial yeasts for the consolidated bioprocessing of starchy biomass to ethanol

PubMed Central

Favaro, Lorenzo; Jooste, Tania; Basaglia, Marina; Rose, Shaunita H.; Saayman, Maryna; Görgens, Johann F.; Casella, Sergio; van Zyl, Willem H.

2013-01-01

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a one step process, is a promising strategy for the effective ethanol production from cheap lignocellulosic and starchy materials. CBP requires a highly engineered microbial strain able to both hydrolyze biomass with enzymes produced on its own and convert the resulting simple sugars into high-titer ethanol. Recently, heterologous production of cellulose and starch-degrading enzymes has been achieved in yeast hosts, which has realized direct processing of biomass to ethanol. However, essentially all efforts aimed at the efficient heterologous expression of saccharolytic enzymes in yeast have involved laboratory strains and much of this work has to be transferred to industrial yeasts that provide the fermentation capacity and robustness desired for large scale bioethanol production. Specifically, the development of an industrial CBP amylolytic yeast would allow the one-step processing of low-cost starchy substrates into ethanol. This article gives insight in the current knowledge and achievements on bioethanol production from starchy materials with industrial engineered S. cerevisiae strains. PMID:22989992

Thermostable microbial xylanases for pulp and paper industries: trends, applications and further perspectives.

PubMed

Kumar, Vishal; Marín-Navarro, Julia; Shukla, Pratyoosh

2016-02-01

Xylanases are enzymes with biotechnological relevance in a number of fields, including food, feed, biofuel, and textile industries. Their most significant application is in the paper and pulp industry, where they are used as a biobleaching agent, showing clear economic and environmental advantages over chemical alternatives. Since this process requires high temperatures and alkali media, the identification of thermostable and alkali stable xylanases represents a major biotechnological goal in this field. Moreover, thermostability is a desirable property for many other applications of xylanases. The review makes an overview of xylanase producing microorganisms and their current implementation in paper biobleaching. Future perspectives are analyzed focusing in the efforts carried out to generate thermostable enzymes by means of modern biotechnological tools, including metagenomic analysis, enzyme molecular engineering and nanotechnology. Furthermore, structural and mutagenesis studies have revealed critical sites for stability of xylanases from glycoside hydrolase families GH10 and GH11, which constitute the main classes of these enzymes. The overall conclusions of these works are summarized here and provide relevant information about putative weak spots within xylanase structures to be targeted in future protein engineering approaches.

Engineering strategies for the fermentative production of plant alkaloids in yeast.

PubMed

Trenchard, Isis J; Smolke, Christina D

2015-07-01

Microbial hosts engineered for the biosynthesis of plant natural products offer enormous potential as powerful discovery and production platforms. However, the reconstruction of these complex biosynthetic schemes faces numerous challenges due to the number of enzymatic steps and challenging enzyme classes associated with these pathways, which can lead to issues in metabolic load, pathway specificity, and maintaining flux to desired products. Cytochrome P450 enzymes are prevalent in plant specialized metabolism and are particularly difficult to express heterologously. Here, we describe the reconstruction of the sanguinarine branch of the benzylisoquinoline alkaloid pathway in Saccharomyces cerevisiae, resulting in microbial biosynthesis of protoberberine, protopine, and benzophenanthridine alkaloids through to the end-product sanguinarine, which we demonstrate can be efficiently produced in yeast in the absence of the associated biosynthetic enzyme. We achieved titers of 676 μg/L stylopine, 548 μg/L cis-N-methylstylopine, 252 μg/L protopine, and 80 μg/L sanguinarine from the engineered yeast strains. Through our optimization efforts, we describe genetic and culture strategies supporting the functional expression of multiple plant cytochrome P450 enzymes in the context of a large multi-step pathway. Our results also provided insight into relationships between cytochrome P450 activity and yeast ER physiology. We were able to improve the production of critical intermediates by 32-fold through genetic techniques and an additional 45-fold through culture optimization. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Design, selection, and characterization of a split chorismate mutase

PubMed Central

Müller, Manuel M; Kries, Hajo; Csuhai, Eva; Kast, Peter; Hilvert, Donald

2010-01-01

Split proteins are versatile tools for detecting protein–protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein–protein interactions. PMID:20306491

Over production of lignocellulosic enzymes of Coriolus versicolor by genetic engineering methodology. Final report

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

Williams, A.L.

1998-07-01

The project seeks to understand the biological and chemical processes involved in the secretion of the enzyme polyphenol oxidase (PPO) by the hyphae, the basic unit of the filamentous fungus Coriolus versicolor. These studies are made to determine rational strategies for enhanced secretion of PPO, both with the use of recombinant DNA techniques and without. This effort focuses on recombinant DNA techniques to enhance enzyme production. The major thrust of this project was two-fold: to mass produce C. versicolor tyrosinase (polyphenol oxidase) by genetic engineering as well as cultural manipulations; and to utilize PPO as a biocatalyst in the processingmore » of lignocellulose as a renewable energy resource. In this study, the assessment of genomic and cDNA recombinant clones with regards to the overproduction of PPO continued. Further, immunocytochemical techniques were employed to assess the mechanism(s) involved in the secretion of PPO by the hyphae. Also, factors influencing PPO secretion were examined.« less

Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering.

PubMed

Der, Bryan S; Edwards, David R; Kuhlman, Brian

2012-05-08

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Engineering strategies for the fermentative production of plant alkaloids in yeast

PubMed Central

Trenchard, Isis J.; Smolke, Christina D.

2015-01-01

Microbial hosts engineered for the biosynthesis of plant natural products offer enormous potential as powerful discovery and production platforms. However, the reconstruction of these complex biosynthetic schemes faces numerous challenges due to the number of enzymatic steps and challenging enzyme classes associated with these pathways, which can lead to issues in metabolic load, pathway specificity, and maintaining flux to desired products. Cytochrome P450 enzymes are prevalent in plant specialized metabolism and are particularly difficult to express heterologously. Here, we describe the reconstruction of the sanguinarine branch of the benzylisoquinoline alkaloid pathway in Saccharomyces cerevisiae, resulting in microbial biosynthesis of protoberberine, protopine, and benzophenanthridine alkaloids through to the end-product sanguinarine, which we demonstrate can be efficiently produced in yeast in the absence of the associated biosynthetic enzyme. We achieved titers of 676 µg/L stylopine, 548 µg/L cis-N-methylstylopine, 252 µg/L protopine, and 80 µg/L sanguinarine from the engineered yeast strains. Through our optimization efforts, we describe genetic and culture strategies supporting the functional expression of multiple plant cytochrome P450 enzymes in the context of a large multi-step pathway. Our results also provided insight into relationships between cytochrome P450 activity and yeast ER physiology. We were able to improve the production of critical intermediates by 32-fold through genetic techniques and an additional 45-fold through culture optimization. PMID:25981946

Natural and engineered polyhydroxyalkanoate (PHA) synthase: key enzyme in biopolyester production.

PubMed

Zou, Huibin; Shi, Mengxun; Zhang, Tongtong; Li, Lei; Li, Liangzhi; Xian, Mo

2017-10-01

With the finite supply of petroleum and increasing concern with environmental issues associated with their harvest and processing, the development of more eco-friendly, sustainable alternative biopolymers that can effectively fill the role of petro-polymers has become a major focus. Polyhydroxyalkanoate (PHA) can be naturally produced by many species of bacteria and the PHA synthase is believed to be key enzyme in this natural pathway. Natural PHA synthases are diverse and can affect the properties of the produced PHAs, such as monomer composition, molecular weights, and material properties. Moreover, recent studies have led to major advances in the searching of PHA synthases that display specific properties, as well as engineering efforts that offer more efficient PHA synthases, increased PHA compound production, or even novel biopolyesters which cannot be naturally produced. In this article, we review the updated information of natural PHA synthases and their engineering strategies for improved performance in polyester production. We also speculate future trends on the development of robust PHA synthases and their application in biopolyester production.

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

None

This is a coordinated program to effect the microbiological degradation of cellulosic biomasses and will focus on the use of anaerobic microorganisms which possess cellulolytic enzyme. The studies will attempt to increase the enzyme levels through genetics, mutation and strain selection. In addition, the direct conversion from cellulosic biomasses to liquid fuel (ethanol) and/or soluble sugars by the cellulolytic, anaerobic organism is also within the scope of this program. Process and engineering scale-up, along with economic analyses, will be performed throughout the course of the program. The second area of our major effort is devoted to the production of chemicalmore » feedstocks. In particular, three fermentations have been identified for exploration. These are: acrylic acid, acetone/butanol and acetic acid. The main efforts in these fermentations will address means for the reduction of the cost of manufacturing for these large volume chemicals.« less

Generation and Functional Evaluation of Designer Monoterpene Synthases.

PubMed

Srividya, N; Lange, I; Lange, B M

2016-01-01

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes. © 2016 Elsevier Inc. All rights reserved.

PanDaTox: A tool for accelerated metabolic engineering

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

Amitai, Gil; Sorek, Rotem

2012-07-18

Metabolic engineering is often facilitated by cloning of genes encoding enzymes from various heterologous organisms into E. coli. Such engineering efforts are frequently hampered by foreign genes that are toxic to the E. coli host. We have developed PanDaTox (www.weizmann.ac.il/pandatox), a web-based resource that provides experimental toxicity information for more than 1.5 million genes from hundreds of different microbial genomes. The toxicity predictions, which were extensively experimentally verified, are based on serial cloning of genes into E. coli as part of the Sanger whole genome shotgun sequencing process. PanDaTox can accelerate metabolic engineering projects by allowing researchers to exclude toxicmore » genes from the engineering plan and verify the clonability of selected genes before the actual metabolic engineering experiments are conducted.« less

Fatty acid biosynthesis revisited: Structure elucidation and metabolic engineering

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

Beld, Joris; Lee, D. John; Burkart, Michael D.

Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understandingmore » of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. Lastly, in this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.« less

Fatty acid biosynthesis revisited: Structure elucidation and metabolic engineering

DOE PAGES

Beld, Joris; Lee, D. John; Burkart, Michael D.

2014-10-20

Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understandingmore » of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. Lastly, in this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.« less

Opportunities for Merging Chemical and Biological Synthesis

PubMed Central

Wallace, Stephen; Balskus, Emily P.

2014-01-01

Organic chemists and metabolic engineers use largely orthogonal technologies to access small molecules like pharmaceuticals and commodity chemicals. As the use of biological catalysts and engineered organisms for chemical production grows, it is becoming increasingly evident that future efforts for chemical manufacture will benefit from the integration and unified expansion of these two fields. This review will discuss approaches that combine chemical and biological synthesis for small molecule production. We highlight recent advances in combining enzymatic and non-enzymatic catalysis in vitro, discuss the application of design principles from organic chemistry for engineering non-biological reactivity into enzymes, and describe the development of biocompatible chemistry that can be interfaced with microbial metabolism. PMID:24747284

Expanding the Scope of Site-Specific Recombinases for Genetic and Metabolic Engineering

PubMed Central

Gaj, Thomas; Sirk, Shannon J.; Barbas, Carlos F.

2014-01-01

Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study. PMID:23982993

Reversed enantioselectivity of diisopropyl fluorophosphatase against organophosphorus nerve agents by rational design.

PubMed

Melzer, Marco; Chen, Julian C-H; Heidenreich, Anne; Gäb, Jürgen; Koller, Marianne; Kehe, Kai; Blum, Marc-Michael

2009-12-02

Diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall enhanced activity against tested nerve agents. The reversed stereochemical preference is explained through modeling studies and the crystal structures of the two mutants. Using the engineered mutants in combination with wild-type DFPase leads to significantly enhanced activity and detoxification, which is especially important for personal decontamination. Our findings may also be of relevance for the structurally related enzyme human paraoxonase (PON), which is of considerable interest as a potential catalytic in vivo scavenger in case of organophosphorus poisoning.

The challenges of sequencing by synthesis.

PubMed

Fuller, Carl W; Middendorf, Lyle R; Benner, Steven A; Church, George M; Harris, Timothy; Huang, Xiaohua; Jovanovich, Stevan B; Nelson, John R; Schloss, Jeffery A; Schwartz, David C; Vezenov, Dmitri V

2009-11-01

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to $100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.

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.

Polypyrrole membranes as scaffolds for biomolecule immobilization

NASA Astrophysics Data System (ADS)

Hery, Travis M.; Satagopan, Sriram; Northcutt, Robert G.; Tabita, F. Robert; Sundaresan, Vishnu-Baba

2016-12-01

Enzymes have evolved over hundreds of years through changes in ecosystems (climate, atmosphere, hydrology, etc). The evolutionary changes driven by the need to survive has led to enzymes with diverse functionality such as reduction of carbon dioxide and methane to other forms of carbon, fixation of nitrogen, and high temperature biochemical processes. While these enzymes have useful properties, engineering a scalable cell-free system with these enzymes will be useful for stable production of desired products without involving the vagaries of cellular metabolism. This article presents various approaches to incorporate ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a conducting polymer (polypyrrole (PPy)) doped with a bulky anion (dodecylbenzenesulfonate (DBS)) in an effort to create functional devices for the conversion of carbon dioxide into precursors for high-value chemicals. We demonstrate that the tailored device creates an environment where the enzyme can retain its function while being protected from denaturing conditions. It is envisioned that the 3-PGA produced by RuBisCO will be converted into value-added products.

Computational characterization of DNA/peptide/nanotube self assembly for bioenergy applications

NASA Astrophysics Data System (ADS)

Ortiz, Vanessa; Araki, Ruriko; Collier, Galen

2012-02-01

Multi-enzyme pathways have become a subject of increasing interest for their role in the engineering of biomimetic systems for applications including biosensors, bioelectronics, and bioenergy. The efficiencies found in natural metabolic pathways partially arise from biomolecular self-assembly of the component enzymes in an effort to avoid transport limitations. The ultimate goal of this effort is to design and build biofuel cells with efficiencies similar to those of native systems by introducing biomimetic structures that immobilize multiple enzymes in specific orientations on a bioelectrode. To achieve site-specific immobilization, the specificity of DNA-binding domains is exploited with an approach that allows any redox enzyme to be modified to site-specifically bind to double stranded (ds) DNA while retaining activity. Because of its many desirable properties, the bioelectrode of choice is single-wall carbon nanotubes (SWNTs), but little is known about dsDNA/SWNT assembly and how this might affect the activity of the DNA-binding domains. Here we evaluate the feasibility of the proposed assembly by performing atomistic molecular dynamics simulations to look at the stability and conformations adopted by dsDNA when bound to a SWNT. We also evaluate the effects of the presence of a SWNT on the stability of the complex formed by a DNA-binding domain and DNA.

Expanding the scope of site-specific recombinases for genetic and metabolic engineering.

PubMed

Gaj, Thomas; Sirk, Shannon J; Barbas, Carlos F

2014-01-01

Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study. © 2013 Wiley Periodicals, Inc.

Strategies for the production of cell wall-deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

DOE PAGES

Park, Sang -Hyuck; Ong, Rebecca Garlock; Sticklen, Mariam

2015-12-02

Microbial cell wall-deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall-deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall-deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yieldsmore » and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue-specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. As a result, we also summarize methods that have been used to access and maintain activity of plant-generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.« less

Strategies for the production of cell wall-deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

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

Park, Sang -Hyuck; Ong, Rebecca Garlock; Sticklen, Mariam

Microbial cell wall-deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall-deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall-deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yieldsmore » and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue-specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. As a result, we also summarize methods that have been used to access and maintain activity of plant-generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.« less

Computational protein design and protein-ligand interaction studies for the improvement of organophosphorus degrading potential of Deinococcus radiodurans.

PubMed

Manoharan, Prabu; Sridhar, J

2018-05-01

The organophosphorus hydrolase enzyme is involved in the catalyzing reaction that involve hydrolysis of organophosphate toxic compounds. An enzyme from Deinococcus radiodurans reported as homologous to phosphotriesterase and show activity against organophosphate. In the past activity of this enzyme is low and efforts made to improve the activity by experimental mutation study. However only very few organophosphates tested against very few catalytic site mutations. In order to improve the catalytic power of the organophosphorus hydrolase enzyme, we carried out systematic functional hotspot based protein engineering strategy. The mutants tested against 46 know organophosphate compounds using molecular docking study. Finally, we carried out an extensive molecular docking study to predict the binding of 46 organophosphate compounds to wild-type protein and mutant organophosphorus hydrolase enzyme. At the end we are able to improve the degrading potential of organophosphorus hydrolase enzyme against organophosphate toxic compounds. This preliminary study and the outcome would be useful guide for the experimental scientist involved in the bioremediation of toxic organophosphate compounds. Copyright © 2018 Elsevier Inc. All rights reserved.

Microbial Engineering for Aldehyde Synthesis

PubMed Central

Kunjapur, Aditya M.

2015-01-01

Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity. PMID:25576610

Molecular engineering of industrial enzymes: recent advances and future prospects.

PubMed

Yang, Haiquan; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Liu, Long; Chen, Jian

2014-01-01

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.

The construction, fouling and enzymatic cleaning of a textile dye surface.

PubMed

Onaizi, Sagheer A; He, Lizhong; Middelberg, Anton P J

2010-11-01

The enzymatic cleaning of a rubisco protein stain bound onto Surface Plasmon Resonance (SPR) biosensor chips having a dye-bound upper layer is investigated. This novel method allowed, for the first time, a detailed kinetic study of rubisco cleanability (defined as fraction of adsorbed protein removed from a surface) from dyed surfaces (mimicking fabrics) at different enzyme concentrations. Analysis of kinetic data using an established mathematical model able to decouple enzyme transfer and reaction processes [Onaizi, He, Middelberg, Chem. Eng. Sci. 64 (2008) 3868] revealed a striking effect of dyeing on enzymatic cleaning performance. Specifically, the absolute rate constants for enzyme transfer to and from a dye-bound rubisco stain were significantly higher than reported previously for un-dyed surfaces. These increased transfer rates resulted in higher surface cleanability. Higher enzyme mobility (i.e., higher enzyme adsorption and desorption rates) at the liquid-dye interface was observed, consistent with previous suggestions that enzyme surface mobility is likely correlated with overall enzyme cleaning performance. Our results show that reaction engineering models of enzymatic action at surfaces may provide insight able to guide the design of better stain-resistant surfaces, and may also guide efforts to improve cleaning formulations. Copyright 2010 Elsevier Inc. All rights reserved.

Biogenesis and Metabolic Maintenance of Rubisco.

PubMed

Bracher, Andreas; Whitney, Spencer M; Hartl, F Ulrich; Hayer-Hartl, Manajit

2017-04-28

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO 2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Rubisco is a remarkably inefficient enzyme, fixing only 2-10 CO 2 molecules per second. Efforts to increase crop yields by bioengineering Rubisco remain unsuccessful, owing in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. The large subunit of Rubisco requires the chaperonin system for folding, and recent studies have shown that assembly of hexadecameric Rubisco is mediated by specific assembly chaperones. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands, including RuBP. Metabolic repair depends on remodeling of Rubisco by the ATP-dependent Rubisco activase and hydrolysis of inhibitory sugar phosphates by specific phosphatases. Here, we review our present understanding of the structure and function of these auxiliary factors and their utilization in efforts to engineer more catalytically efficient Rubisco enzymes.

Heparin and related polysaccharides: Synthesis using recombinant enzymes and metabolic engineering

PubMed Central

Suflita, Matthew; Fu, Li; He, Wenqin; Koffas, Mattheos; Linhardt, Robert J.

2015-01-01

Glycosaminoglycans are linear anionic polysaccharides that exhibit a number of important biological and pharmacological activities. The two most prominent members of this class of polysaccharides are heparin/heparan sulfate and the chondroitin sulfates (including dermatan sulfate). These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of most animal cells. The chemical synthesis of these glycosaminoglycans is precluded by their structural complexity. Today, we depend on food animal tissues for their isolation and commercial production. Ton quantities of these glycosaminoglycans are used annually as pharmaceuticals and nutraceuticals. The variability of animal-sourced glycosaminoglycans, their inherent impurities, the limited availability of source tissues, the poor control of these source materials, and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans. This mini-review focuses on the use of recombinant enzymes and metabolic engineering for the production of heparin and chondroitin sulfates. PMID:26219501

Computational methods in metabolic engineering for strain design.

PubMed

Long, Matthew R; Ong, Wai Kit; Reed, Jennifer L

2015-08-01

Metabolic engineering uses genetic approaches to control microbial metabolism to produce desired compounds. Computational tools can identify new biological routes to chemicals and the changes needed in host metabolism to improve chemical production. Recent computational efforts have focused on exploring what compounds can be made biologically using native, heterologous, and/or enzymes with broad specificity. Additionally, computational methods have been developed to suggest different types of genetic modifications (e.g. gene deletion/addition or up/down regulation), as well as suggest strategies meeting different criteria (e.g. high yield, high productivity, or substrate co-utilization). Strategies to improve the runtime performances have also been developed, which allow for more complex metabolic engineering strategies to be identified. Future incorporation of kinetic considerations will further improve strain design algorithms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biocatalysts: application and engineering for industrial purposes.

PubMed

Jemli, Sonia; Ayadi-Zouari, Dorra; Hlima, Hajer Ben; Bejar, Samir

2016-01-01

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

NREL Explains the Higher Cellulolytic Activity of a Vital Microorganism

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

The discovery of a new mode of action by C. thermocellum to convert biomass to biofuels is significant because the bacterium is already recognized as one of the most effective in the biosphere. Researchers found that, in addition to using common cellulase degradation mechanisms attached to cells, C. thermocellum also uses a new category of cell-free scaffolded enzymes. The new discovery will influence the strategies used to improve the cellulolytic activity of biomass degrading microbes going forward. Better understanding of this bacterium could lead to cheaper production of ethanol and drop-in fuels. Also, this discovery demonstrates that nature's biomass conversionmore » behaviors are not fully understood and remain as opportunities for future microbial/enzyme engineering efforts.« less

76 FR 8707 - Syngenta Seeds, Inc.; Determination of Nonregulated Status for Corn Genetically Engineered To...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-15

... for Corn Genetically Engineered To Produce an Enzyme That Facilitates Ethanol Production AGENCY... event 3272, which has been genetically engineered to produce a microbial enzyme that facilitates ethanol... transformation event 3272, which has been genetically engineered to produce a microbial enzyme that facilitates...

Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

PubMed

Hadd, Andrew; Perona, John J

2014-12-19

We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

Natural and engineered biosynthesis of fluorinated natural products.

PubMed

Walker, Mark C; Chang, Michelle C Y

2014-09-21

Both natural products and synthetic organofluorines play important roles in the discovery and design of pharmaceuticals. The combination of these two classes of molecules has the potential to be useful in the ongoing search for new bioactive compounds but our ability to produce site-selectively fluorinated natural products remains limited by challenges in compatibility between their high structural complexity and current methods for fluorination. Living systems provide an alternative route to chemical fluorination and could enable the production of organofluorine natural products through synthetic biology approaches. While the identification of biogenic organofluorines has been limited, the study of the native organisms and enzymes that utilize these compounds can help to guide efforts to engineer the incorporation of this unusual element into complex pharmacologically active natural products. This review covers recent advances in understanding both natural and engineered production of organofluorine natural products.

Nanoscale Engineering of Designer Cellulosomes.

PubMed

Gunnoo, Melissabye; Cazade, Pierre-André; Galera-Prat, Albert; Nash, Michael A; Czjzek, Mirjam; Cieplak, Marek; Alvarez, Beatriz; Aguilar, Marina; Karpol, Alon; Gaub, Hermann; Carrión-Vázquez, Mariano; Bayer, Edward A; Thompson, Damien

2016-07-01

Biocatalysts showcase the upper limit obtainable for high-speed molecular processing and transformation. Efforts to engineer functionality in synthetic nanostructured materials are guided by the increasing knowledge of evolving architectures, which enable controlled molecular motion and precise molecular recognition. The cellulosome is a biological nanomachine, which, as a fundamental component of the plant-digestion machinery from bacterial cells, has a key potential role in the successful development of environmentally-friendly processes to produce biofuels and fine chemicals from the breakdown of biomass waste. Here, the progress toward so-called "designer cellulosomes", which provide an elegant alternative to enzyme cocktails for lignocellulose breakdown, is reviewed. Particular attention is paid to rational design via computational modeling coupled with nanoscale characterization and engineering tools. Remaining challenges and potential routes to industrial application are put forward. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Recent advances in rational approaches for enzyme engineering

PubMed Central

Steiner, Kerstin; Schwab, Helmut

2012-01-01

Enzymes are an attractive alternative in the asymmetric syntheses of chiral building blocks. To meet the requirements of industrial biotechnology and to introduce new functionalities, the enzymes need to be optimized by protein engineering. This article specifically reviews rational approaches for enzyme engineering and de novo enzyme design involving structure-based approaches developed in recent years for improvement of the enzymes’ performance, broadened substrate range, and creation of novel functionalities to obtain products with high added value for industrial applications. PMID:24688651

Engineering Novel and Improved Biocatalysts by Cell Surface Display

PubMed Central

Smith, Mason R.; Khera, Eshita; Wen, Fei

2017-01-01

Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts. PMID:29056821

Structure-function mapping of key determinants for hydrocarbon biosynthesis by squalene and squalene synthase-like enzymes from the green alga Botryococcus braunii race B.

PubMed

Bell, Stephen A; Niehaus, Thomas D; Nybo, S Eric; Chappell, Joseph

2014-12-09

Squalene and botryococcene are branched-chain, triterpene compounds that arise from the head-to-head condensation of two molecules of farnesyl diphosphate to yield 1'-1 and 1'-3 linkages, respectively. The enzymes that catalyze their formation have attracted considerable interest from the medical field as potential drug targets and the renewable energy sector for metabolic engineering efforts. Recently, the enzymes responsible for botryococcene and squalene biosynthesis in the green alga Botryococcus braunii race B were characterized. To better understand how the specificity for the 1'-1 and 1'-3 linkages was controlled, we attempted to identify the functional residues and/or domains responsible for this step in the catalytic cascade. Existing crystal structures for the mammalian squalene synthase and Staphylococcus dehydrosqualene synthase enzymes were exploited to develop molecular models for the B. braunii botryococcene and squalene synthase enzymes. Residues within the active sites that could mediate catalytic specificity were identified, and reciprocal mutants were created in an attempt to interconvert the reaction product specificity of the enzymes. We report here the identification of several amino acid positions contributing to the rearrangement of the cyclopropyl intermediate to squalene, but these same positions do not appear to be sufficient to account for the cyclopropyl rearrangement to give botryococcene.

Enzymes useful for chiral compound synthesis: structural biology, directed evolution, and protein engineering for industrial use.

PubMed

Kataoka, Michihiko; Miyakawa, Takuya; Shimizu, Sakayu; Tanokura, Masaru

2016-07-01

Biocatalysts (enzymes) have many advantages as catalysts for the production of useful compounds as compared to chemical catalysts. The stereoselectivity of the enzymes is one advantage, and thus the stereoselective production of chiral compounds using enzymes is a promising approach. Importantly, industrial application of the enzymes for chiral compound production requires the discovery of a novel useful enzyme or enzyme function; furthermore, improving the enzyme properties through protein engineering and directed evolution approaches is significant. In this review, the significance of several enzymes showing stereoselectivity (quinuclidinone reductase, aminoalcohol dehydrogenase, old yellow enzyme, and threonine aldolase) in chiral compound production is described, and the improvement of these enzymes using protein engineering and directed evolution approaches for further usability is discussed. Currently, enzymes are widely used as catalysts for the production of chiral compounds; however, for further use of enzymes in chiral compound production, improvement of enzymes should be more essential, as well as discovery of novel enzymes and enzyme functions.

Identification and microbial production of a terpene-based advanced biofuel

PubMed Central

Peralta-Yahya, Pamela P.; Ouellet, Mario; Chan, Rossana; Mukhopadhyay, Aindrila; Keasling, Jay D.; Lee, Taek Soon

2011-01-01

Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in bisabolene titers in Escherichia coli to >900 mg l−1. We produce bisabolene in Saccharomyces cerevisiae (>900 mg l−1), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels. PMID:21952217

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

PubMed

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-10

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Enzymes from solvent-tolerant microbes: useful biocatalysts for non-aqueous enzymology.

PubMed

Gupta, Anshu; Khare, S K

2009-01-01

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent's toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.

Biocatalysis engineering: the big picture.

PubMed

Sheldon, Roger A; Pereira, Pedro C

2017-05-22

In this tutorial review we describe a holistic approach to the invention, development and optimisation of biotransformations utilising isolated enzymes. Increasing attention to applied biocatalysis is motivated by its numerous economic and environmental benefits. Biocatalysis engineering concerns the development of enzymatic systems as a whole, which entails engineering its different components: substrate engineering, medium engineering, protein (enzyme) engineering, biocatalyst (formulation) engineering, biocatalytic cascade engineering and reactor engineering.

Spatially resolved metabolic analysis reveals a central role for transcriptional control in carbon allocation to wood.

PubMed

Roach, Melissa; Arrivault, Stéphanie; Mahboubi, Amir; Krohn, Nicole; Sulpice, Ronan; Stitt, Mark; Niittylä, Totte

2017-06-15

The contribution of transcriptional and post-transcriptional regulation to modifying carbon allocation to developing wood of trees is not well defined. To clarify the role of transcriptional regulation, the enzyme activity patterns of eight central primary metabolism enzymes across phloem, cambium, and developing wood of aspen (Populus tremula L.) were compared with transcript levels obtained by RNA sequencing of sequential stem sections from the same trees. Enzymes were selected on the basis of their importance in sugar metabolism and in linking primary metabolism to lignin biosynthesis. Existing enzyme assays were adapted to allow measurements from ~1 mm3 sections of dissected stem tissue. These experiments provided high spatial resolution of enzyme activity changes across different stages of wood development, and identified the gene transcripts probably responsible for these changes. In most cases, there was a clear positive relationship between transcripts and enzyme activity. During secondary cell wall formation, the increases in transcript levels and enzyme activities also matched with increased levels of glucose, fructose, hexose phosphates, and UDP-glucose, emphasizing an important role for transcriptional regulation in carbon allocation to developing aspen wood. These observations corroborate the efforts to increase carbon allocation to wood by engineering gene regulatory networks. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Predicting novel substrates for enzymes with minimal experimental effort with active learning.

PubMed

Pertusi, Dante A; Moura, Matthew E; Jeffryes, James G; Prabhu, Siddhant; Walters Biggs, Bradley; Tyo, Keith E J

2017-11-01

Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes, developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of ~80% using ~33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Bioelectrochemical interface engineering: toward the fabrication of electrochemical biosensors, biofuel cells, and self-powered logic biosensors.

PubMed

Zhou, Ming; Dong, Shaojun

2011-11-15

Over the past decade, researchers have devoted considerable attention to the integration of living organisms with electronic elements to yield bioelectronic devices. Not only is the integration of DNA, enzymes, or whole cells with electronics of scientific interest, but it has many versatile potential applications. Researchers are using these ideas to fabricate biosensors for analytical applications and to assemble biofuel cells (BFCs) and biomolecule-based devices. Other research efforts include the development of biocomputing systems for information processing. In this Account, we focus on our recent progress in engineering at the bioelectrochemical interface (BECI) for the rational design and construction of important bioelectronic devices, ranging from electrochemical (EC-) biosensors to BFCs, and self-powered logic biosensors. Hydrogels and sol-gels provide attractive materials for the immobilization of enzymes because they make EC-enzyme biosensors stable and even functional in extreme environments. We use a layer-by-layer (LBL) self-assembly technique to fabricate multicomponent thin films on the BECI at the nanometer scale. Additionally, we demonstrate how carbon nanomaterials have paved the way for new and improved EC-enzyme biosensors. In addition to the widely reported BECI-based electrochemical impedance spectroscopy (EIS)-type aptasensors, we integrate the LBL technique with our previously developed "solid-state probe" technique for redox probes immobilization on electrode surfaces to design and fabricate BECI-based differential pulse voltammetry (DPV)-type aptasensors. BFCs can directly harvest energy from ambient biofuels as green energy sources, which could lead to their application as simple, flexible, and portable power sources. Porous materials provide favorable microenvironments for enzyme immobilization, which can enhance BFC power output. Furthermore, by introducing aptamer-based logic systems to BFCs, such systems could be applied as self-powered and intelligent aptasensors for the logic detection. We have developed biocomputing keypad lock security systems which can be also used for intelligent medical diagnostics. BECI engineering provides a simple but effective approach toward the design and fabrication of EC-biosensors, BFCs, and self-powered logic biosensors, which will make essential contributions in the development of creative and practical bioelectronic devices. The exploration of novel interface engineering applications and the creation of new fabrication concepts or methods merit further attention.

Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an α-Gal epitope

PubMed Central

2010-01-01

Background Oligosaccharides containing a terminal Gal-α1,3-Gal moiety are collectively known as α-Gal epitopes. α-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the α-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the α-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the α-Gal epitope, Gal-α1,3-Lac. Results Agrobacterium sp. ATCC 31749 was engineered to produce Gal-α1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:α1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the α-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting α-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-α1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-α1,3-Lac. Conclusions The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of α-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the α-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides. PMID:20067629

Developments in biotechnological research in Austria

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

Kubicek, C.P.

1996-12-01

Austria is a small European country with a small number of universities and biotechnological industries, but with great efforts in the implementation of environmental consciousness and corresponding legal standards. This review attempts to describe the biotechnological landscape of Austria, thereby focusing on the highlights in research by industry, universities, and research laboratories, as published during 1990 to early 1995. These will include microbial metabolite (organic acids, antibiotics) and biopolymer (polyhydroxibutyrate, S-layers) production; enzyme (cellulases, hemicellulases, ligninases) technology and biocatalysis; environmental biotechnology; plant breeding and plant protection; mammalian cell products; fermenter design; and bioprocess engineering. 234 refs.

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

Yuzawa, Satoshi; Keasling, Jay D.; Katz, Leonard

Complex polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from large enzyme complexes named type I modular polyketide synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel polyketides of predicted structure. Finally, we have repurposed PKSs to produce a number of short-chain mono- and di-carboxylic acids and ketones that could have applications as fuels or industrial chemicals.

Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

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

Agarwal, Pratul K

2012-01-01

Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted thatmore » mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.« less

Can enzyme engineering benefit from the modulation of protein motions? Lessons learned from NMR relaxation dispersion experiments.

PubMed

Doucet, Nicolas

2011-04-01

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant 'invisible states' adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing 'structure-function' analyses with 'flexibility-function' investigations. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

PubMed Central

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-01

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

Expanding P450 catalytic reaction space through evolution and engineering

PubMed Central

McIntosh, John A.; Farwell, Christopher C.; Arnold, Frances H.

2014-01-01

Advances in protein and metabolic engineering have led to wider use of enzymes to synthesize important molecules. However, many desirable transformations are not catalyzed by any known enzyme, driving interest in understanding how new enzymes can be created. The cytochrome P450 enzyme family, whose members participate in xenobiotic metabolism and natural products biosynthesis, catalyzes an impressive range of difficult chemical reactions that continues to grow as new enzymes are characterized. Recent work has revealed that P450-derived enzymes can also catalyze useful reactions previously accessible only to synthetic chemistry. The evolution and engineering of these enzymes provides an excellent case study for how to genetically encode new chemistry and expand biology’s reaction space. PMID:24658056

Mechanisms of fatty acid synthesis in marine fungus-like protists.

PubMed

Xie, Yunxuan; Wang, Guangyi

2015-10-01

Thraustochytrids are unicellular fungus-like protists and are well known for their ability to produce interesting nutraceutical compounds. Significant efforts have been made to improve their efficient production of important fatty acids (FAs), mostly by optimizing fermentation conditions and selecting highly productive thraustochytrid strains. Furthermore, noticeable improvements have been made in understanding the mechanism of FA biosynthesis, allowing for a better understanding of how thraustochytrids assemble these unique metabolites and how their biosynthesis is coupled with other related pathways. This review summarizes recent achievements on two major FA biosynthesis pathways, the standard pathway and the polyketide synthase pathway, and detail features of individual enzymes involved in FA biosynthesis, biotechnological advances in pathway engineering and enzyme characterization, and the discovery of other pathways that affect the efficiency of FA accumulation. Perspectives of biotechnological potential application of thraustochytrids are also discussed.

Physiological Effects of Free Fatty Acid Production in Genetically Engineered Synechococcus elongatus PCC 7942

PubMed Central

Ruffing, Anne M.; Jones, Howland D.T.

2012-01-01

The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production. PMID:22473793

A paradigm shift in biomass technology from complete to partial cellulose hydrolysis: lessons learned from nature.

PubMed

Chen, Rachel

2015-01-01

A key characteristic of current biomass technology is the requirement for complete hydrolysis of cellulose and hemicellulose, which stems from the inability of microbial strains to use partially hydrolyzed cellulose, or cellodextrin. The complete hydrolysis paradigm has been practiced over the past 4 decades with major enzyme companies perfecting their cellulase mix for maximal yield of monosaccharides, with corresponding efforts in strain development focus almost solely on the conversion of monosaccharides, not cellodextrin, to products. While still in its nascent infancy, a new paradigm requiring only partial hydrolysis has begun to take hold, promising a shift in the biomass technology at its fundamental core. The new paradigm has the potential to reduce the requirement for cellulase enzymes in the hydrolysis step and provides new strategies for metabolic engineers, synthetic biologists and alike in engineering fermenting organisms. Several recent publications reveal that microorganisms engineered to metabolize cellodextrins, rather than monomer glucose, can reap significant energy gains in both uptake and subsequent phosphorylation. These energetic benefits can in turn be directed for enhanced robustness and increased productivity of a bioprocess. Furthermore, the new cellodextrin metabolism endows the biocatalyst the ability to evade catabolite repression, a cellular regulatory mechanism that is hampering rapid conversion of biomass sugars to products. Together, the new paradigm offers significant advantages over the old and promises to overcome several critical barriers in biomass technology. More research, however, is needed to realize these promises, especially in discovery and engineering of cellodextrin transporters, in developing a cost-effective method for cellodextrin generation, and in better integration of cellodextrin metabolism to endogenous glycolysis.

In-vitro engineering of novel bioactivity in the natural enzymes

NASA Astrophysics Data System (ADS)

Tiwari, Vishvanath

2016-10-01

Enzymes catalyze various biochemical functions with high efficiency and specificity. In-vitro design of the enzyme leads to novel bioactivity in this natural biomolecule that give answers of some vital questions like crucial residues in binding with substrate, molecular evolution, cofactor specificity etc. Enzyme engineering technology involves directed evolution, rational designing, semi-rational designing and structure-based designing using chemical modifications. Similarly, combined computational and in-vitro evolution approaches together help in artificial designing of novel bioactivity in the natural enzyme. DNA shuffling, error prone PCR and staggered extension process are used to artificially redesign active site of enzyme, which can alter its efficiency and specificity. Modifications of the enzyme can lead to the discovery of new path of molecular evolution, designing of efficient enzymes, locating active sites and crucial residues, shift in substrate and cofactor specificity. The methods and thermodynamics of in-vitro designing of the enzyme are also discussed. Similarly, engineered thermophilic and psychrophilic enzymes attain substrate specificity and activity of mesophilic enzymes that may also be beneficial for industry and therapeutics.

Methods for improving enzymatic trans-glycosylation for synthesis of human milk oligosaccharide biomimetics.

PubMed

Zeuner, Birgitte; Jers, Carsten; Mikkelsen, Jørn Dalgaard; Meyer, Anne S

2014-10-08

Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is a novel field of research in advanced food ingredient chemistry, involving the use of rare enzymes, which have until now mainly been studied for their biochemical significance, not for targeted biosynthesis applications. For the enzymatic synthesis of biofunctional glycans reaction parameter optimization to promote "reverse" catalysis with glycosidases is currently preferred over the use of glycosyl transferases. Numerous methods exist for minimizing the undesirable glycosidase-catalyzed hydrolysis and for improving the trans-glycosylation yields. This review provides an overview of the approaches and data available concerning optimization of enzymatic trans-glycosylation for novel synthesis of complex bioactive carbohydrates using sialidases, α-l-fucosidases, and β-galactosidases as examples. The use of an adequately high acceptor/donor ratio, reaction time control, continuous product removal, enzyme recycling, and/or the use of cosolvents may significantly improve trans-glycosylation and biocatalytic productivity of the enzymatic reactions. Protein engineering is also a promising technique for obtaining high trans-glycosylation yields, and proof-of-concept for reversing sialidase activity to trans-sialidase action has been established. However, the protein engineering route currently requires significant research efforts in each case because the structure-function relationship of the enzymes is presently poorly understood.

Subcellular Targeting of Methylmercury Lyase Enhances Its Specific Activity for Organic Mercury Detoxification in Plants1

PubMed Central

Bizily, Scott P.; Kim, Tehryung; Kandasamy, Muthugapatti K.; Meagher, Richard B.

2003-01-01

Methylmercury is an environmental pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals. In an effort to remove this toxin in situ, we have been engineering plants that express the bacterial mercury resistance enzymes organomercurial lyase MerB and mercuric ion reductase MerA. In vivo kinetics experiments suggest that the diffusion of hydrophobic organic mercury to MerB limits the rate of the coupled reaction with MerA (Bizily et al., 2000). To optimize reaction kinetics for organic mercury compounds, the merB gene was engineered to target MerB for accumulation in the endoplasmic reticulum and for secretion to the cell wall. Plants expressing the targeted MerB proteins and cytoplasmic MerA are highly resistant to organic mercury and degrade organic mercury at 10 to 70 times higher specific activity than plants with the cytoplasmically distributed wild-type MerB enzyme. MerB protein in endoplasmic reticulum-targeted plants appears to accumulate in large vesicular structures that can be visualized in immunolabeled plant cells. These results suggest that the toxic effects of organic mercury are focused in microenvironments of the secretory pathway, that these hydrophobic compartments provide more favorable reaction conditions for MerB activity, and that moderate increases in targeted MerB expression will lead to significant gains in detoxification. In summary, to maximize phytoremediation efficiency of hydrophobic pollutants in plants, it may be beneficial to target enzymes to specific subcellular environments. PMID:12586871

ECUT (Energy Conversion and Utilization Technologies Program). Biocatalysis Project

NASA Technical Reports Server (NTRS)

1986-01-01

Presented are the FY 1985 accomplishments, activities, and planned research efforts of the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Program. The Project's technical activities were organized as follows: In the Molecular Modeling and Applied Genetics work element, research focused on (1) modeling and simulation studies to establish the physiological basis of high temperature tolerance in a selected enzyme and the catalytic mechanisms of three species of another enzyme, and (2) determining the degree of plasmid amplification and stability of several DNA bacterial strains. In the Bioprocess Engineering work element, research focused on (1) studies of plasmid propagation and the generation of models, (2) developing methods for preparing immobilized biocatalyst beads, and (3) developing an enzyme encapsulation method. In the Process Design and Analysis work element, research focused on (1) further refinement of a test case simulation of the economics and energy efficiency of alternative biocatalyzed production processes, (2) developing a candidate bioprocess to determine the potential for reduced energy consumption and facility/operating costs, and (3) a techno-economic assessment of potential advancements in microbial ammonia production.

Genetically Engineered Materials for Biofuels Production

NASA Astrophysics Data System (ADS)

Raab, Michael

2012-02-01

Agrivida, Inc., is an agricultural biotechnology company developing industrial crop feedstocks for the fuel and chemical industries. Agrivida's crops have improved processing traits that enable efficient, low cost conversion of the crops' cellulosic components into fermentable sugars. Currently, pretreatment and enzymatic conversion of the major cell wall components, cellulose and hemicellulose, into fermentable sugars is the most expensive processing step that prevents widespread adoption of biomass in biofuels processes. To lower production costs we are consolidating pretreatment and enzyme production within the crop. In this strategy, transgenic plants express engineered cell wall degrading enzymes in an inactive form, which can be reactivated after harvest. We have engineered protein elements that disrupt enzyme activity during normal plant growth. Upon exposure to specific processing conditions, the engineered enzymes are converted into their active forms. This mechanism significantly lowers pretreatment costs and enzyme loadings (>75% reduction) below those currently available to the industry.

Rapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilm.

PubMed

Tong, Xiaoxue; Barberi, Tania Triscari; Botting, Catherine H; Sharma, Sunil V; Simmons, Mark J H; Overton, Tim W; Goss, Rebecca J M

2016-10-21

Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm's extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme's longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. Here we provide the first evidence of a recombinant enzyme's regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a "plug-and-play" fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals.

Rewiring Algae's Catalytic Circuits - Continuum Magazine | NREL

Science.gov Websites

with labels depicting the engineering of hydrogen-producing enzyme to create a hydrogen production circuit to increase hydrogen during photosynthesis. Engineering of the hydrogen-producing enzyme to create circuits, or pathways. To do so, they would replace the normal hydrogen-producing enzyme, hydrogenase

The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown.

PubMed

Seppälä, Susanna; Wilken, St Elmo; Knop, Doriv; Solomon, Kevin V; O'Malley, Michelle A

2017-11-01

A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that are currently in biotechnological use, and explore more basal, non-conventional fungi and their underexploited biomass-degrading mechanisms as promising agents in the transition towards a bio-based society. Of special interest are anaerobic fungi like the Neocallimastigomycota, which were recently found to harbor the largest diversity of biomass-degrading enzymes among the fungal kingdom. Enzymes sourced from these basal fungi have been used to metabolically engineer substrate utilization in yeast, and may offer new paths to lignin breakdown and tunneled biocatalysis. We also contrast classic enzymology approaches with emerging 'omics'-based tools to decipher function within novel fungal isolates and identify new promising enzymes. Recent developments in genome editing are expected to accelerate discovery and metabolic engineering within these systems, yet are still limited by a lack of high-resolution genomes, gene regulatory regions, and even appropriate culture conditions. Finally, we present new opportunities to harness the biomass-degrading potential of undercharacterized fungi via heterologous expression and engineered microbial consortia. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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.

A study of over production and enhanced secretion of enzymes. Quarterly report 1

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

Dashek, W.V.

1992-12-28

The current project is concerned with the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. The project is divided into two segments: over-production of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electron microscopical techniques. The former approach employs recombinant DNA procedures, ligation of appropriate nuclease generated DNA fragments into a vector and the subsequent transformation of Escherichia coli to yield E. coli harboring a C. versicolor DNA insert. The biochemistry/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO inhibitors to elevate C.versicolor`s ability to synthesizemore » and secrete lignocellulosic enzymes. In this connection, cell fractionation/kinetic analysis, TEM immunoelectron microscopic localization and TEM substrate localization of PPO are being employed to assess the route of secretion. Both approaches will culminate in the batch culture of either E. coli or C. versicolor, in a fermentor with the subsequent development of rapid isolation and purification procedures to yield elevated quantities of pure lignocellulosic enzymes. During the past year, research effort were directed toward determining the route of polyphenol oxidase (PPO) secretion by the wood-decay fungus, Coriolus versicolor. In addition, research activities were continued to over-produce and to purify PPO as well as define the time-dependent intra- and extra-cellular appearances of C. versicolor ligninases and cellulases.« less

Accelerating the Rate-Limiting Step in Novel Enzymatic Carbohydrate-to-Hydrogen Technology by Enzyme Engineering

DTIC Science & Technology

2011-10-30

stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low- cost high-capacity celiulosic...NOVEL ENZYMATIC CARBOHYDRATE-TO-HYDROGEN TECHNOLOGY BY ENZYME ENGINEERING Grant/Contract Number: FA9550-08-1-0145 Program Manager: Dr. Walt...bbtransformation (SyPaB) is the implementation of complicated biochemical reactions by in vitro assembly of enzyme and coenzymes. Different from in vivo

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus.

PubMed

Lian, Hong; Zeldes, Benjamin M; Lipscomb, Gina L; Hawkins, Aaron B; Han, Yejun; Loder, Andrew J; Nishiyama, Declan; Adams, Michael W W; Kelly, Robert M

2016-12-01

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO 3 - and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO 2 . Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO 2 -sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO 2 into 3HP production in metabolically engineered P. furiosus, and hint at the important role that CA and BPL likely play in the native 3HP/4HB pathway in M. sedula. Biotechnol. Bioeng. 2016;113: 2652-2660. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Probing Enzyme-Surface Interactions via Protein Engineering and Single-Molecule Techniques

DTIC Science & Technology

2017-06-26

SECURITY CLASSIFICATION OF: The overall objective of this research was to exploit protein engineering and fluorescence single-molecule methods to... Engineering and Single-Molecule Techniques The views, opinions and/or findings contained in this report are those of the author(s) and should not...Status: Technology Transfer: Report Date: 1 FINAL REPORT Project Title: Probing Enzyme-Surface Interactions via Protein Engineering and

Maximizing the efficiency of multienzyme process by stoichiometry optimization.

PubMed

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

2014-09-05

Multienzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of the stoichiometry of the biocatalysts. Here we present a workflow for maximizing the efficiency of a three-enzyme system catalyzing a five-step chemical conversion. Kinetic models of pathways with wild-type or engineered enzymes were built, and the enzyme stoichiometry of each pathway was optimized. Mathematical modeling and one-pot multienzyme experiments provided detailed insights into pathway dynamics, enabled the selection of a suitable engineered enzyme, and afforded high efficiency while minimizing biocatalyst loadings. Optimizing the stoichiometry in a pathway with an engineered enzyme reduced the total biocatalyst load by an impressive 56 %. Our new workflow represents a broadly applicable strategy for optimizing multienzyme processes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes.

PubMed

Amrein, Beat A; Bauer, Paul; Duarte, Fernanda; Janfalk Carlsson, Åsa; Naworyta, Agata; Mowbray, Sherry L; Widersten, Mikael; Kamerlin, Shina C L

2015-10-02

Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broad range of substrates. The enzyme can be engineered to increase the yield of optically pure products as a result of changes in both enantio- and regioselectivity. It is thus highly attractive in biocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals. The present work aims to establish the principles underlying the activity and selectivity of the enzyme through a combined computational, structural, and kinetic study using the substrate trans -stilbene oxide as a model system. Extensive empirical valence bond simulations have been performed on the wild-type enzyme together with several experimentally characterized mutants. We are able to computationally reproduce the differences between the activities of different stereoisomers of the substrate and the effects of mutations of several active-site residues. In addition, our results indicate the involvement of a previously neglected residue, H104, which is electrostatically linked to the general base H300. We find that this residue, which is highly conserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonated form in order to provide charge balance in an otherwise negatively charged active site. Our data show that unless the active-site charge balance is correctly treated in simulations, it is not possible to generate a physically meaningful model for the enzyme that can accurately reproduce activity and selectivity trends. We also expand our understanding of other catalytic residues, demonstrating in particular the role of a noncanonical residue, E35, as a "backup base" in the absence of H300. Our results provide a detailed view of the main factors driving catalysis and regioselectivity in this enzyme and identify targets for subsequent enzyme design efforts.

Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

PubMed

Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

2015-01-01

We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular polyketide synthases: opportunities and challenges.

PubMed

Yuzawa, Satoshi; Keasling, Jay D; Katz, Leonard

2017-04-01

Complex polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from large enzyme complexes named type I modular polyketide synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel polyketides of predicted structure. We have repurposed PKSs to produce a number of short-chain mono- and di-carboxylic acids and ketones that could have applications as fuels or industrial chemicals.

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

PubMed

Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

2017-03-17

Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

PubMed Central

Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

2013-01-01

There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

Selective production of decanoic acid from iterative reversal of β-oxidation pathway.

PubMed

Kim, Seohyoung; Gonzalez, Ramon

2018-05-01

Decanoic acid is a valuable compound used as precursor for industrial chemicals, pharmaceuticals, and biofuels. Despite efforts to produce it from renewables, only limited achievements have been reported. Here, we report an engineered cell factory able to produce decanoic acid as a major product from glycerol, and abundant and renewable feedstock. We exploit the overlapping chain-length specificity of β-oxidation reversal (r-BOX) and thioesterase enzymes to selectively generate decanoic acid. This was achieved by selecting r-BOX enzymes that support the synthesis of acyl-CoA of up to 10 carbons (thiolase BktB and enoyl-CoA reductase EgTER) and a thioesterase that exhibited high activity toward decanoyl-CoA and longer-chain acyl-CoAs (FadM). Combined chromosomal and episomal expression of r-BOX core enzymes such as enoyl-CoA reductase and thiolase (in the presence of E. coli thioesterase FadM) increased titer and yield of decanoic acid, respectively. The carbon flux toward decanoic acid was substantially increased by the use of an organic overlay, which decreased its intracellular accumulation and presumably increased its concentration gradient across cell membrane, suggesting that decanoic acid transport to the extracellular medium might be a major bottleneck. When cultivated in the presence of a n-dodecane overlay, the final engineered strain produced 2.1 g/L of decanoic acid with a yield of 0.1 g/g glycerol. Collectively, our data suggests that r-BOX can be used as a platform to selectively produce decanoic acid and its derivatives at high yield, titer and productivity. © 2018 Wiley Periodicals, Inc.

Current Developments and Challenges in the Search for a Naturally Selected Diels-Alderase

PubMed Central

Kim, Hak Joong; Ruszczycky, Mark W.; Liu, Hung-wen

2012-01-01

Only a very few examples of enzymes known to catalyze pericyclic reactions have been reported, and presently no enzyme has been demonstrated unequivocally to catalyze a Diels-Alder reaction. Nevertheless, research into secondary metabolism has led to the discovery of numerous natural products exhibiting the structural hallmarks of [4+2] cycloadditions, prompting efforts to characterize the responsible enzymatic processes. These efforts have resulted in a growing collection of enzymes believed to catalyze pericyclic [4+2] cycloaddition reactions; however, in each case the complexity of the substrates and catalytic properties of these enzymes poses significant challenges in substantiating these hypotheses. Herein we consider the principles motivating these efforts and the enzymological systems currently under investigation. PMID:22260931

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

Trends in bacterial trehalose metabolism and significant nodes of metabolic pathway in the direction of trehalose accumulation

PubMed Central

Ruhal, Rohit; Kataria, Rashmi; Choudhury, Bijan

2013-01-01

Summary The current knowledge of trehalose biosynthesis under stress conditions is incomplete and needs further research. Since trehalose finds industrial and pharmaceutical applications, enhanced accumulation of trehalose in bacteria seems advantageous for commercial production. Moreover, physiological role of trehalose is a key to generate stress resistant bacteria by metabolic engineering. Although trehalose biosynthesis requires few metabolites and enzyme reactions, it appears to have a more complex metabolic regulation. Trehalose biosynthesis in bacteria is known through three pathways – OtsAB, TreYZ and TreS. The interconnections of in vivo synthesis of trehalose, glycogen or maltose were most interesting to investigate in recent years. Further, enzymes at different nodes (glucose-6-P, glucose-1-P and NDP-glucose) of metabolic pathways influence enhancement of trehalose accumulation. Most of the study of trehalose biosynthesis was explored in medically significant Mycobacterium, research model Escherichia coli, industrially applicable Corynebacterium and food and probiotic interest Propionibacterium freudenreichii. Therefore, the present review dealt with the trehalose metabolism in these bacteria. In addition, an effort was made to recognize how enzymes at different nodes of metabolic pathway can influence trehalose accumulation. PMID:23302511

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

DOE PAGES

Price, J. Vincent; Chen, Long; Whitaker, W. Brian; ...

2016-10-24

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

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

Price, J. Vincent; Chen, Long; Whitaker, W. Brian

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

PubMed Central

Price, J. Vincent; Chen, Long; Whitaker, W. Brian; Papoutsakis, Eleftherios; Chen, Wilfred

2016-01-01

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. PMID:27791059

Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering.

PubMed

Karimova, Madina; Splith, Victoria; Karpinski, Janet; Pisabarro, M Teresa; Buchholz, Frank

2016-07-22

Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. aB, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets.

Engineering education research: Impacts of an international network of female engineers on the persistence of Liberian undergraduate women studying engineering

NASA Astrophysics Data System (ADS)

Rimer, Sara; Reddivari, Sahithya; Cotel, Aline

2015-11-01

As international efforts to educate and empower women continue to rise, engineering educators are in a unique position to be a part of these efforts by encouraging and supporting women across the world at the university level through STEM education and outreach. For the past two years, the University of Michigan has been a part of a grassroots effort to encourage and support the persistence of engineering female students at University of Liberia. This effort has led to the implementation of a leadership camp this past August for Liberian engineering undergraduate women, meant to: (i) to empower engineering students with the skills, support, and inspiration necessary to become successful and well-rounded engineering professionals in a global engineering market; and (ii) to strengthen the community of Liberian female engineers by building cross-cultural partnerships among students resulting in a international network of women engineers. This session will present qualitative research findings on the impact of this grassroots effort on Liberian female students? persistence in engineering, and the future directions of this work.

Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning

PubMed Central

Bacik, John-Paul; Wrenbeck, Emily E.; Michalczyk, Ryszard; Whitehead, Timothy A.

2017-01-01

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications. PMID:28196882

Effect of Additives on the Selectivity and Reactivity of Enzymes.

PubMed

Liang, Yi-Ru; Wu, Qi; Lin, Xian-Fu

2017-01-01

Enzymes have been widely used as efficient, eco-friendly, and biodegradable catalysts in organic chemistry due to their mild reaction conditions and high selectivity and efficiency. In recent years, the catalytic promiscuity of many enzymes in unnatural reactions has been revealed and studied by chemists and biochemists, which has expanded the application potential of enzymes. To enhance the selectivity and activity of enzymes in their natural or promiscuous reactions, many methods have been recommended, such as protein engineering, process engineering, and media engineering. Among them, the additive approach is very attractive because of its simplicity to use and high efficiency. In this paper, we will review the recent developments about the applications of additives to improve the catalytic performances of enzymes in their natural and promiscuous reactions. These additives include water, organic bases, water mimics, cosolvents, crown ethers, salts, surfactants, and some particular molecular additives. © 2017 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Therapeutic Enzymes: Applications and Approaches to Pharmacological Improvement.

PubMed

Yari, Maryam; Ghoshoon, Mohammad B; Vakili, Bahareh; Ghasemi, Younes

2017-01-01

Among therapeutic proteins, enzymes represent small and of course profitable market. They can be used to treat important, rare, and deadly diseases. Enzyme therapy is the only available treatment for certain disorders. Here, pharmaceutical enzymes are reviewed. They are categorized in four main groups, enzymes in replacement therapy, enzymes in cancer treatment, enzymes for fibrinolysis, and finally enzymes that are used topically for various treatments. Furthermore, enzyme gene therapy and future perspective of therapeutic enzymes are mentioned in brief. There are many important approved enzymes in pharmaceutical market. Several approaches such as point mutation, fusion protein designing, glycoengineering, and PEGylation were used to achieve improved enzymes. Although sometimes enzymes were engineered to facilitate production and purification process, appropriate delivery to target sites, extending half-life, and reducing immunogenicity are among the main goals of engineering approaches. Overall, enzymes play a critical role in treatment of common and rare diseases. Evaluation of new enzymes as well as improvement of approved enzymes are of the most important challenges in biotechnology. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Enzyme dynamics and engineering: one step at a time.

PubMed

Tokuriki, Nobuhiko; Jackson, Colin J

2014-10-23

Although protein dynamics are accepted as being essential for enzyme function, their effects are not fully understood. In this issue of Chemistry and Biology, Gobeil and coworkers describe how engineered changes in the millisecond motions of a mutant TEM-1 β-lactamase do not significantly affect substrate turnover. This mutational robustness has implications for protein engineering and design strategies.

Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

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

Tomasic, Ivan B.; Metcalf, Matthew C.; Guce, Abigail I.

2010-09-03

The human lysosomal enzymes {alpha}-galactosidase ({alpha}-GAL, EC 3.2.1.22) and {alpha}-N-acetylgalactosaminidase ({alpha}-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of {alpha}-GAL and {alpha}-NAGAL. The engineered {alpha}-GAL (which we call {alpha}-GALSA) retains the antigenicity of {alpha}-GAL but has acquired the enzymatic specificity of {alpha}-NAGAL. Conversely, the engineered {alpha}-NAGAL (which we call {alpha}-NAGAL{sup EL}) retains the antigenicity of {alpha}-NAGAL but has acquired themore » enzymatic specificity of the {alpha}-GAL enzyme. Comparison of the crystal structures of the designed enzyme {alpha}-GAL{sup SA} to the wild-type enzymes shows that active sites of {alpha}-GAL{sup SA} and {alpha}-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.« less

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

PubMed

Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

2017-06-16

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching.

PubMed

Boonyapakron, Katewadee; Jaruwat, Aritsara; Liwnaree, Benjamas; Nimchua, Thidarat; Champreda, Verawat; Chitnumsub, Penchit

2017-10-10

In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the k cat of Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pK a and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO 2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus

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

Lian, Hong; Zeldes, Benjamin M.; Lipscomb, Gina L.

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO 3 – and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO 2. Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, whilemore » the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO 2-sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. Here, while further efforts to improve 3HP production by regulating gene dosage,« less

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO 2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus

DOE PAGES

Lian, Hong; Zeldes, Benjamin M.; Lipscomb, Gina L.; ...

2016-06-18

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO 3 – and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO 2. Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, whilemore » the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO 2-sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. Here, while further efforts to improve 3HP production by regulating gene dosage,« less

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges.

PubMed

Chánique, Andrea M; Parra, Loreto P

2018-01-01

Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa . Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.

Directed evolution: an approach to engineer enzymes.

PubMed

Kaur, Jasjeet; Sharma, Rohit

2006-01-01

Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.

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.

An Example for Integrated Gas Turbine Engine Testing and Analysis Using Modeling and Simulation

DTIC Science & Technology

2006-12-01

USAF Academy in a joint test and analysis effort of the F109 turbofan engine. This process uses a swirl investigation as a vehicle to exercise and...test and analysis effort of the F109 turbofan engine. This process uses a swirl investigation as a vehicle to exercise and demonstrate the approach...test and analysis effort of the F109 turbofan engine, an effort which uses a swirl investigation as a vehicle to exercise and demonstrate the

Protein Design for Pathway Engineering

PubMed Central

Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

2013-01-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

Protein design for pathway engineering.

PubMed

Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

2014-02-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. Copyright © 2013 Elsevier Inc. All rights reserved.

Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Challenges and Opportunities.

PubMed

Heinisch, Tillmann; Ward, Thomas R

2016-09-20

The biotin-streptavidin technology offers an attractive means to engineer artificial metalloenzymes (ArMs). Initiated over 50 years ago by Bayer and Wilchek, the biotin-(strept)avidin techonology relies on the exquisite supramolecular affinity of either avidin or streptavidin for biotin. This versatile tool, commonly referred to as "molecular velcro", allows nearly irreversible anchoring of biotinylated probes within a (strept)avidin host protein. Building upon a visionary publication by Whitesides from 1978, several groups have been exploiting this technology to create artificial metalloenzymes. For this purpose, a biotinylated organometallic catalyst is introduced within (strept)avidin to afford a hybrid catalyst that combines features reminiscent of both enzymes and organometallic catalysts. Importantly, ArMs can be optimized by chemogenetic means. Combining a small collection of biotinylated organometallic catalysts with streptavidin mutants allows generation of significant diversity, thus allowing optimization of the catalytic performance of ArMs. Pursuing this strategy, the following reactions have been implemented: hydrogenation, alcohol oxidation, sulfoxidation, dihydroxylation, allylic alkylation, transfer hydrogenation, Suzuki cross-coupling, C-H activation, and metathesis. In this Account, we summarize our efforts in the latter four reactions. X-ray analysis of various ArMs based on the biotin-streptavidin technology reveals the versatility and commensurability of the biotin-binding vestibule to accommodate and interact with transition states of the scrutinized organometallic transformations. In particular, streptavidin residues at positions 112 and 121 recurrently lie in close proximity to the biotinylated metal cofactor. This observation led us to develop a streamlined 24-well plate streptavidin production and screening platform to optimize the performance of ArMs. To date, most of the efforts in the field of ArMs have focused on the use of purified protein samples. This seriously limits the throughput of the optimization process. With the ultimate goal of complementing natural enzymes in the context of synthetic and chemical biology, we outline the milestones required to ultimately implement ArMs within a cellular environment. Indeed, we believe that ArMs may allow signficant expansion of the natural enzymes' toolbox to access new-to-nature reactivities in vivo. With this ambitious goal in mind, we report on our efforts to (i) activate the biotinylated catalyst precursor upon incorporation within streptavidin, (ii) minimize the effect of the cellular environment on the ArM's performance, and (iii) demonstrate the compatibility of ArMs with natural enzymes in cascade reactions.

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution.

PubMed

Ye, Lidan; Yang, Chengcheng; Yu, Hongwei

2018-01-01

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

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.

History and future of genetically engineered food animal regulation: an open request.

PubMed

Wells, Kevin D

2016-06-01

Modern biotechnology resulted from of a series of incremental improvements in the understanding of DNA and the enzymes that nature evolved to manipulate it. As the potential impact of genetic engineering became apparent, scientists began the process of trying to identify the potential unintended consequences. Restrictions to recombinant DNA experimentation were at first self-imposed. Collaborative efforts between scientists and lawyers formalized an initial set of guidelines. These guidelines have been used to promulgate regulations around world. However, the initial guidelines were only intended as a starting point and were motivated by a specific set of concerns. As new data became available, the guidelines and regulations should have been adapted to the new knowledge. Instead, other social drivers drove the development of regulations. For most species and most applications, the framework that was established has slowly allowed some products to reach the market. However, genetically engineered livestock that are intended for food have been left in a regulatory state of limbo. To date, no genetically engineered food animal is available in the marketplace. A short history and a U.S.-based genetic engineer's perspective are presented. In addition, a request to regulatory agencies is presented for consideration as regulation continues to evolve. Regulators appear to have shown preference for the slow, random progression of evolution over the efficiency of intentional design.

Enzyme Engineering for In Situ Immobilization.

PubMed

Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

2016-10-14

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

Designing Improved Enzymes of industrial application from marine microorganisms using Protein Engineering

NASA Astrophysics Data System (ADS)

Prajapati, A. S.; Panchal, K.; Subramanian, R. B.; Patel, D. H.; Sudhir, A. P.; Dave, B. R.

2015-12-01

Global demand for energy has grown with the development of new industries, requiring constant improvement and search for new sources of energy. One of the challenges today is releasing the energy of glucose that nature has cleverly locked into lignocellulosic biomass. Potent and efficient enzyme preparations need to be developed for the enzymatic saccharification process to be more economical. Approaches like enzyme engineering, reconstitution of enzyme mixtures and bioprospecting for superior enzymes are gaining importance. The ocean is considered to be a great reservoir of biodiversity. Because enzymes have unequalled advantages, many industries are keenly interested in adapting enzymatic methods for their processes. Microbial communities in marine environments are ecologically relevant as intermediaries of energy and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. The exploitation of marine bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. Several industrial enzymes are derived from terrestrial sources, whereas, marine environment which encompasses about 71 percent of the earth's surface and a vast resources for useful enzymes, remain unexplored. Marine microorganisms take active part in the mineralization of complex organic matter through degradative pathways of their metabolism. Bacteria from marine environments secrete different enzymes based on their habitat and their ecological functions. Therefore marine microbial enzymes have become the focal point of interest. Even though many of these enzymes are being isolated, the efficiency of hydrolysis is very poor. This could be overcome by altering the substrate specificity of lignocellulases. Protein engineering could prove to be useful to improve the catalytic function these enzymes.

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges

PubMed Central

Chánique, Andrea M.; Parra, Loreto P.

2018-01-01

Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa. Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity. PMID:29491854

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

PubMed

Wieczorek, Andrew S; Martin, Vincent J J

2012-12-15

The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study, chimeric protein scaffolds consisting of type 1 and type 2 cohesins anchored on the surface of L. lactis allowed for the controlled positioning of dockerin-fused reporter enzymes onto the scaffolds. By binding single enzymes or enzyme pairs to the scaffolds, our data also suggest that the size and relative positions of enzymes can affect the catalytic profiles of the resulting complexes. These insights will be of great value as we engineer more advanced scaffold-guided protein complexes to optimize biochemical pathways.

Design of biomimetic catalysts by molecular imprinting in synthetic polymers: the role of transition state stabilization.

PubMed

Wulff, Günter; Liu, Junqiu

2012-02-21

The impressive efficiency and selectivity of biological catalysts has engendered a long-standing effort to understand the details of enzyme action. It is widely accepted that enzymes accelerate reactions through their steric and electronic complementarity to the reactants in the rate-determining transition states. Thus, tight binding to the transition state of a reactant (rather than to the corresponding substrate) lowers the activation energy of the reaction, providing strong catalytic activity. Debates concerning the fundamentals of enzyme catalysis continue, however, and non-natural enzyme mimics offer important additional insight in this area. Molecular structures that mimic enzymes through the design of a predetermined binding site that stabilizes the transition state of a desired reaction are invaluable in this regard. Catalytic antibodies, which can be quite active when raised against stable transition state analogues of the corresponding reaction, represent particularly successful examples. Recently, synthetic chemistry has begun to match nature's ability to produce antibody-like binding sites with high affinities for the transition state. Thus, synthetic, molecularly imprinted polymers have been engineered to provide enzyme-like specificity and activity, and they now represent a powerful tool for creating highly efficient catalysts. In this Account, we review recent efforts to develop enzyme models through the concept of transition state stabilization. In particular, models for carboxypeptidase A were prepared through the molecular imprinting of synthetic polymers. On the basis of successful experiments with phosphonic esters as templates to arrange amidinium groups in the active site, the method was further improved by combining the concept of transition state stabilization with the introduction of special catalytic moieties, such as metal ions in a defined orientation in the active site. In this way, the imprinted polymers were able to provide both an electrostatic stabilization for the transition state through the amidinium group as well as a synergism of transition state recognition and metal ion catalysis. The result was an excellent catalyst for carbonate hydrolysis. These enzyme mimics represent the most active catalysts ever prepared through the molecular imprinting strategy. Their catalytic activity, catalytic efficiency, and catalytic proficiency clearly surpass those of the corresponding catalytic antibodies. The active structures in natural enzymes evolve within soluble proteins, typically by the refining of the folding of one polypeptide chain. To incorporate these characteristics into synthetic polymers, we used the concept of transition state stabilization to develop soluble, nanosized carboxypeptidase A models using a new polymerization method we term the "post-dilution polymerization method". With this methodology, we were able to prepare soluble, highly cross-linked, single-molecule nanoparticles. These particles have controlled molecular weights (39 kDa, for example) and, on average, one catalytically active site per particle. Our strategies have made it possible to obtain efficient new enzyme models and further advance the structural and functional analogy with natural enzymes. Moreover, this bioinspired design based on molecular imprinting in synthetic polymers offers further support for the concept of transition state stabilization in catalysis.

Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase.

PubMed

Gemperlein, Katja; Zipf, Gregor; Bernauer, Hubert S; Müller, Rolf; Wenzel, Silke C

2016-01-01

Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Structure, function, and engineering of enzymes in isoflavonoid biosynthesis.

PubMed

Wang, Xiaoqiang

2011-03-01

Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.

Directing enzyme devolution for biosynthesis of alkanols and 1,n-alkanediols from natural polyhydroxy compounds.

PubMed

Dai, Lu; Tao, Fei; Tang, Hongzhi; Guo, Yali; Shen, Yaling; Xu, Ping

2017-11-01

Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as "specialists". However, some enzymes are "generalists" that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols, including 1,2-butanediol, 1,2,4-butanetriol, erythritol, 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays, and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Engineering fungal morphology for enhanced production of hydrolytic enzymes by Aspergillus oryzae SBS50 using microparticles.

PubMed

Singh, Bijender

2018-06-01

Effect of microparticles and silver nanoparticles was studied on the production of hydrolytic enzymes by a potent phytase-producing mould, Aspergillus oryzae SBS50. Addition of microparticles, viz. talc powder and aluminum oxide enhanced phytase production from 2894 to 3903 and 2847 to 4204 U/L, cellulase from 2529 to 4931 and 2455 to 3444 U/L, xylanase from 9067 to 9642 and 9994 to 14,783 U/L, amylase from 5880 to 11,000 and 6130 to 13,145 U/L, respectively. Fungal morphology was also engineered by the use of microparticles. Fungal pellet size was significantly reduced (~ 90%) by the addition of microparticles. Fermentation time was reduced from 4 to 3 days after the addition of microparticles, thus increasing the productivity of the enzymes significantly. These results confirmed the importance of microparticles in engineering fungal morphology for enhanced production of hydrolytic enzymes.

De novo active sites for resurrected Precambrian enzymes

NASA Astrophysics Data System (ADS)

Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

2017-07-01

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

Biological production of organic compounds

DOEpatents

Yu, Jianping; Wang, Bo; Paddock, Troy; Carrieri, Damian; Maness, Pin-Ching; Seibert, Michael

2018-03-13

Methods of producing ethylene oxide and ethylene glycol are disclosed herein. Ethylene produced by cyanobacteria engineered to express ethylene-forming enzymes may be converted to ethylene oxide by bacteria engineered to express a monooxygenase enzyme. Ethylene oxide may be converted to ethylene glycol by exposure to an acidic solution. The methods may be performed in a bioreactor.

Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

PubMed

Munawar, Nayla; Engel, Paul C

2013-01-01

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

Genetic engineering of grass cell wall polysaccharides for biorefining.

PubMed

Bhatia, Rakesh; Gallagher, Joe A; Gomez, Leonardo D; Bosch, Maurice

2017-09-01

Grasses represent an abundant and widespread source of lignocellulosic biomass, which has yet to fulfil its potential as a feedstock for biorefining into renewable and sustainable biofuels and commodity chemicals. The inherent recalcitrance of lignocellulosic materials to deconstruction is the most crucial limitation for the commercial viability and economic feasibility of biomass biorefining. Over the last decade, the targeted genetic engineering of grasses has become more proficient, enabling rational approaches to modify lignocellulose with the aim of making it more amenable to bioconversion. In this review, we provide an overview of transgenic strategies and targets to tailor grass cell wall polysaccharides for biorefining applications. The bioengineering efforts and opportunities summarized here rely primarily on (A) reprogramming gene regulatory networks responsible for the biosynthesis of lignocellulose, (B) remodelling the chemical structure and substitution patterns of cell wall polysaccharides and (C) expressing lignocellulose degrading and/or modifying enzymes in planta. It is anticipated that outputs from the rational engineering of grass cell wall polysaccharides by such strategies could help in realizing an economically sustainable, grass-derived lignocellulose processing industry. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Engineering Escherichia coli for Conversion of Glucose to Medium-Chain ω-Hydroxy Fatty Acids and α,ω-Dicarboxylic Acids.

PubMed

Bowen, Christopher H; Bonin, Jeff; Kogler, Anna; Barba-Ostria, Carlos; Zhang, Fuzhong

2016-03-18

In search of sustainable approaches to plastics production, many efforts have been made to engineer microbial conversions of renewable feedstock to short-chain (C2-C8) bifunctional polymer precursors (e.g., succinic acid, cadaverine, 1,4-butanediol). Less attention has been given to medium-chain (C12-C14) monomers such as ω-hydroxy fatty acids (ω-OHFAs) and α,ω-dicarboxylic acids (α,ω-DCAs), which are precursors to high performance polyesters and polyamides. Here we engineer a complete microbial conversion of glucose to C12 and C14 ω-OHFAs and α,ω-DCAs, with precise control of product chain length. Using an expanded bioinformatics approach, we screen a wide range of enzymes across phyla to identify combinations that yield complete conversion of intermediates to product α,ω-DCAs. Finally, through optimization of culture conditions, we enhance production titer of C12 α,ω-DCA to nearly 600 mg/L. Our results indicate potential for this microbial factory to enable commercially relevant, renewable production of C12 α,ω-DCA-a valuable precursor to the high-performance plastic, nylon-6,12.

Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering

PubMed Central

Karimova, Madina; Splith, Victoria; Karpinski, Janet; Pisabarro, M. Teresa; Buchholz, Frank

2016-01-01

Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. aB, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets. PMID:27444945

Determination of enzyme thermal parameters for rational enzyme engineering and environmental/evolutionary studies.

PubMed

Lee, Charles K; Monk, Colin R; Daniel, Roy M

2013-01-01

Of the two independent processes by which enzymes lose activity with increasing temperature, irreversible thermal inactivation and rapid reversible equilibration with an inactive form, the latter is only describable by the Equilibrium Model. Any investigation of the effect of temperature upon enzymes, a mandatory step in rational enzyme engineering and study of enzyme temperature adaptation, thus requires determining the enzymes' thermodynamic parameters as defined by the Equilibrium Model. The necessary data for this procedure can be collected by carrying out multiple isothermal enzyme assays at 3-5°C intervals over a suitable temperature range. If the collected data meet requirements for V max determination (i.e., if the enzyme kinetics are "ideal"), then the enzyme's Equilibrium Model parameters (ΔH eq, T eq, ΔG (‡) cat, and ΔG (‡) inact) can be determined using a freely available iterative model-fitting software package designed for this purpose.Although "ideal" enzyme reactions are required for determination of all four Equilibrium Model parameters, ΔH eq, T eq, and ΔG (‡) cat can be determined from initial (zero-time) rates for most nonideal enzyme reactions, with substrate saturation being the only requirement.

Teaching Engineering Ethics to PhD Students: A Berkeley-Delft Initiative : Commentary on "Ethics Across the Curriculum: Prospects for Broader (and Deeper) Teaching and Learning in Research and Engineering Ethics".

PubMed

Taebi, Behnam; Kastenberg, William E

2016-07-13

A joint effort by the University of California at Berkeley and Delft University of Technology to develop a graduate engineering ethics course for PhD students encountered two types of challenges: academic and institutional. Academically, long-term collaborative research efforts between engineering and philosophy faculty members might be needed before successful engineering ethics courses can be initiated; the teaching of ethics to engineering graduate students and collaborative research need to go hand-in-hand. Institutionally, both bottom-up approaches at the level of the faculty and as a joint research and teaching effort, and top-down approaches that include recognition by a University's administration and the top level of education management, are needed for successful and sustainable efforts to teach engineering ethics.

Advantages of a distant cellulase catalytic base.

PubMed

Burgin, Tucker; Ståhlberg, Jerry; Mayes, Heather B

2018-03-30

The inverting glycoside hydrolase Trichoderma reesei ( Hypocrea jecorina ) Cel6A is a promising candidate for protein engineering for more economical production of biofuels. Until recently, its catalytic mechanism had been uncertain: The best candidate residue to serve as a catalytic base, Asp-175, is farther from the glycosidic cleavage site than in other glycoside hydrolase enzymes. Recent unbiased transition path sampling simulations revealed the hydrolytic mechanism for this more distant base, employing a water wire; however, it is not clear why the enzyme employs a more distant catalytic base, a highly conserved feature among homologs across different kingdoms. In this work, we describe molecular dynamics simulations designed to uncover how a base with a longer side chain, as in a D175E mutant, affects procession and active site alignment in the Michaelis complex. We show that the hydrogen bond network is tuned to the shorter aspartate side chain, and that a longer glutamate side chain inhibits procession as well as being less likely to adopt a catalytically productive conformation. Furthermore, we draw comparisons between the active site in Trichoderma reesei Cel6A and another inverting, processive cellulase to deduce the contribution of the water wire to the overall enzyme function, revealing that the more distant catalytic base enhances product release. Our results can inform efforts in the study and design of enzymes by demonstrating how counterintuitive sacrifices in chemical reactivity can have worthwhile benefits for other steps in the catalytic cycle. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Flight Test Engineering

NASA Technical Reports Server (NTRS)

Pavlock, Kate Maureen

2013-01-01

Although the scope of flight test engineering efforts may vary among organizations, all point to a common theme: flight test engineering is an interdisciplinary effort to test an asset in its operational flight environment. Upfront planning where design, implementation, and test efforts are clearly aligned with the flight test objective are keys to success. This chapter provides a top level perspective of flight test engineering for the non-expert. Additional research and reading on the topic is encouraged to develop a deeper understanding of specific considerations involved in each phase of flight test engineering.

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering.

PubMed

Nagao, Jun-ichi; Aso, Yuji; Shioya, Kouki; Nakayama, Jiro; Sonomoto, Kenji

2007-01-01

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.

EFFECTS OF X-IRRADIATION ON THE HEXOBARBITAL METABOLIZING ENZYME SYSTEM OF RAT LIVER MICROSOMES.

DTIC Science & Technology

RADIATION EFFECTS , *ENZYME INHIBITORS, *HYPNOTICS AND SEDATIVES, ENZYMES, BIOSYNTHESIS, METABOLISM, DETOXIFICATION, BARBITURATES, OXIDATION...MICROSOMES, LIVER, REGENERATION(ENGINEERING), EXCISION, SUBLETHAL DOSAGE, TOXICITY , HYPNOSIS, SLEEP, HEAD(ANATOMY), MALES, FEMALES, RATS.

Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus

PubMed Central

Wang, Yupeng; Khan, Iram F.; Boissel, Sandrine; Jarjour, Jordan; Pangallo, Joseph; Thyme, Summer; Baker, David; Scharenberg, Andrew M.; Rawlings, David J.

2014-01-01

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20–22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)—a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering. PMID:24682825

Structure, inhibition, and regulation of essential lipid A enzymes.

PubMed

Zhou, Pei; Zhao, Jinshi

2017-11-01

The Raetz pathway of lipid A biosynthesis plays a vital role in the survival and fitness of Gram-negative bacteria. Research efforts in the past three decades have identified individual enzymes of the pathway and have provided a mechanistic understanding of the action and regulation of these enzymes at the molecular level. This article reviews the discovery, biochemical and structural characterization, and regulation of the essential lipid A enzymes, as well as continued efforts to develop novel antibiotics against Gram-negative pathogens by targeting lipid A biosynthesis. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

Energy Efficient Engine (E3) combustion system component technology performance report

NASA Technical Reports Server (NTRS)

Burrus, D. L.; Chahrour, C. A.; Foltz, H. L.; Sabla, P. E.; Seto, S. P.; Taylor, J. R.

1984-01-01

The Energy Efficient Engine (E3) combustor effort was conducted as part of the overall NASA/GE E3 Program. This effort included the selection of an advanced double-annular combustion system design. The primary intent of this effort was to evolve a design that meets the stringent emissions and life goals of the E3, as well as all of the usual performance requirements of combustion systems for modern turbofan engines. Numerous detailed design studies were conducted to define the features of the combustion system design. Development test hardware was fabricated, and an extensive testing effort was undertaken to evaluate the combustion system subcomponents in order to verify and refine the design. Technology derived from this effort was incorporated into the engine combustion hardware design. The advanced engine combustion system was then evaluated in component testing to verify the design intent. What evolved from this effort was an advanced combustion system capable of satisfying all of the combustion system design objectives and requirements of the E3.

New Developments in Vaccines, Inhibitors of Anthrax Toxins, and Antibiotic Therapeutics for Bacillus anthracis

PubMed Central

Beierlein, J.M.; Anderson, A.C.

2013-01-01

Bacillus anthracis, the causative agent responsible for anthrax infections, poses a significant biodefense threat. There is a high mortality rate associated with untreated anthrax infections; specifically, inhalation anthrax is a particularly virulent form of infection with mortality rates close to 100%, even with aggressive treatment. Currently, a vaccine is not available to the general public and few antibiotics have been approved by the FDA for the treatment of inhalation anthrax. With the threat of natural or engineered bacterial resistance to antibiotics and the limited population for whom the current drugs are approved, there is a clear need for more effective treatments against this deadly infection. A comprehensive review of current research in drug discovery is presented in this article, including efforts to improve the purity and stability of vaccines, design inhibitors targeting the anthrax toxins, and identify inhibitors of novel enzyme targets. High resolution structural information for the anthrax toxins and several essential metabolic enzymes has played a significant role in aiding the structure-based design of potent and selective antibiotics. PMID:22050756

Reducing Enzyme Costs Increases Market Potential of Biofuels; The Spectrum of Clean Energy Innovation (Fact Sheet)

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

Not Available

Fact sheet describing NREL's work with enzyme producers Novozymes and Genencor to engineer new cellulase enzymes to breakdown cellulosic ethanol into fermentable sugars that can be converted into biofuels.

Immobilization, stabilization and patterning techniques for enzyme based sensor systems.

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

Flounders, A.W.; Carichner, S.C.; Singh, A.K.

1997-01-01

Sandia National Laboratories has recently opened the Chemical and Radiation Detection Laboratory (CRDL) in Livermore CA to address the detection needs of a variety of government agencies (e.g., Department of Energy, Environmental Protection Agency, Department of Agriculture) as well as provide a fertile environment for the cooperative development of new industrial technologies. This laboratory consolidates a variety of existing chemical and radiation detection efforts and enables Sandia to expand into the novel area of biochemically based sensors. One aspect of this biosensor effort is further development and optimization of enzyme modified field effect transistors (EnFETs). Recent work has focused uponmore » covalent attachment of enzymes to silicon dioxide and silicon nitride surfaces for EnFET fabrication. They are also investigating methods to pattern immobilized proteins; a critical component for development of array-based sensor systems. Novel enzyme stabilization procedures are key to patterning immobilized enzyme layers while maintaining enzyme activity. Results related to maximized enzyme loading, optimized enzyme activity and fluorescent imaging of patterned surfaces will be presented.« less

Biochemical Engineering Fundamentals

ERIC Educational Resources Information Center

Bailey, J. E.; Ollis, D. F.

1976-01-01

Discusses a biochemical engineering course that is offered as part of a chemical engineering curriculum and includes topics that influence the behavior of man-made or natural microbial or enzyme reactors. (MLH)

Preliminary engineering cost trends for highway projects.

DOT National Transportation Integrated Search

2011-10-21

Preliminary engineering (PE) for a highway project encompasses two efforts: planning to minimize the physical, social, and human environmental impacts of projects and engineering design to deliver the best alternative. PE efforts begin years in advan...

de novo computational enzyme design.

PubMed

Zanghellini, Alexandre

2014-10-01

Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthetic and systems biology for microbial production of commodity chemicals.

PubMed

Chubukov, Victor; Mukhopadhyay, Aindrila; Petzold, Christopher J; Keasling, Jay D; Martín, Héctor García

2016-01-01

The combination of synthetic and systems biology is a powerful framework to study fundamental questions in biology and produce chemicals of immediate practical application such as biofuels, polymers, or therapeutics. However, we cannot yet engineer biological systems as easily and precisely as we engineer physical systems. In this review, we describe the path from the choice of target molecule to scaling production up to commercial volumes. We present and explain some of the current challenges and gaps in our knowledge that must be overcome in order to bring our bioengineering capabilities to the level of other engineering disciplines. Challenges start at molecule selection, where a difficult balance between economic potential and biological feasibility must be struck. Pathway design and construction have recently been revolutionized by next-generation sequencing and exponentially improving DNA synthesis capabilities. Although pathway optimization can be significantly aided by enzyme expression characterization through proteomics, choosing optimal relative protein expression levels for maximum production is still the subject of heuristic, non-systematic approaches. Toxic metabolic intermediates and proteins can significantly affect production, and dynamic pathway regulation emerges as a powerful but yet immature tool to prevent it. Host engineering arises as a much needed complement to pathway engineering for high bioproduct yields; and systems biology approaches such as stoichiometric modeling or growth coupling strategies are required. A final, and often underestimated, challenge is the successful scale up of processes to commercial volumes. Sustained efforts in improving reproducibility and predictability are needed for further development of bioengineering.

Synthetic and systems biology for microbial production of commodity chemicals

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

Chubukov, Victor; Mukhopadhyay, Aindrila; Petzold, Christopher J.

The combination of synthetic and systems biology is a powerful framework to study fundamental questions in biology and produce chemicals of immediate practical application such as biofuels, polymers, or therapeutics. However, we cannot yet engineer biological systems as easily and precisely as we engineer physical systems. In this review, we describe the path from the choice of target molecule to scaling production up to commercial volumes. We present and explain some of the current challenges and gaps in our knowledge that must be overcome in order to bring our bioengineering capabilities to the level of other engineering disciplines. Challenges startmore » at molecule selection, where a difficult balance between economic potential and biological feasibility must be struck. Pathway design and construction have recently been revolutionized by next-generation sequencing and exponentially improving DNA synthesis capabilities. Although pathway optimization can be significantly aided by enzyme expression characterization through proteomics, choosing optimal relative protein expression levels for maximum production is still the subject of heuristic, non-systematic approaches. Toxic metabolic intermediates and proteins can significantly affect production, and dynamic pathway regulation emerges as a powerful but yet immature tool to prevent it. Host engineering arises as a much needed complement to pathway engineering for high bioproduct yields; and systems biology approaches such as stoichiometric modeling or growth coupling strategies are required. A final, and often underestimated, challenge is the successful scale up of processes to commercial volumes. Sustained efforts in improving reproducibility and predictability are needed for further development of bioengineering.« less

Synthetic and systems biology for microbial production of commodity chemicals

DOE PAGES

Chubukov, Victor; Mukhopadhyay, Aindrila; Petzold, Christopher J.; ...

2016-04-07

The combination of synthetic and systems biology is a powerful framework to study fundamental questions in biology and produce chemicals of immediate practical application such as biofuels, polymers, or therapeutics. However, we cannot yet engineer biological systems as easily and precisely as we engineer physical systems. In this review, we describe the path from the choice of target molecule to scaling production up to commercial volumes. We present and explain some of the current challenges and gaps in our knowledge that must be overcome in order to bring our bioengineering capabilities to the level of other engineering disciplines. Challenges startmore » at molecule selection, where a difficult balance between economic potential and biological feasibility must be struck. Pathway design and construction have recently been revolutionized by next-generation sequencing and exponentially improving DNA synthesis capabilities. Although pathway optimization can be significantly aided by enzyme expression characterization through proteomics, choosing optimal relative protein expression levels for maximum production is still the subject of heuristic, non-systematic approaches. Toxic metabolic intermediates and proteins can significantly affect production, and dynamic pathway regulation emerges as a powerful but yet immature tool to prevent it. Host engineering arises as a much needed complement to pathway engineering for high bioproduct yields; and systems biology approaches such as stoichiometric modeling or growth coupling strategies are required. A final, and often underestimated, challenge is the successful scale up of processes to commercial volumes. Sustained efforts in improving reproducibility and predictability are needed for further development of bioengineering.« less

Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity

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

Johnson, Adam J.; Brown, Melissa N.; Black, Margaret E., E-mail: blackm@vetmed.wsu.edu

2011-12-09

Highlights: Black-Right-Pointing-Pointer Goal was to enhance dFdC cytotoxicity by the creation of a UCMK/dCK fusion enzyme. Black-Right-Pointing-Pointer The UCMK/dCK fusion enzyme possesses both native activities. Black-Right-Pointing-Pointer The fusion renders cells equally sensitive to dFdC relative to dCK expression alone. Black-Right-Pointing-Pointer Dual activities of fusion not sufficient to augment cell dFdC sensitivity in vitro. Black-Right-Pointing-Pointer Data may warrant the implementation of UCMK mutagenesis studies. -- Abstract: While gemcitabine (2 Prime -2 Prime -difluoro-2 Prime -deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhancemore » dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.« less

Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered β-galactosidase.

PubMed

Estevinho, Berta N; Samaniego, Nuria; Talens-Perales, David; Fabra, Maria José; López-Rubio, Amparo; Polaina, Julio; Marín-Navarro, Julia

2018-08-01

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C. Copyright © 2018 Elsevier B.V. All rights reserved.

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

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.

Biotechnological advances and perspectives of gamma-aminobutyric acid production.

PubMed

Xu, Ning; Wei, Liang; Liu, Jun

2017-03-01

Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that is widely distributed among various organisms. Since GABA has several well-known physiological functions, such as mediating neurotransmission and hypotensive activity, as well as having tranquilizer effects, it is commonly used as a bioactive compound in the food, pharmaceutical and feed industries. The major pathway of GABA biosynthesis is the irreversible decarboxylation of L-glutamate catalyzed by glutamate decarboxylase (GAD), which develops a safe, sustainable and environmentally friendly alternative in comparison with traditional chemical synthesis methods. To date, several microorganisms have been successfully engineered for high-level GABA biosynthesis by overexpressing exogenous GADs. However, the activity of almost all reported microbial GADs sharply decreases at physiological near-neutral pH, which in turn provokes negative effects on the application of these GADs in the recombinant strains for GABA production. Therefore, ongoing efforts in the molecular evolution of GADs, in combination with high-throughput screening and metabolic engineering of particular producer strains, offer fascinating new prospects for effective, environmentally friendly and economically viable GABA biosynthesis. In this review, we briefly introduce the applications in which GABA is used, and summarize the most important methods associated with GABA production. The major achievements and present challenges in the biotechnological synthesis of GABA, focusing on screening and enzyme engineering of GADs, as well as metabolic engineering strategy for one-step GABA biosynthesis, will be extensively discussed.

Genetic engineering approach to toxic waste management: case study for organophosphate waste treatment.

PubMed

Coppella, S J; DelaCruz, N; Payne, G F; Pogell, B M; Speedie, M K; Karns, J S; Sybert, E M; Connor, M A

1990-01-01

Currently, there has been limited use of genetic engineering for waste treatment. In this work, we are developing a procedure for the in situ treatment of toxic organophosphate wastes using the enzyme parathion hydrolase. Since this strategy is based on the use of an enzyme and not viable microorganisms, recombinant DNA technology could be used without the problems associated with releasing genetically altered microorganisms into the environment. The gene coding for parathion hydrolase was cloned into a Streptomyces lividans, and this transformed bacterium was observed to express and excrete this enzyme. Subsequently, fermentation conditions were developed to enhance enzyme production, and this fermentation was scaled-up to the pilot scale. The cell-free culture fluid (i.e., a nonpurified enzyme solution) was observed to be capable of effectively hydrolyzing organophosphate compounds under laboratory and simulated in situ conditions.

Biocatalysis in the Pharmaceutical Industry: The Need for Speed

PubMed Central

2017-01-01

The use of biocatalysis in the pharmaceutical industry continues to expand as a result of increased access to enzymes and the ability to engineer those enzymes to meet the demands of industrial processes. However, we are still just scratching the surface of potential biocatalytic applications. The time pressures present in pharmaceutical process development are incompatible with the long lead times required for engineering a suitable biocatalyst. Dramatic increases in the speed of protein engineering are needed to deliver on the ever increasing opportunities for industrial biocatalytic processes. PMID:28523096

Biocatalysis in the Pharmaceutical Industry: The Need for Speed.

PubMed

Truppo, Matthew D

2017-05-11

The use of biocatalysis in the pharmaceutical industry continues to expand as a result of increased access to enzymes and the ability to engineer those enzymes to meet the demands of industrial processes. However, we are still just scratching the surface of potential biocatalytic applications. The time pressures present in pharmaceutical process development are incompatible with the long lead times required for engineering a suitable biocatalyst. Dramatic increases in the speed of protein engineering are needed to deliver on the ever increasing opportunities for industrial biocatalytic processes.

Towards efficient chemical synthesis via engineering enzyme catalysis in biomimetic nanoreactors.

PubMed

Liu, Jia; Yang, Qihua; Li, Can

2015-09-18

Biocatalysis with immobilized enzymes as catalysts holds enormous promise in developing more efficient and sustainable processes for the synthesis of fine chemicals, chiral pharmaceuticals and biomass feedstocks. Despite the appealing potentials, nowadays the industrial-scale application of biocatalysts is still quite modest in comparison with that of traditional chemical catalysts. A critical issue is that the catalytic performance of enzymes, the sophisticated and vulnerable catalytic machineries, strongly depends on their intracellular working environment; however the working circumstances provided by the support matrix are radically different from those in cells. This often leads to various adverse consequences on enzyme conformation and dynamic properties, consequently decreasing the overall performance of immobilized enzymes with regard to their activity, selectivity and stability. Engineering enzyme catalysis in support nanopores by mimicking the physiological milieu of enzymes in vivo and investigating how the interior microenvironment of nanopores imposes an influence on enzyme behaviors in vitro are of paramount significance to modify and improve the catalytic functions of immobilized enzymes. In this feature article, we have summarized the recent advances in mimicking the working environment and working patterns of intracellular enzymes in nanopores of mesoporous silica-based supports. Especially, we have demonstrated that incorporation of polymers into silica nanopores could be a valuable approach to create the biomimetic microenvironment for enzymes in the immobilized state.

Expression of the Aspergillus niger InuA gene in Saccharomyces cerevisiae permits growth on the plant storage carbohydrate inulin at low enzymatic concentrations

DOE PAGES

Close, Dan

2015-01-01

The plant storage carbohydrate inulin represents an attractive biomass feedstock for fueling industrial scale bioconversion processes due to its low cost, ability for cultivation on arid and semi-arid lands, and amenability to consolidated bioprocessing applications. As a result, increasing efforts are emerging towards engineering industrially relevant microorganisms, such as yeast, to efficiently ferment inulin into high value fuels and chemicals. Although some strains of the industrially relevant yeast model Saccharomyces cerevisiae can naturally ferment inulin, the efficiency of this process is often supplemented through expression of exogenous inulinase enzymes that externally convert inulin into its more easily fermentable component monomericmore » sugars. Here, the effects of overexpressing the Aspergillus niger InuA inulinase enzyme in an S. cerevisiae strain incapable of endogenously fermenting inulin were evaluated to determine their impact on growth. Expression of the A. niger InuA inulinase enzyme permitted growth on otherwise intractable inulin substrates from both Dahlia tubers and Chicory root. Despite being in the top 10 secreted proteins, growth on inulin was not observed until 120 h post-inoculation and required the addition of 0.1 g fructose/l to initiate enzyme production in the absence of endogenous inulinase activity. High temperature/pressure pre-treatment of inulin prior to fermentation decreased this time to 24 h and removed the need for fructose addition. The pre-growth lag time on untreated inulin was attributed primarily to low enzymatic efficiency, with a maximum value of 0.13 0.02 U InuA/ml observed prior to the peak culture density of 2.65 0.03 g/l. Nevertheless, a minimum excreted enzymatic activity level of only 0.03 U InuA/ml was found to be required for sustained growth under laboratory conditions, suggesting that future metabolic engineering strategies can likely redirect carbon flow away from inulinase production and reorient it towards product production or cellular growth in order to optimize strain development.« less

Expression of the Aspergillus niger InuA gene in Saccharomyces cerevisiae permits growth on the plant storage carbohydrate inulin at low enzymatic concentrations

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

Close, Dan

The plant storage carbohydrate inulin represents an attractive biomass feedstock for fueling industrial scale bioconversion processes due to its low cost, ability for cultivation on arid and semi-arid lands, and amenability to consolidated bioprocessing applications. As a result, increasing efforts are emerging towards engineering industrially relevant microorganisms, such as yeast, to efficiently ferment inulin into high value fuels and chemicals. Although some strains of the industrially relevant yeast model Saccharomyces cerevisiae can naturally ferment inulin, the efficiency of this process is often supplemented through expression of exogenous inulinase enzymes that externally convert inulin into its more easily fermentable component monomericmore » sugars. Here, the effects of overexpressing the Aspergillus niger InuA inulinase enzyme in an S. cerevisiae strain incapable of endogenously fermenting inulin were evaluated to determine their impact on growth. Expression of the A. niger InuA inulinase enzyme permitted growth on otherwise intractable inulin substrates from both Dahlia tubers and Chicory root. Despite being in the top 10 secreted proteins, growth on inulin was not observed until 120 h post-inoculation and required the addition of 0.1 g fructose/l to initiate enzyme production in the absence of endogenous inulinase activity. High temperature/pressure pre-treatment of inulin prior to fermentation decreased this time to 24 h and removed the need for fructose addition. The pre-growth lag time on untreated inulin was attributed primarily to low enzymatic efficiency, with a maximum value of 0.13 0.02 U InuA/ml observed prior to the peak culture density of 2.65 0.03 g/l. Nevertheless, a minimum excreted enzymatic activity level of only 0.03 U InuA/ml was found to be required for sustained growth under laboratory conditions, suggesting that future metabolic engineering strategies can likely redirect carbon flow away from inulinase production and reorient it towards product production or cellular growth in order to optimize strain development.« less

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 heterozygous deletions and the improved yeast strain reported here will provide a useful tool for the production of numerous other isoprenoids.

Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii.

PubMed

Boyle, Nanette R; Morgan, John A

2009-01-07

Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved. A model algal species, C. reinhardtii, was chosen and the metabolic network reconstructed. Intracellular fluxes were then calculated using flux balance analysis (FBA). The metabolic network of primary metabolism for a green alga, C. reinhardtii, was reconstructed using genomic and biochemical information. The reconstructed network accounts for the intracellular localization of enzymes to three compartments and includes 484 metabolic reactions and 458 intracellular metabolites. Based on BLAST searches, one newly annotated enzyme (fructose-1,6-bisphosphatase) was added to the Chlamydomonas reinhardtii database. FBA was used to predict metabolic fluxes under three growth conditions, autotrophic, heterotrophic and mixotrophic growth. Biomass yields ranged from 28.9 g per mole C for autotrophic growth to 15 g per mole C for heterotrophic growth. The flux balance analysis model of central and intermediary metabolism in C. reinhardtii is the first such model for algae and the first model to include three metabolically active compartments. In addition to providing estimates of intracellular fluxes, metabolic reconstruction and modelling efforts also provide a comprehensive method for annotation of genome databases. As a result of our reconstruction, one new enzyme was annotated in the database and several others were found to be missing; implying new pathways or non-conserved enzymes. The use of FBA to estimate intracellular fluxes also provides flux values that can be used as a starting point for rational engineering of C. reinhardtii. From these initial estimates, it is clear that aerobic heterotrophic growth on acetate has a low yield on carbon, while mixotrophically and autotrophically grown cells are significantly more carbon efficient.

Structure-based Engineering of a Plant-Fungal Hybrid Peroxidase for Enhanced Temperature and pH Tolerance.

PubMed

Kohler, Amanda C; Simmons, Blake A; Sale, Kenneth L

2018-04-28

In an age of ever-increasing biotechnological and industrial demand for new and specialized biocatalysts, rational protein engineering offers a direct approach to enzyme design and innovation. Heme peroxidases, as indispensable oxidative biocatalysts, provide a relatively mild alternative to the traditional harsh, and often toxic, chemical catalysts, and subsequently, have found widespread application throughout industry. However, the potential for these enzymes is far greater than their present use, as processes are currently restricted to the more stable, but less catalytically powerful, subset of peroxidases. Here we describe the structure-guided, rational engineering of a plant-fungal hybrid peroxidase built to overcome the application barrier of these high-reduction potential peroxidases. This engineered enzyme has the catalytic versatility and oxidative ability of a high-reduction potential versatile peroxidase, with enhanced temperature and pH tolerance similar to that of a highly stable plant peroxidase. Copyright © 2018 Elsevier Ltd. All rights reserved.

Rapidly Degradable Pyrotechnic System

DTIC Science & Technology

2009-02-01

casing. The second material and primary focus of the program effort was the improvement of a newly emerging enzyme , a thermophilic hydrolase formed...12 3.2.4.1 Details of Selected Enzyme (Description, Production and Environmental Impact) ......................14 4. MATERIALS AND METHODS...24 5.3 Improvement of extracelular enzyme secretion

Problems Associated with a Lack of Cohesive Policy in K-12 Pre-College Engineering

ERIC Educational Resources Information Center

Chandler, John; Fontenot, A. Dean; Tate, Derrick

2011-01-01

This article identifies a number of issues associated with current STEM education reform efforts, especially with regard to efforts to integrate engineering education into the K-12 curriculum. Precollege engineering is especially problematic in STEM reform since there is no well-established tradition of engineering in the K-12 curriculum. This…

Advanced liquid-cooled, turbocharged and intercooled stratified charge rotary engines for aircraft

NASA Technical Reports Server (NTRS)

Mount, Robert E.; Bartel, John; Hady, William F.

1987-01-01

Developments concerning stratified-charge rotary (SCR) engines over the past 10 years are reviewed. Aircraft engines being developed using SCR technology are shown and described, and the ability of such technology to meet general aviation engine needs is considered. Production timing and availability of SCR technology for the development of aviation rotary engines are discussed, and continuing efforts toward improving this technology, including NASA efforts, are described.

Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

NASA Technical Reports Server (NTRS)

Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

2016-01-01

Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

Synthetic control of a fitness tradeoff in yeast nitrogen metabolism

PubMed Central

Bayer, Travis S; Hoff, Kevin G; Beisel, Chase L; Lee, Jack J; Smolke, Christina D

2009-01-01

Background Microbial communities are involved in many processes relevant to industrial and medical biotechnology, such as the formation of biofilms, lignocellulosic degradation, and hydrogen production. The manipulation of synthetic and natural microbial communities and their underlying ecological parameters, such as fitness, evolvability, and variation, is an increasingly important area of research for synthetic biology. Results Here, we explored how synthetic control of an endogenous circuit can be used to regulate a tradeoff between fitness in resource abundant and resource limited environments in a population of Saccharomyces cerevisiae. We found that noise in the expression of a key enzyme in ammonia assimilation, Gdh1p, mediated a tradeoff between growth in low nitrogen environments and stress resistance in high ammonia environments. We implemented synthetic control of an endogenous Gdh1p regulatory network to construct an engineered strain in which the fitness of the population was tunable in response to an exogenously-added small molecule across a range of ammonia environments. Conclusion The ability to tune fitness and biological tradeoffs will be important components of future efforts to engineer microbial communities. PMID:19118500

Engineering Cellulases for Biorefinery

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

Kumar, Manoj

2010-06-27

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Homing endonucleases from mobile group I introns: discovery to genome engineering

PubMed Central

2014-01-01

Homing endonucleases are highly specific DNA cleaving enzymes that are encoded within genomes of all forms of microbial life including phage and eukaryotic organelles. These proteins drive the mobility and persistence of their own reading frames. The genes that encode homing endonucleases are often embedded within self-splicing elements such as group I introns, group II introns and inteins. This combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. Crystallographic analyses of representatives from all known homing endonuclease families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. Several homing endonucleases have been completely redesigned and used for a variety of genome engineering applications. Recent efforts to augment homing endonucleases with auxiliary DNA recognition elements and/or nucleic acid processing factors has further accelerated their use for applications that demand exceptionally high specificity and activity. PMID:24589358

In vitro metabolic engineering for the salvage synthesis of NAD(.).

PubMed

Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

2016-05-01

Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

PubMed Central

Chung, Daehwan; Cha, Minseok; Guss, Adam M.; Westpheling, Janet

2014-01-01

Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169–172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production. PMID:24889625

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii.

PubMed

Chung, Daehwan; Cha, Minseok; Guss, Adam M; Westpheling, Janet

2014-06-17

Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169-172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.

Network reconstruction and systems analysis of plant cell wall deconstruction by Neurospora crassa.

PubMed

Samal, Areejit; Craig, James P; Coradetti, Samuel T; Benz, J Philipp; Eddy, James A; Price, Nathan D; Glass, N Louise

2017-01-01

Plant biomass degradation by fungal-derived enzymes is rapidly expanding in economic importance as a clean and efficient source for biofuels. The ability to rationally engineer filamentous fungi would facilitate biotechnological applications for degradation of plant cell wall polysaccharides. However, incomplete knowledge of biomolecular networks responsible for plant cell wall deconstruction impedes experimental efforts in this direction. To expand this knowledge base, a detailed network of reactions important for deconstruction of plant cell wall polysaccharides into simple sugars was constructed for the filamentous fungus Neurospora crassa . To reconstruct this network, information was integrated from five heterogeneous data types: functional genomics, transcriptomics, proteomics, genetics, and biochemical characterizations. The combined information was encapsulated into a feature matrix and the evidence weighted to assign annotation confidence scores for each gene within the network. Comparative analyses of RNA-seq and ChIP-seq data shed light on the regulation of the plant cell wall degradation network, leading to a novel hypothesis for degradation of the hemicellulose mannan. The transcription factor CLR-2 was subsequently experimentally shown to play a key role in the mannan degradation pathway of N. crassa . Here we built a network that serves as a scaffold for integration of diverse experimental datasets. This approach led to the elucidation of regulatory design principles for plant cell wall deconstruction by filamentous fungi and a novel function for the transcription factor CLR-2. This expanding network will aid in efforts to rationally engineer industrially relevant hyper-production strains.

Engineering yeast consortia for surface-display of complex cellulosome structures

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

Chen, Wilfred

As our society marches toward a more technologically advanced future, energy and environmental sustainability are some of the most challenging problems we face today. Biomass is one of the most abundant renewable-feedstock for sustainable production of biofuels. However, the main technological obstacle to more widespread uses of this resource is the lack of low-cost technologies to overcome the recalcitrant nature of the cellulosic structure, especially the hydrolysis step on highly ordered celluloses. In this proposal, we successfully engineered several efficient and inexpensive whole-cell biocatalysts in an effort to produce economically compatible and sustainable biofuels, namely cellulosic ethanol. Our approach wasmore » to display of a highly efficient cellulolytic enzyme complex, named cellulosome, on the surface of a historical ethanol producer Saccharomyces cerevisiae for the simultaneous and synergistic saccharification and fermentation of cellulose to ethanol. We first demonstrated the feasibility of assembling a mini-cellulosome by incubating E. coli lysates expressing three different cellulases. Resting cells displaying mini-cellulosomes produced 4-fold more ethanol from phosphoric acid-swollen cellulose (PASC) than cultures with only added enzymes. The flexibility to assemble the mini-cellulosome structure was further demonstrated using a synthetic yeast consortium through intracellular complementation. Direct ethanol production from PASC was demonstrated with resting cell cultures. To create a microorganism suitable for a more cost-effective process, called consolidated bioprocessing (CBP), a synthetic consortium capable of displaying mini-cellulosomes on the cell surface via intercellular complementation was created. To further improve the efficiency, a new adaptive strategy of employing anchoring and adaptor scaffoldins to amplify the number of enzymatic subunits was developed, resulting in the creation of an artificial tetravalent cellulosome on the yeast surface and a significant improvement in cellulosic ethanol production. Although this adaptive strategy is ideal for assembling more complex cellulosome for large-scale production of cellulosic ethanol, a substantially larger number of enzymes (up to 10 to 12) is needed to better mimic the natural cellulosome structures for practical usage of the technology.« less

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.

Engineering chemical interactions in microbial communities.

PubMed

Kenny, Douglas J; Balskus, Emily P

2018-03-05

Microbes living within host-associated microbial communities (microbiotas) rely on chemical communication to interact with surrounding organisms. These interactions serve many purposes, from supplying the multicellular host with nutrients to antagonizing invading pathogens, and breakdown of chemical signaling has potentially negative consequences for both the host and microbiota. Efforts to engineer microbes to take part in chemical interactions represent a promising strategy for modulating chemical signaling within these complex communities. In this review, we discuss prominent examples of chemical interactions found within host-associated microbial communities, with an emphasis on the plant-root microbiota and the intestinal microbiota of animals. We then highlight how an understanding of such interactions has guided efforts to engineer microbes to participate in chemical signaling in these habitats. We discuss engineering efforts in the context of chemical interactions that enable host colonization, promote host health, and exclude pathogens. Finally, we describe prominent challenges facing this field and propose new directions for future engineering efforts.

STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

PubMed

Daugherty, Ashley B; Horton, John R; Cheng, Xiaodong; Lutz, Stefan

2015-02-06

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for ( S )-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

PubMed Central

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra

2017-01-01

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications. PMID:28455329

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

PubMed

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra; Miceli, Cristina

2017-07-01

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii ( Ef Amy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, Ef Amy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active Ef Amy with improved thermostability and catalytic efficiency at low temperatures. We engineered two Ef Amy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of Ef Amy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named Ef Amy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel Ef Amy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications. Copyright © 2017 Yang et al.

Reducing Enzyme Costs Increases the Market Potential of Biofuels (Fact Sheet)

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

Not Available

Cellulosic ethanol prices depend heavily on the cost of the cellulase enzymes used to break down the biomass into fermentable sugars. To reduce these costs, NREL partnered with two leading enzyme companies, Novozymes and Genencor, to engineer new cellulase enzymes that are exceptionally good at breaking down cellulose. Genencor is now part of DuPont Industrial Biosciences.

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

PubMed Central

2012-01-01

Background Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites. PMID:23148661

Engineered enzymatically active bacteriophages and methods of uses thereof

DOEpatents

Collins, James J [Newton, MA; Kobayashi, Hideki [Yokohama, JP; Kearn, Mads [Ottawa, CA; Araki, Michihiro [Minatoku, JP; Friedland, Ari [Boston, MA; Lu, Timothy Kuan-Ta [Palo Alto, CA

2012-05-22

The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

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.

Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

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

Brown, Margaret E.; Mukhopadhyay, Aindrila; Keasling, Jay D.

In this paper, we report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradationmore » pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. Finally, in the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.« less

Energy Efficient Engine combustor test hardware detailed design report

NASA Technical Reports Server (NTRS)

Burrus, D. L.; Chahrour, C. A.; Foltz, H. L.; Sabla, P. E.; Seto, S. P.; Taylor, J. R.

1984-01-01

The Energy Efficient Engine (E3) Combustor Development effort was conducted as part of the overall NASA/GE E3 Program. This effort included the selection of an advanced double-annular combustion system design. The primary intent was to evolve a design which meets the stringent emissions and life goals of the E3 as well as all of the usual performance requirements of combustion systems for modern turbofan engines. Numerous detailed design studies were conducted to define the features of the combustion system design. Development test hardware was fabricated, and an extensive testing effort was undertaken to evaluate the combustion system subcomponents in order to verify and refine the design. Technology derived from this development effort will be incorporated into the engine combustion system hardware design. This advanced engine combustion system will then be evaluated in component testing to verify the design intent. What is evolving from this development effort is an advanced combustion system capable of satisfying all of the combustion system design objectives and requirements of the E3. Fuel nozzle, diffuser, starting, and emissions design studies are discussed.

Bioenergy News | Bioenergy | NREL

Science.gov Websites

. April 13, 2018 News Release: Research Team Engineers a Better Plastic-Degrading Enzyme A breakthrough in enzyme research led by the U.S. Department of Energy's National Renewable Energy Laboratory (NREL) and the United Kingdom's University of Portsmouth has led to an improved variant of an enzyme that can

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Molecular Bases of Enantioselectivity of Haloalkane Dehalogenase DbjA

NASA Astrophysics Data System (ADS)

Sato, Yukari; Natsume, Ryo; Prokop, Zbynek; Brezovsky, Jan; Chaloupkova, Radka; Damborsky, Jiri; Nagata, Yuji; Senda, Toshiya

Enzymes are widely used for the synthesis of pharmaceuticals, agrochemicals, and food additives because they can catalyze high enantioselective transformations. In order to construct selective enzymes by protein engineering, it is important to understand the molecular basis of enzyme-substrate interactions that contribute to enantioselectivity. The haloalkane dehalogenase DbjA showed high enantioselectivity for two racemic mixtures: α-bromoesters and β-bromoalkanes. Thermodynamic analysis, protein crystallography, and computer simulations indicated that DbjA carries two bases for the enantiodiscrimination of each racemic mixture. This study helps us understand the molecular basis of the enantioselectivity and opens up new possibilities for constructing enantiospecific biocatalysts through protein engineering.

Engineering Cellulase Enzymes for Bioenergy

NASA Astrophysics Data System (ADS)

Atreya, Meera Elizabeth

Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for follow-up mutational studies. My goal was to improve the effective catalytic activity of cellulase enzymes in industrially-relevant conditions (such as in the presence of high concentrations of cellobiose or at elevated temperatures). The insights gained from my work on enzyme inhibition may inform future efforts to address this important issue. More efficient enzymes should reduce the amount of these proteins needed to break down cellulose to glucose. This, in turn, should decrease the price of the resulting biofuel making it more cost-competitive with fossil fuels and thus encouraging adoption of renewable transportation fuels that reduce our greenhouse gas emissions.

Military Engineer Contribution to Operational Art: The Hybrid Threat Environment

DTIC Science & Technology

2015-05-22

engineering are capabilities and activities that support the maneuver force, to include mobility , countermobility, and survivability tasks.12 These may...effort or “ways” engineers support the pursuit of the operational desired state. The engineer lines of effort (LOE) include assure mobility , enhance...protection, enable force projection and logistics, and building partner capacity and develop infrastructure.16 The assured mobility LOE seeks to

The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design.

PubMed

Kim, Su Jin; Lee, Jeong Ah; Joo, Jeong Chan; Yoo, Young Je; Kim, Yong Hwan; Song, Bong Keun

2010-01-01

Protein thermostability is a crucial issue in the practical application of enzymes, such as inorganic synthesis and enzymatic polymerization of phenol derivatives. Much attention has been focused on the enhancement and numerous successes have been achieved through protein engineering methods. Despite fruitful results based on random mutagenesis, it was still necessary to develop a novel strategy that can reduce the time and effort involved in this process. In this study, a rapid and effective strategy is described for increasing the thermal stability of a protein. Instead of random mutagenesis, a rational strategy was adopted to theoretically stabilize the thermo labile residues of a protein using computational methods. Protein residues with high flexibility can be thermo labile due to their large range of movement. Here, residue B factor values were used to identify putatively thermo labile residues and the RosettaDesign program was applied to search for stable sequences. Coprinus cinereus (CiP) heme peroxidase was selected as a model protein for its importance in commercial applications, such as the polymerization of phenolic compounds. Eleven CiP residues with the highest B factor values were chosen as target mutation sites for thermostabilization, and then redesigned using RosettaDesign to identify sequences. Eight mutants based on the redesigns, were produced as functional enzymes and two of these (S323Y and E328D) showed increased thermal stability over the wild-type in addition to conserved catalytic activity. Thus, this strategy can be used as a rapid and effective in silico design tool for obtaining thermostable proteins. (c) 2010 American Institute of Chemical Engineers

A status report on the Energy Efficient Engine Project

NASA Technical Reports Server (NTRS)

Macioce, L. E.; Schaefer, J. W.; Saunders, N. T.

1980-01-01

The Energy Efficient Engine (E3) Project is directed at providing, by 1984, the advanced technologies which could be used for a new generation of fuel conservative turbofan engines. This paper summarizes the scope of the entire project and the current status of these efforts. Included is a description of the preliminary designs of the fully developed engines, the potential benefits of these advanced engines, and highlights of some of the component technology efforts conducted to date.

[Fermentation production of microbial catalase and its application in textile industry].

PubMed

Zhang, Dongxu; Du, Guocheng; Chen, Jian

2010-11-01

Microbial catalase is an important industrial enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen. This enzyme has great potential of application in food, textile and pharmaceutical industries. The production of microbial catalase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering. In this paper, we review the progresses in fermentation production of microbial catalase and its application in textile industry. Among these progresses, we will highlight strain isolation, substrate and environment optimization, enzyme induction, construction of engineering strains and application process optimization. Meanwhile, we also address future research trends for microbial catalase production and its application in textile industry. Molecular modification (site-directed mutagenesis and directed revolution) will endue catalase with high pH and temperature stabilities. Improvement of catalase production, based on the understanding of induction mechanism and the process control of recombinant stain fermentation, will further accelerate the application of catalase in textile industry.

Integrated cellulosic enzymes hydrolysis and fermentative advanced yeast bioconversion solution ready for biomass biorefineries

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

Kumar, Manoj

2011-05-04

These are slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme,more » as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Development of a Commerical Enzyme System for Lignocellulosic Biomass Saccharification

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

Kumar, Manoj

2011-02-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Fully Integrated Lignocellulosic Biorefinery with Onsite Production of Enzymes and Yeast

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

Kumar, Manoj

2010-06-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Biochemical Conversion Processes of Lignocellulosic Biomass to Fuels and Chemicals - A Review.

PubMed

Brethauer, Simone; Studer, Michael H

2015-01-01

Lignocellulosic biomass - such as wood, agricultural residues or dedicated energy crops - is a promising renewable feedstock for production of fuels and chemicals that is available at large scale at low cost without direct competition for food usage. Its biochemical conversion in a sugar platform biorefinery includes three main unit operations that are illustrated in this review: the physico-chemical pretreatment of the biomass, the enzymatic hydrolysis of the carbohydrates to a fermentable sugar stream by cellulases and finally the fermentation of the sugars by suitable microorganisms to the target molecules. Special emphasis in this review is put on the technology, commercial status and future prospects of the production of second-generation fuel ethanol, as this process has received most research and development efforts so far. Despite significant advances, high enzyme costs are still a hurdle for large scale competitive lignocellulosic ethanol production. This could be overcome by a strategy termed 'consolidated bioprocessing' (CBP), where enzyme production, enzymatic hydrolysis and fermentation is integrated in one step - either by utilizing one genetically engineered superior microorganism or by creating an artificial co-culture. Insight is provided on both CBP strategies for the production of ethanol as well as of advanced fuels and commodity chemicals.

Crystallization and preliminary crystallographic analysis of l-asparaginase from Erwinia carotovora

PubMed Central

Wikman, Linnea E. K.; Krasotkina, Julya; Kuchumova, Anastasia; Sokolov, Nikolay N.; Papageorgiou, Anastassios C.

2005-01-01

Bacterial l-asparaginases have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia for over 30 y. However, their use is limited owing to the glutaminase activity of the administered enzymes, which results in serious side effects. In contrast, l-asparaginase from Erwinia carotovora exhibits low glutaminase activity at physiological concentrations of l-asparagine and l-glutamine in the blood. Recombinant Er. carotovora l-­asparaginase was crystallized in the presence of l-glutamate by the hanging-drop vapour-diffusion method using 10 mg ml−1 purified enzyme, 16–18%(w/v) PEG 3350 and 0.2 M NaF. X-ray diffraction data were collected to 2.6 Å at 293 K using an in-house rotating-anode generator. The crystals belong to the monoclinic P21 space group, with unit-cell parameters a = 78.0, b = 112.3, c = 78.7 Å, β = 101.9° and a homotetramer in the crystallographic asymmetric unit. A molecular-replacement solution has been found and refinement is currently in progress. The crystal structure may provide leads towards protein-engineering efforts aimed at safer asparaginase administration in leukaemia treatment. PMID:16511054

Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability.

PubMed

Moraïs, Sarah; Stern, Johanna; Kahn, Amaranta; Galanopoulou, Anastasia P; Yoav, Shahar; Shamshoum, Melina; Smith, Matthew A; Hatzinikolaou, Dimitris G; Arnold, Frances H; Bayer, Edward A

2016-01-01

The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.Graphical abstractConversion of conventional designer cellulosomes into thermophilic designer cellulosomes.

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae.

PubMed

Kwak, Suryang; Kim, Soo Rin; Xu, Haiqing; Zhang, Guo-Chang; Lane, Stephan; Kim, Heejin; Jin, Yong-Su

2017-11-01

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Layered Systems Engineering Engines

NASA Technical Reports Server (NTRS)

Breidenthal, Julian C.; Overman, Marvin J.

2009-01-01

A notation is described for depicting the relationships between multiple, contemporaneous systems engineering efforts undertaken within a multi-layer system-of-systems hierarchy. We combined the concepts of remoteness of activity from the end customer, depiction of activity on a timeline, and data flow to create a new kind of diagram which we call a "Layered Vee Diagram." This notation is an advance over previous notations because it is able to be simultaneously precise about activity, level of granularity, product exchanges, and timing; these advances provide systems engineering managers a significantly improved ability to express and understand the relationships between many systems engineering efforts. Using the new notation, we obtain a key insight into the relationship between project duration and the strategy selected for chaining the systems engineering effort between layers, as well as insights into the costs, opportunities, and risks associated with alternate chaining strategies.

'Unknown' proteins and 'orphan' enzymes: the missing half of the engineering parts list--and how to find it.

PubMed

Hanson, Andrew D; Pribat, Anne; Waller, Jeffrey C; de Crécy-Lagard, Valérie

2009-12-14

Like other forms of engineering, metabolic engineering requires knowledge of the components (the 'parts list') of the target system. Lack of such knowledge impairs both rational engineering design and diagnosis of the reasons for failures; it also poses problems for the related field of metabolic reconstruction, which uses a cell's parts list to recreate its metabolic activities in silico. Despite spectacular progress in genome sequencing, the parts lists for most organisms that we seek to manipulate remain highly incomplete, due to the dual problem of 'unknown' proteins and 'orphan' enzymes. The former are all the proteins deduced from genome sequence that have no known function, and the latter are all the enzymes described in the literature (and often catalogued in the EC database) for which no corresponding gene has been reported. Unknown proteins constitute up to about half of the proteins in prokaryotic genomes, and much more than this in higher plants and animals. Orphan enzymes make up more than a third of the EC database. Attacking the 'missing parts list' problem is accordingly one of the great challenges for post-genomic biology, and a tremendous opportunity to discover new facets of life's machinery. Success will require a co-ordinated community-wide attack, sustained over years. In this attack, comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes. Furthermore, it is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.

Biocatalyst Enhancement

EPA Science Inventory

The increasing availability of enzyme collections has assisted attempts by pharmaceutical producers to adopt green chemistry approaches to manufacturing. A joint effort between an enzyme producer and a pharmaceutical manufacturer has been enhanced over the past three years by ena...

RNA as an Enzyme.

ERIC Educational Resources Information Center

Cech, Thomas R.

1986-01-01

Reviews current findings that explain RNA's function as an enzyme in addition to being an informational molecule. Highlights recent research efforts and notes changes in the information base on RNA activity. Includes models and diagrams of RNA activity. (ML)

Student Interest in Engineering Design-Based Science

ERIC Educational Resources Information Center

Selcen Guzey, S.; Moore, Tamara J.; Morse, Gillian

2016-01-01

Current reform efforts in science education around the world call on teachers to use integrated approaches to teach science. As a part of such reform efforts in the United States, engineering practices and engineering design have been identified in K-12 science education standards. However, there is relatively little is known about effective ways…

What are the Limitations of Enzymes in Synthetic Organic Chemistry?

PubMed

Reetz, Manfred T

2016-12-01

Enzymes have been used in organic chemistry and biotechnology for 100 years, but their widespread application has been prevented by a number of limitations, including the often-observed limited thermostability, narrow substrate scope, and low or wrong stereo- and/or regioselectivity. Directed evolution provides a means to address and generally solve these problems, especially since recent methodology development has made this protein engineering method faster, more efficient, and more reliable than in the past. This Darwinian approach to asymmetric catalysis has led to a number of industrial applications. Metabolic-pathway engineering, mutasynthesis, and fermentation are likewise enzyme-based techniques that enrich chemistry. This account outlines the scope, and particularly, the limitations, of biocatalysis. The complementary nature of enzymes and man-made catalysts is emphasized. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes.

PubMed

Kürten, Charlotte; Syrén, Per-Olof

2016-01-16

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Directed evolution: tailoring biocatalysts for industrial applications.

PubMed

Kumar, Ashwani; Singh, Suren

2013-12-01

Current challenges and promises of white biotechnology encourage protein engineers to use a directed evolution approach to generate novel and useful biocatalysts for various sets of applications. Different methods of enzyme engineering have been used in the past in an attempt to produce enzymes with improved functions and properties. Recent advancement in the field of random mutagenesis, screening, selection and computational design increased the versatility and the rapid development of enzymes under strong selection pressure with directed evolution experiments. Techniques of directed evolution improve enzymes fitness without understanding them in great detail and clearly demonstrate its future role in adapting enzymes for use in industry. Despite significant advances to date regarding biocatalyst improvement, there still remains a need to improve mutagenesis strategies and development of easy screening and selection tools without significant human intervention. This review covers fundamental and major development of directed evolution techniques, and highlights the advances in mutagenesis, screening and selection methods with examples of enzymes developed by using these approaches. Several commonly used methods for creating molecular diversity with their advantages and disadvantages including some recently used strategies are also discussed.

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme.

PubMed

Watkins, Daniel W; Jenkins, Jonathan M X; Grayson, Katie J; Wood, Nicola; Steventon, Jack W; Le Vay, Kristian K; Goodwin, Matthew I; Mullen, Anna S; Bailey, Henry J; Crump, Matthew P; MacMillan, Fraser; Mulholland, Adrian J; Cameron, Gus; Sessions, Richard B; Mann, Stephen; Anderson, J L Ross

2017-08-25

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 . The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 .

Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

PubMed

Fujishima, Kosuke; Wang, Kendrick M; Palmer, Jesse A; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J

2018-01-29

Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

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

Engineering Ligninolytic Consortium for Bioconversion of Lignocelluloses to Ethanol and Chemicals.

PubMed

Bilal, Muhammad; Nawaz, Muhammad Zohaib; Iqbal, Hafiz M N; Hou, Jialin; Mahboob, Shahid; Al-Ghanim, Khalid A; Cheng, Hairong

2018-01-01

Rising environmental concerns and recent global scenario of cleaner production and consumption are leading to the design of green industrial processes to produce alternative fuels and chemicals. Although bioethanol is one of the most promising and eco-friendly alternatives to fossil fuels yet its production from food and feed has received much negative criticism. The main objective of this study was to present the noteworthy potentialities of lignocellulosic biomass as an enormous and renewable biological resource. The particular focus was also given on engineering ligninolytic consortium for bioconversion of lignocelluloses to ethanol and chemicals on sustainable and environmentally basis. Herein, an effort has been made to extensively review, analyze and compile salient information related to the topic of interest. Several authentic bibliographic databases including PubMed, Scopus, Elsevier, Springer, Bentham Science and other scientific databases were searched with utmost care, and inclusion/ exclusion criterion was adopted to appraise the quality of retrieved peer-reviewed research literature. Bioethanol production from lignocellulosic biomass can largely satisfy the possible inconsistency of first-generation ethanol since it utilizes inedible lignocellulosic feedstocks, primarily sourced from agriculture and forestry wastes. Two major polysaccharides in lignocellulosic biomass namely, cellulose and hemicellulose constitute a complex lignocellulosic network by connecting with lignin, which is highly recalcitrant to depolymerization. Several attempts have been made to reduce the cost involved in the process through improving the pretreatment process. While, the ligninolytic enzymes of white rot fungi (WRF) including laccase, lignin peroxidase (LiP), and manganese peroxidase (MnP) have appeared as versatile biocatalysts for delignification of several lignocellulosic residues. The first part of the review is mainly focused on engineering ligninolytic consortium. In the second part, WRF and its unique ligninolytic enzyme-based bio-delignification of lignocellulosic biomass, enzymatic hydrolysis, and fermentation of hydrolyzed feedstock are discussed. The metabolic engineering, enzymatic engineering, synthetic biology aspects for ethanol production and platform chemicals production are comprehensively reviewed in the third part. Towards the end information is also given on futuristic viewpoints. In conclusion, given the present unpredicted scenario of energy and fuel crisis accompanied by global warming, lignocellulosic bioethanol holds great promise as an alternative to petroleum. Apart from bioethanol, the simultaneous production of other value-added products may improve the economics of lignocellulosic bioethanol bioconversion process. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Overview of NASA Lewis Research Center free-piston Stirling engine activities

NASA Technical Reports Server (NTRS)

Slaby, J. G.

1984-01-01

A generic free-piston Stirling technology project is being conducted to develop technologies generic to both space power and terrestrial heat pump applications in a cooperative, cost-shared effort. The generic technology effort includes extensive parametric testing of a 1 kW free-piston Stirling engine (RE-1000), development of a free-piston Stirling performance computer code, design and fabrication under contract of a hydraulic output modification for RE-1000 engine tests, and a 1000-hour endurance test, under contract, of a 3 kWe free-piston Stirling/alternator engine. A newly initiated space power technology feasibility demonstration effort addresses the capability of scaling a free-piston Stirling/alternator system to about 25 kWe; developing thermodynamic cycle efficiency or equal to 70 percent of Carnot at temperature ratios in the order of 1.5 to 2.0; achieving a power conversion unit specific weight of 6 kg/kWe; operating with noncontacting gas bearings; and dynamically balancing the system. Planned engine and component design and test efforts are described.

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

PubMed

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

2016-04-01

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

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.

Enzyme as catalytic wheel powered by a Markovian engine: conformational coupling and barrier surfing models

NASA Astrophysics Data System (ADS)

Tsong, Tian Yow; Chang, Cheng-Hung

2005-05-01

We examine a typical Michaelis-Menten Enzyme (MME) and redress it to form a transducer of free energy, and electric, acoustic, or other types of energy. This amendment and extension is necessary in lieu of recent experiments in which enzymes are shown to perform pump, motor, and locomotion functions resembling their macroscopic counterparts. Classical textbook depicts enzyme, or an MME, as biocatalyst which can enhance the rate of a chemical reaction by lowering the activation barrier but cannot shift the thermodynamic equilibrium of the biochemical reaction. An energy transducer, on the other hand, must also be able to harvest, store, or divert energy and in doing so alter the chemical equilibrium, change the energy form, fuel an energy consuming process, or perform all these functions stepwise in one catalytic turnover. The catalytic wheel presented in this communication is both a catalyst and an energy transducer and can perform all these tasks with ease. A Conformational Coupling Model for the rotary motors and a Barrier Surfing Model for the track-guided stepping motors and transporters, are presented and compared. It is shown that the core engine of the catalytic wheel, or a Brownian motor, is a Markovian engine. It remains to be seen if this core engine is the basic mechanism for a wide variety of bio-molecular energy transducers, as well as certain other dynamic systems, for example, the Parrondo's Games.

An air-breathing enzymatic cathode with extended lifetime by continuous laccase supply.

PubMed

Kipf, Elena; Sané, Sabine; Morse, Daniel; Messinger, Thorsten; Zengerle, Roland; Kerzenmacher, Sven

2018-04-22

We present a novel concept of an air-breathing enzymatic biofuel cell cathode combined with continuous supply of unpurified laccase-containing supernatant of the white-rot fungus Trametes versicolor for extended lifetime. The air-breathing cathode design obviates the need for energy-intensive active aeration. In a corresponding long-term experiment at a constant current density of 50 µA cm -2 , we demonstrated an increased lifetime of 33 days (cathode potential above 0.430 V vs. SCE), independent of enzyme degradation. The obtained data suggest that theoretically a longer lifetime is feasible. However, further engineering efforts are required to prevent clogging and fouling of the supply tubes. These results represent an important step towards the realization of enzymatic biofuel cell cathodes with extended lifetime and enhanced performance. Copyright © 2018 Elsevier Ltd. All rights reserved.

ECUT (Energy Conversion and Utilization Technologies) program: Biocatalysis Project

NASA Technical Reports Server (NTRS)

1988-01-01

Fiscal year 1987 research activities and accomplishments for the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Division are presented. The project's technical activities were organized into three work elements. The Molecular Modeling and Applied Genetics work element includes modeling and simulation studies to verify a dynamic model of the enzyme carboxypeptidase; plasmid stabilization by chromosomal integration; growth and stability characteristics of plasmid-containing cells; and determination of optional production parameters for hyper-production of polyphenol oxidase. The Bioprocess Engineering work element supports efforts in novel bioreactor concepts that are likely to lead to substantially higher levels of reactor productivity, product yields, and lower separation energetics. The Bioprocess Design and Assessment work element attempts to develop procedures (via user-friendly computer software) for assessing the economics and energetics of a given biocatalyst process.

Research status and development of application fields in enzyme technology

NASA Astrophysics Data System (ADS)

Ji, Y. B.; Wang, S. W.; Yu, M.; Ru, X.; Wei, C.; Zhu, H. J.; Li, Z. Y.; Zhao, H.; Qiao, A. N.; Guo, S. Z.; Lu, L.

2018-01-01

Biological enzymes are catalyzed by living cells, most of which are proteins, and very few are RNA. Biological engineering as a new high-tech has been rapid development, Enzyme manufacturing and application areas are gradually expanding, In this paper, the status and progress of the application of enzyme technology are reviewed by reviewing the literature. and aims to provide reference for the application of enzyme technology and provide scientific basis for its future research and development in new field.

Industrial applications of enzyme biocatalysis: Current status and future aspects.

PubMed

Choi, Jung-Min; Han, Sang-Soo; Kim, Hak-Sung

2015-11-15

Enzymes are the most proficient catalysts, offering much more competitive processes compared to chemical catalysts. The number of industrial applications for enzymes has exploded in recent years, mainly owing to advances in protein engineering technology and environmental and economic necessities. Herein, we review recent progress in enzyme biocatalysis, and discuss the trends and strategies that are leading to broader industrial enzyme applications. The challenges and opportunities in developing biocatalytic processes are also discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein engineering in designing tailored enzymes and microorganisms for biofuels production

PubMed Central

Wen, Fei; Nair, Nikhil U; Zhao, Huimin

2009-01-01

Summary Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. However, the recalcitrant nature of lignocellulose poses technical hurdles to an economically viable biorefinery. Low enzymatic hydrolysis efficiency and low productivity, yield, and titer of biofuels are among the top cost contributors. Protein engineering has been used to improve the performances of lignocellulose-degrading enzymes, as well as proteins involved in biofuel synthesis pathways. Unlike its great success seen in other industrial applications, protein engineering has achieved only modest results in improving the lignocellulose-to-biofuels efficiency. This review will discuss the unique challenges that protein engineering faces in the process of converting lignocellulose to biofuels and how they are addressed by recent advances in this field. PMID:19660930

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues.

PubMed

Ben Taher, Imen; Fickers, Patrick; Chniti, Sofien; Hassouna, Mnasser

2017-03-01

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L -1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L -1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397-406, 2017. © 2017 American Institute of Chemical Engineers.

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

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.

An overview of the NASA rotary engine research program

NASA Technical Reports Server (NTRS)

Meng, P. R.; Hady, W. F.

1984-01-01

A brief overview and technical highlights of the research efforts and studies on rotary engines over the last several years at the NASA Lewis Research Center are presented. The test results obtained from turbocharged rotary engines and preliminary results from a high performance single rotor engine were discussed. Combustion modeling studies of the rotary engine and the use of a Laser Doppler Velocimeter to confirm the studies were examined. An in-house program in which a turbocharged rotary engine was installed in a Cessna Skymaster for ground test studies was reviewed. Details are presented on single rotor stratified charge rotary engine research efforts, both in-house and on contract.

RubisCO selection using the vigorously aerobic and metabolically versatile bacterium Ralstonia eutropha.

PubMed

Satagopan, Sriram; Tabita, F Robert

2016-08-01

Recapturing atmospheric CO2 is key to reducing global warming and increasing biological carbon availability. Ralstonia eutropha is a biotechnologically useful aerobic bacterium that uses the Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) for CO2 utilization, suggesting that it may be a useful host to bioselect RubisCO molecules with improved CO2 -capture capabilities. A host strain of R. eutropha was constructed for this purpose after deleting endogenous genes encoding two related RubisCOs. This strain could be complemented for CO2 -dependent growth by introducing native or heterologous RubisCO genes. Mutagenesis and suppressor selection identified amino acid substitutions in a hydrophobic region that specifically influences RubisCO's interaction with its substrates, particularly O2 , which competes with CO2 at the active site. Unlike most RubisCOs, the R. eutropha enzyme has evolved to retain optimal CO2 -fixation rates in a fast-growing host, despite the presence of high levels of competing O2 . Yet its structure-function properties resemble those of several commonly found RubisCOs, including the higher plant enzymes, allowing strategies to engineer analogous enzymes. Because R. eutropha can be cultured rapidly under harsh environmental conditions (e.g., with toxic industrial flue gas), in the presence of near saturation levels of oxygen, artificial selection and directed evolution studies in this organism could potentially impact efforts toward improving RubisCO-dependent biological CO2 utilization in aerobic environments. d-ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39; phosphoribulokinase, EC 2.7.1.19. © 2016 Federation of European Biochemical Societies.

Enhancing the light-driven production of D-lactate by engineering cyanobacterium using a combinational strategy

NASA Astrophysics Data System (ADS)

Li, Chao; Tao, Fei; Ni, Jun; Wang, Yu; Yao, Feng; Xu, Ping

2015-05-01

It is increasingly attractive to engineer cyanobacteria for bulk production of chemicals from CO2. However, cofactor bias of cyanobacteria is different from bacteria that prefer NADH, which hampers cyanobacterial strain engineering. In this study, the key enzyme D-lactate dehydrogenase (LdhD) from Lactobacillus bulgaricus ATCC11842 was engineered to reverse its favored cofactor from NADH to NADPH. Then, the engineered enzyme was introduced into Synechococcus elongatus PCC7942 to construct an efficient light-driven system that produces D-lactic acid from CO2. Mutation of LdhD drove a fundamental shift in cofactor preference towards NADPH, and increased D-lactate productivity by over 3.6-fold. We further demonstrated that introduction of a lactic acid transporter and bubbling CO2-enriched air also enhanced D-lactate productivity. Using this combinational strategy, increased D-lactate concentration and productivity were achieved. The present strategy may also be used to engineer cyanobacteria for producing other useful chemicals.

An airline study of advanced technology requirements for advanced high speed commercial engines. 3: Propulsion system requirements

NASA Technical Reports Server (NTRS)

Sallee, G. P.

1973-01-01

The advanced technology requirements for an advanced high speed commercial transport engine are presented. The results of the phase 3 effort cover the requirements and objectives for future aircraft propulsion systems. These requirements reflect the results of the Task 1 and 2 efforts and serve as a baseline for future evaluations, specification development efforts, contract/purchase agreements, and operational plans for future subsonic commercial engines. This report is divided into five major sections: (1) management objectives for commercial propulsion systems, (2) performance requirements for commercial transport propulsion systems, (3) design criteria for future transport engines, (4) design requirements for powerplant packages, and (5) testing.

Engineering cells for cell culture bioprocessing--physiological fundamentals.

PubMed

Seth, Gargi; Hossler, Patrick; Yee, Joon Chong; Hu, Wei-Shou

2006-01-01

In the past decade, we have witnessed a tremendous increase in the number of mammalian cell-derived therapeutic proteins with clinical applications. The success of making these life-saving biologics available to the public is partly due to engineering efforts to enhance process efficiency. To further improve productivity, much effort has been devoted to developing metabolically engineered producing cells, which possess characteristics favorable for large-scale bioprocessing. In this article we discuss the fundamental physiological basis for cell engineering. Different facets of cellular mechanisms, including metabolism, protein processing, and the balancing pathways of cell growth and apoptosis, contribute to the complex traits of favorable growth and production characteristics. We present our assessment of the current state of the art by surveying efforts that have already been undertaken in engineering cells for a more robust process. The concept of physiological homeostasis as a key determinant and its implications on cell engineering is emphasized. Integrating the physiological perspective with cell culture engineering will facilitate attainment of dream cells with superlative characteristics.

Genetically Engineered Theranostic Mesenchymal Stem Cells for the Evaluation of the Anticancer Efficacy of Enzyme/Prodrug Systems

PubMed Central

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-01-01

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TKSR39 mutants), yeast cytosine deaminase: uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs’ bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. PMID:25575867

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

PubMed Central

Capone, Simona; Ćorajević, Lejla; Bonifert, Günther; Murth, Patrick; Maresch, Daniel; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

2015-01-01

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins. PMID:26404235

The energy efficient engine project

NASA Technical Reports Server (NTRS)

Macioce, L. E.; Schaefer, J. W.; Saunders, N. T.

1980-01-01

The Energy Efficient Engine Project is directed at providing, by 1984, the advanced technologies which could be used for a generation of fuel conservative turbofan engines. The project is conducted through contracts with the General Electric Company and Pratt and Whitney Aircraft. The scope of the entire project and the current status of these efforts are summarized. A description of the preliminary designs of the fully developed engines is included and the potential benefits of these advanced engines, as well as highlights of some of the component technology efforts conducted to date, are discussed.

Optimization of yeast-based production of medicinal protoberberine alkaloids.

PubMed

Galanie, Stephanie; Smolke, Christina D

2015-09-16

Protoberberine alkaloids are bioactive molecules abundant in plant preparations for traditional medicines. Yeast engineered to express biosynthetic pathways for fermentative production of these compounds will further enable investigation of the medicinal properties of these molecules and development of alkaloid-based drugs with improved efficacy and safety. Here, we describe the optimization of a biosynthetic pathway in Saccharomyces cerevisiae for conversion of rac-norlaudanosoline to the protoberberine alkaloid (S)-canadine. This yeast strain is engineered to express seven heterologous enzymes, resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. The seven enzymes include three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase. A number of strategies were implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization, that led to an over 70-fold increase in canadine titer up to 1.8 mg/L. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines, for the first time in a microbial host. We also demonstrate that this strain is viable at pilot scale. By applying metabolic engineering and synthetic biology strategies for increased conversion of simple benzylisoquinoline alkaloids to complex protoberberine alkaloids, this work will facilitate chemoenzymatic synthesis or de novo biosynthesis of these and other high-value compounds using a microbial cell factory.

The synthesis of starch from carbon dioxide using isolubilized stabilized enzymes

NASA Technical Reports Server (NTRS)

Bassham, J. A.; Bearden, L.; Wilke, C.; Carroad, P.; Mitra, G.; Ige, R.

1972-01-01

Systems for artificial manufacture of starch and for delineation of technological areas, and the rationale for studying them are considered. A discussion of the enzyme-catalyzed routes of synthesis available and a choice as to the most promising route are presented. A discussion of the enzymes involved, of enzyme insolubilization technology, and of possible engineering approaches, with examples in the form of model calculations for both reactors and separators, are also presented.

Overview of the 1986 free-piston Stirling activities at NASA Lewis Research Center

NASA Technical Reports Server (NTRS)

Alger, Donald L.

1986-01-01

An overview of the NASA Lewis Research Center's free-piston Stirling engine research is presented, including efforts to improve and advance its design for use in specific space power applications. These efforts are a part of the SP-100 program being conducted to support the Department of Defense (DOD), Department of Energy (DOE) and NASA. Such efforts include: (1) the testing and improvement of 25 kWe Stirling Space Power Demonstrator Engine (SPDE); (2) the preliminary design of 25 kWe single-cylinder Experimental stirling Space Engine (ESSE); and, (3) a study to determine the feasibility of scaling a single-cylinder free-piston Stirling engine/linear alternator to 150 kWe. Other NASA Lewis free-piston Stirling engine activities will be described, directed toward the advancement of general free-piston Stirling engine technology and its application in specific terrestrial applications. One such effort, supported by DOE/Oak Ridge National Laboratory (DRNL), is the development of a free-piston Stirling engine which produces hydraulic power. Finally, a terrestrial solar application involving a conceptual design of a 25 kWe Solar Advanced Stirling Conversion System (ASCS) capable of delivering power to an electric utility grid will be discussed. The latter work is supported by DOE/Sandia National Laboratory (SNLA).

Investigating a Bio-Engineered Enzyme.

ERIC Educational Resources Information Center

Bullerwell, Lornie; And Others

1994-01-01

Describes science experiments with the enzyme lactose, which is available commercially as Lactaid and Dairy Ease. Experiments show how the rate of reaction of lactose converted to glucose and galactose is influenced by temperature, pH, and substrate concentration. (PR)

Cellulase recycling in biorefineries--is it possible?

PubMed

Gomes, Daniel; Rodrigues, Ana Cristina; Domingues, Lucília; Gama, Miguel

2015-05-01

On a near future, bio-based economy will assume a key role in our lives. Lignocellulosic materials (e.g., agroforestry residues, industrial/solid wastes) represent a cheaper and environmentally friendly option to fossil fuels. Indeed, following suitable processing, they can be metabolized by different microorganisms to produce a wide range of compounds currently obtained by chemical synthesis. However, due to the recalcitrant nature of these materials, they cannot be directly used by microorganisms, the conversion of polysaccharides into simpler sugars being thus required. This conversion, which is usually undertaken enzymatically, represents a significant part on the final cost of the process. This fact has driven intense efforts on the reduction of the enzyme cost following different strategies. Here, we describe the fundamentals of the enzyme recycling technology, more specifically, cellulase recycling. We focus on the main strategies available for the recovery of both the liquid- and solid-bound enzyme fractions and discuss the relevant operational parameters (e.g., composition, temperature, additives, and pH). Although the efforts from the industry and enzyme suppliers are primarily oriented toward the development of enzyme cocktails able to quickly and effectively process biomass, it seems clear by now that enzyme recycling is technically possible.

Microbial synthesis of medium-chain chemicals from renewables.

PubMed

Sarria, Stephen; Kruyer, Nicholas S; Peralta-Yahya, Pamela

2017-12-01

Linear, medium-chain (C8-C12) hydrocarbons are important components of fuels as well as commodity and specialty chemicals. As industrial microbes do not contain pathways to produce medium-chain chemicals, approaches such as overexpression of endogenous enzymes or deletion of competing pathways are not available to the metabolic engineer; instead, fatty acid synthesis and reversed β-oxidation are manipulated to synthesize medium-chain chemical precursors. Even so, chain lengths remain difficult to control, which means that purification must be used to obtain the desired products, titers of which are typically low and rarely exceed milligrams per liter. By engineering the substrate specificity and activity of the pathway enzymes that generate the fatty acyl intermediates and chain-tailoring enzymes, researchers can boost the type and yield of medium-chain chemicals. Development of technologies to both manipulate chain-tailoring enzymes and to assay for products promises to enable the generation of g/L yields of medium-chain chemicals.

Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases.

PubMed

Matuskova, Miroslava; Kozovska, Zuzana; Toro, Lenka; Durinikova, Erika; Tyciakova, Silvia; Cierna, Zuzana; Bohovic, Roman; Kucerova, Lucia

2015-04-09

Metastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP. Human adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination. We demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases. Combined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy in vivo.

Recent Stirling engine loss-understanding results

NASA Technical Reports Server (NTRS)

Tew, Roy C.; Thieme, Lanny G.; Dudenhoefer, James E.

1990-01-01

For several years, NASA and other U.S. government agencies have been funding experimental and analytical efforts to improve the understanding of Stirling thermodynamic losses. NASA's objective is to improve Stirling engine design capability to support the development of new engines for space power. An overview of these efforts was last given at the 1988 IECEC. Recent results of this research are reviewed.

Design, development, and evaluation of a second generation interactive Simulator for Engineering Ethics Education (SEEE2).

PubMed

Alfred, Michael; Chung, Christopher A

2012-12-01

This paper describes a second generation Simulator for Engineering Ethics Education. Details describing the first generation activities of this overall effort are published in Chung and Alfred (Sci Eng Ethics 15:189-199, 2009). The second generation research effort represents a major development in the interactive simulator educational approach. As with the first generation effort, the simulator places students in first person perspective scenarios involving different types of ethical situations. Students must still gather data, assess the situation, and make decisions. The approach still requires students to develop their own ability to identify and respond to ethical engineering situations. However, were as, the generation one effort involved the use of a dogmatic model based on National Society of Professional Engineers' Code of Ethics, the new generation two model is based on a mathematical model of the actual experiences of engineers involved in ethical situations. This approach also allows the use of feedback in the form of decision effectiveness and professional career impact. Statistical comparisons indicate a 59 percent increase in overall knowledge and a 19 percent improvement in teaching effectiveness over an Internet Engineering Ethics resource based approach.

The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization.

PubMed

Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

2016-11-01

Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified.

Biofuel cells for biomedical applications: colonizing the animal kingdom.

PubMed

Falk, Magnus; Narváez Villarrubia, Claudia W; Babanova, Sofia; Atanassov, Plamen; Shleev, Sergey

2013-07-22

Interdisciplinary research has combined the efforts of many scientists and engineers to gain an understanding of biotic and abiotic electrochemical processes, materials properties, biomedical, and engineering approaches for the development of alternative power-generating and/or energy-harvesting devices, aiming to solve health-related issues and to improve the quality of human life. This review intends to recapitulate the principles of biofuel cell development and the progress over the years, thanks to the contribution of cross-disciplinary researchers that have combined knowledge and innovative ideas to the field. The emergence of biofuel cells, as a response to the demand of electrical power devices that can operate under physiological conditions, are reviewed. Implantable biofuel cells operating inside living organisms have been envisioned for over fifty years, but few reports of implanted devices have existed up until very recently. The very first report of an implanted biofuel cell (implanted in a grape) was published only in 2003 by Adam Heller and his coworkers. This work was a result of earlier scientific efforts of this group to "wire" enzymes to the electrode surface. The last couple of years have, however, seen a multitude of biofuel cells being implanted and operating in different living organisms, including mammals. Herein, the evolution of the biofuel concept, the understanding and employment of catalyst and biocatalyst processes to mimic biological processes, are explored. These potentially green technology biodevices are designed to be applied for biomedical applications to power nano- and microelectronic devices, drug delivery systems, biosensors, and many more. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Steps towards the synthetic biology of polyketide biosynthesis.

PubMed

Cummings, Matthew; Breitling, Rainer; Takano, Eriko

2014-02-01

Nature is providing a bountiful pool of valuable secondary metabolites, many of which possess therapeutic properties. However, the discovery of new bioactive secondary metabolites is slowing down, at a time when the rise of multidrug-resistant pathogens and the realization of acute and long-term side effects of widely used drugs lead to an urgent need for new therapeutic agents. Approaches such as synthetic biology are promising to deliver a much-needed boost to secondary metabolite drug development through plug-and-play optimized hosts and refactoring novel or cryptic bacterial gene clusters. Here, we discuss this prospect focusing on one comprehensively studied class of clinically relevant bioactive molecules, the polyketides. Extensive efforts towards optimization and derivatization of compounds via combinatorial biosynthesis and classical engineering have elucidated the modularity, flexibility and promiscuity of polyketide biosynthetic enzymes. Hence, a synthetic biology approach can build upon a solid basis of guidelines and principles, while providing a new perspective towards the discovery and generation of novel and new-to-nature compounds. We discuss the lessons learned from the classical engineering of polyketide synthases and indicate their importance when attempting to engineer biosynthetic pathways using synthetic biology approaches for the introduction of novelty and overexpression of products in a controllable manner. © 2013 The Authors FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

DOE PAGES

Welner, Ditte Hededam; Shin, David; Tomaleri, Giovani P.; ...

2017-06-09

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, coexpression partners, purification methods, and optimization of protein solubility and stability. Hence researchersmore » are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble: Insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.« less

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

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

Welner, Ditte Hededam; Shin, David; Tomaleri, Giovani P.

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, coexpression partners, purification methods, and optimization of protein solubility and stability. Hence researchersmore » are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble: Insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.« less

"Trojan Horse" strategy for deconstruction of biomass for biofuels production.

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

Sinclair, Michael B.; Hadi, Masood Z.; Timlin, Jerilyn Ann

2008-08-01

Production of renewable biofuels to displace fossil fuels currently consumed in the transportation sector is a pressing multi-agency national priority. Currently, nearly all fuel ethanol is produced from corn-derived starch. Dedicated 'energy crops' and agricultural waste are preferred long-term solutions for renewable, cheap, and globally available biofuels as they avoid some of the market pressures and secondary greenhouse gas emission challenges currently facing corn ethanol. These sources of lignocellulosic biomass are converted to fermentable sugars using a variety of chemical and thermochemical pretreatments, which disrupt cellulose and lignin cross-links, allowing exogenously added recombinant microbial enzymes to more efficiently hydrolyze themore » cellulose for 'deconstruction' into glucose. This process is plagued with inefficiencies, primarily due to the recalcitrance of cellulosic biomass, mass transfer issues during deconstruction, and low activity of recombinant deconstruction enzymes. Costs are also high due to the requirement for enzymes and reagents, and energy-intensive and cumbersome pretreatment steps. One potential solution to these problems is found in synthetic biology; they propose to engineer plants that self-produce a suite of cellulase enzymes targeted to the apoplast for cleaving the linkages between lignin and cellulosic fibers; the genes encoding the degradation enzymes, also known as cellulases, are obtained from extremophilic organisms that grow at high temperatures (60-100 C) and acidic pH levels (<5). These enzymes will remain inactive during the life cycle of the plant but become active during hydrothermal pretreatment i.e., elevated temperatures. Deconstruction can be integrated into a one-step process, thereby increasing efficiency (cellulose-cellulase mass-transfer rates) and reducing costs. The proposed disruptive technologies address biomass deconstruction processes by developing transgenic plants encoding a suite of enzymes used in cellulosic deconstruction. The unique aspects of this technology are the rationally engineered, highly productive extremophilic enzymes, targeted to specific cellular locations (apoplast) and their dormancy during normal plant proliferation, which become Trojan horses during pretreatment conditions. They have been leveraging established Sandia's enzyme-engineering and imaging capabilities. Their technical approach not only targets the recalcitrance and mass-transfer problem during biomass degradation but also eliminates the costs associated with industrial-scale production of microbial enzymes added during processing.« less

Engineering self-assembled bioreactors from protein microcompartments

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

Savage, David

2016-10-12

The goals of this research are to understand how organisms such as bacteria segregate certain metabolic processes inside of specific structures, or “microcompartments,” in the cell and apply this knowledge to develop novel engineered microcompartments for use in nanotechnology and metabolic engineering. For example, in some bacteria, self-assembling protein microcompartments called carboxysomes encapsulate the enzymes involved in carbon fixation, enabling the cell to utilize carbon dioxide more effectively than if the enzymes were free in the cell. The proposed research will determine how structures such as carboxysomes assemble and function in bacteria and develop a means for creating novel, syntheticmore » microcompartments for optimizing production of specific energy-rich compounds.« less

Approaches for improving thermostability characteristics in cellulases.

PubMed

Anbar, Michael; Bayer, Edward A

2012-01-01

Many efforts have been invested to reduce the cost of biofuel production to substitute renewable sources of energy for fossil-based fuels. At the forefront of these efforts are the initiatives to convert plant-derived cellulosic material to biofuels. Although significant improvements have been achieved recently in cellulase engineering in both efficiency and cost reduction, complete degradation of lignocellulosic material still requires very long periods of time and high enzyme loads. Thermostable cellulases offer many advantages in the bioconversion process, which include increase in specific activity, higher levels of stability, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess. Besides rational design methods, which require deep understanding of protein structure-function relationship, two of the major methods for improvement in specific cellulase properties are directed evolution and knowledge-based library design based on multiple sequence alignments. In this chapter, we provide protocols for constructing and screening of improved thermostable cellulases. Modifications of these protocols may also be used for screening for other improved properties of cellulases such as pH tolerance, high salt, and more. Copyright © 2012 Elsevier Inc. All rights reserved.

Engineering Technology Of Fish Farming Floating Nets Cages On Polka Dot Grouper (Cromileptes Altivelis) Used Artificial Feed Enriched Phytase Enzyme

NASA Astrophysics Data System (ADS)

Samidjan, Istiyanto; Rachmawati, Diana

2018-02-01

One solution is to utilize engineering technology cultivation floating cage net polka dot grouper (ducker grouper), which is given artificial feed enriched with phytase enzymes. The objectives of this study was to examine the use of technology engineering floating net on ducker grouper on artificial feed that is enriched with different dose phytase enzymes to accelerate growth and survival. The research method used ducker grouper fish size 15,5 ± 0,5 cm in the net cages unit (1 m x 1 m x 1 m), 250 fish per cage, using 12 cages. Each net-cages was made of polyethylens netting, mesh size 12.5 mm. with complete randomized design (CRD) 4 treatment and 3 replication were feed Artificial enriched of phytase enzyme with the doses of A (0 FTU · kg-1 diet), B (200 FTU · kg-1 diet), C (500 FTU · kg-1 diet), and D (800 FTU · kg-1 diet) phytase enzyme. Feed was given 2 times a day in the morning and afternoon with 5% biomass per day. Data includes the growth of absolute weight polka dot grouper, FCR, and survival rate analyzed variety and Test Tukey.The result of the research showed that the difference of artificial feeding enriched phytase enzyme significantly (P <0,05) to growth, food conversion ratio (FCR), survival rete of polka dot grouper. The best treatment at C (500 mg / kg of feed) increase growth of absolute weight of 128.75 g, 1.75 (FCR), and a survival rate of 93.5%.

Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

PubMed

Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

2016-12-01

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

Structure-Based Virtual Screening and Biochemical Evaluation for the Identification of Novel Trypanosoma Brucei Aldolase Inhibitors.

PubMed

Ferreira, Leonardo L G; Ferreira, Rafaela S; Palomino, David L; Andricopulo, Adriano D

2018-04-27

The glycolytic enzyme fructose-1,6-bisphosphate aldolase is a validated molecular target in human African trypanosomiasis (HAT) drug discovery, a neglected tropical disease (NTD) caused by the protozoan Trypanosoma brucei. Herein, a structure-based virtual screening (SBVS) approach to the identification of novel T. brucei aldolase inhibitors is described. Distinct molecular docking algorithms were used to screen more than 500,000 compounds against the X-ray structure of the enzyme. This SBVS strategy led to the selection of a series of molecules which were evaluated for their activity on recombinant T. brucei aldolase. The effort led to the discovery of structurally new ligands able to inhibit the catalytic activity the enzyme. The predicted binding conformations were additionally investigated in molecular dynamics simulations, which provided useful insights into the enzyme-inhibitor intermolecular interactions. The molecular modeling results along with the enzyme inhibition data generated practical knowledge to be explored in further structure-based drug design efforts in HAT drug discovery. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Identification of GATC- and CCGG- recognizing Type II REases and their putative specificity-determining positions using Scan2S—a novel motif scan algorithm with optional secondary structure constraints

PubMed Central

Niv, Masha Y.; Skrabanek, Lucy; Roberts, Richard J.; Scheraga, Harold A.; Weinstein, Harel

2008-01-01

Restriction endonucleases (REases) are DNA-cleaving enzymes that have become indispensable tools in molecular biology. Type II REases are highly divergent in sequence despite their common structural core, function and, in some cases, common specificities towards DNA sequences. This makes it difficult to identify and classify them functionally based on sequence, and has hampered the efforts of specificity-engineering. Here, we define novel REase sequence motifs, which extend beyond the PD-(D/E)XK hallmark, and incorporate secondary structure information. The automated search using these motifs is carried out with a newly developed fast regular expression matching algorithm that accommodates long patterns with optional secondary structure constraints. Using this new tool, named Scan2S, motifs derived from REases with specificity towards GATC- and CGGG-containing DNA sequences successfully identify REases of the same specificity. Notably, some of these sequences are not identified by standard sequence detection tools. The new motifs highlight potential specificity-determining positions that do not fully overlap for the GATC- and the CCGG-recognizing REases and are candidates for specificity re-engineering. PMID:17972284

Identification of GATC- and CCGG-recognizing Type II REases and their putative specificity-determining positions using Scan2S--a novel motif scan algorithm with optional secondary structure constraints.

PubMed

Niv, Masha Y; Skrabanek, Lucy; Roberts, Richard J; Scheraga, Harold A; Weinstein, Harel

2008-05-01

Restriction endonucleases (REases) are DNA-cleaving enzymes that have become indispensable tools in molecular biology. Type II REases are highly divergent in sequence despite their common structural core, function and, in some cases, common specificities towards DNA sequences. This makes it difficult to identify and classify them functionally based on sequence, and has hampered the efforts of specificity-engineering. Here, we define novel REase sequence motifs, which extend beyond the PD-(D/E)XK hallmark, and incorporate secondary structure information. The automated search using these motifs is carried out with a newly developed fast regular expression matching algorithm that accommodates long patterns with optional secondary structure constraints. Using this new tool, named Scan2S, motifs derived from REases with specificity towards GATC- and CGGG-containing DNA sequences successfully identify REases of the same specificity. Notably, some of these sequences are not identified by standard sequence detection tools. The new motifs highlight potential specificity-determining positions that do not fully overlap for the GATC- and the CCGG-recognizing REases and are candidates for specificity re-engineering.

Determination of the Glycogen Content in Cyanobacteria.

PubMed

De Porcellinis, Alice; Frigaard, Niels-Ulrik; Sakuragi, Yumiko

2017-07-17

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Rational approaches for engineering novel functionalities in carbon-carbon bond forming enzymes

PubMed Central

Baker, Perrin; Seah, Stephen Y. K.

2012-01-01

Enzymes that catalyze carbon-carbon bond formation can be exploited as biocatalyst for synthetic organic chemistry. However, natural enzymes frequently do not possess the required properties or specificities to catalyze industrially useful transformations. This mini-review describes recent work using knowledge-guided site-specific mutagenesis of key active site residues to alter substrate specificity, stereospecificity and reaction specificity of these enzymes. In addition, examples of de novo designed enzymes that catalyze C-C bond reactions not found in nature will be discussed. PMID:24688644

Strategies for discovery and improvement of enzyme function: state of the art and opportunities

PubMed Central

Kaul, Praveen; Asano, Yasuhisa

2012-01-01

Summary Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non‐conventional media, using new substrates for old enzymes, active‐site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. PMID:21883976

Strategies for discovery and improvement of enzyme function: state of the art and opportunities.

PubMed

Kaul, Praveen; Asano, Yasuhisa

2012-01-01

Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non-conventional media, using new substrates for old enzymes, active-site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. © 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Co-Optimization of Fuels and Engines (Co-Optima) -- Introduction

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

Farrell, John T; Wagner, Robert; Holladay, John

The Co-Optimization of Fuels and Engines (Co-Optima) initiative is a U.S. Department of Energy (DOE) effort funded by both the Vehicle and Bioenergy Technology Offices. The overall goal of the effort is to identify the combinations of fuel properties and engine characteristics that maximize efficiency, independent of production pathway or fuel composition, and accelerate commercialization of these technologies. Multiple research efforts are underway focused on both spark-ignition and compression-ignition strategies applicable across the entire light, medium, and heavy-duty fleet. A key objective of Co-Optima's research is to identify new blendstocks that enhance current petroleum blending components, increase blendstock diversity, andmore » provide refiners with increased flexibility to blend fuels with the key properties required to optimize advanced internal combustion engines. In addition to fuels and engines R&D, the initiative is guided by analyses assessing the near-term commercial feasibility of new blendstocks based on economics, environmental performance, compatibility, and large-scale production viability. This talk will provide an overview of the Co-Optima effort.« less

Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates.

PubMed

Addington, Trevor; Calisto, Barbara; Alfonso-Prieto, Mercedes; Rovira, Carme; Fita, Ignasi; Planas, Antoni

2011-02-01

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity. © 2010 Wiley-Liss, Inc.

Research and technology 1995 annual report

NASA Technical Reports Server (NTRS)

1995-01-01

As the NASA Center responsible for assembly, checkout, servicing, launch, recovery, and operational support of Space Transportation System elements and payloads, the John F. Kennedy Space Center is placing increasing emphasis on its advanced technology development program. This program encompasses the efforts of the Engineering Development Directorate laboratories, most of the KSC operations contractors, academia, and selected commercial industries - all working in a team effort within their own areas of expertise. This edition of the Kennedy Space Center Research and Technology 1995 Annual Report covers efforts of all these contributors to the KSC advanced technology development program, as well as technology transfer activities. Major areas of research include environmental engineering, automation, robotics, advanced software, materials science, life sciences, mechanical engineering, nondestructive evaluation, and industrial engineering.

Self-Assembling Protein Nanostructures - Towards Active Functionality

DTIC Science & Technology

2011-04-22

the (alpha-beta)8 barrel. A model thermophilic enzyme , natively a tetramer, was engineered as a monomer, a dimer (submitted to Biochimica et...problem. Objective one successfully built high surface area nanoscaffolds from amyloid fibrils, and demonstrated that enzyme activity could be attached...quaternary structure, amyloid fibril, organophosphorous hydrolase, enzyme Juliet Gerrard University of Canterbury 20 Kirkwood Ave Ilam 8041 - REPORT

Self-Reporting and Detoxifying Materials Based on Extremophilic Proteins

DTIC Science & Technology

2010-01-01

thermosome (Therm), a chaperonin from the thermophilic organism Thermoplasma acidophilum, with the spectral properties of fluorescent...We have developed a new approach to enzyme immobilization/stabilization in which an enzyme - chaperone chimera is engineered to attach a functional...chaperone domain (in this case, a subunit of the recombinant thermosome from Methanocaldococcus jannaschii) to the enzyme of interest (the model

Promiscuous activities of heterologous enzymes lead to unintended metabolic rerouting in Saccharomyces cerevisiae engineered to assimilate various sugars from renewable biomass.

PubMed

Yun, Eun Ju; Oh, Eun Joong; Liu, Jing-Jing; Yu, Sora; Kim, Dong Hyun; Kwak, Suryang; Kim, Kyoung Heon; Jin, Yong-Su

2018-01-01

Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae . Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites. Unbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively. The heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases.

PubMed

Oh, Doo-Byoung

2015-08-01

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy.

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.

Discovery of a diazo-forming enzyme in cremeomycin biosynthesis.

PubMed

Waldman, Abraham J; Balskus, Emily P

2018-05-17

The molecular architectures and potent bioactivities of diazo-containing natural products have attracted the interest of synthetic and biological chemists. Despite this attention, the biosynthetic enzymes involved in diazo group construction have not been identified. Here, we show the ATP-dependent enzyme CreM installs the diazo group in cremeomycin via late-stage N-N bond formation using nitrite. This finding should inspire efforts to use diazo-forming enzymes in biocatalysis and synthetic biology and enable genome-based discovery of new diazo-containing metabolites.

Effects of tung oilseed FAD2 and DGAT2 genes on unsaturated fatty acid accumulation in Rhodotorula glutinis and Arabidopsis thaliana.

PubMed

Chen, Yicun; Cui, Qinqin; Xu, Yongjie; Yang, Susu; Gao, Ming; Wang, Yangdong

2015-08-01

Genetic engineering to produce valuable lipids containing unsaturated fatty acids (UFAs) holds great promise for food and industrial applications. Efforts to genetically modify plants to produce desirable UFAs with single enzymes, however, have had modest success. The key enzymes fatty acid desaturase (FAD) and diacylglycerol acyltransferase (DGAT) are responsible for UFA biosynthesis (a push process) and assembling fatty acids into lipids (a pull process) in plants, respectively. To examine their roles in UFA accumulation, VfFAD2 and VfDGAT2 genes cloned from Vernicia fordii (tung tree) oilseeds were conjugated and transformed into Rhodotorula glutinis and Arabidopsis thaliana via Agrobacterium tumefaciens. Real-time quantitative PCR revealed variable gene expression levels in the transformants, with a much higher level of VfDGAT2 than VfFAD2. The relationship between VfFAD2 expression and linoleic acid (C18:2) increases in R. glutinis (R (2) = 0.98) and A. thaliana (R (2) = 0.857) transformants was statistically linear. The VfDGAT2 expression level was statistically correlated with increased total fatty acid content in R. glutinis (R (2) = 0.962) and A. thaliana (R (2) = 0.8157) transformants. With a similar expression level between single- and two-gene transformants, VfFAD2-VfDGAT2 co-transformants showed a higher linolenic acid (C18:3) yield in R. glutinis (174.36 % increase) and A. thaliana (14.61 % increase), and eicosatrienoic acid (C20:3) was enriched (17.10 % increase) in A. thaliana. Our data suggest that VfFAD2-VfDGAT2 had a synergistic effect on UFA metabolism in R. glutinis, and to a lesser extent, A. thaliana. These results show promise for further genetic engineering of plant lipids to produce desirable UFAs.

Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

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

Deckert, Katelyn; Budiardjo, S. Jimmy; Brunner, Luke C.

2012-08-07

Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492Gmore » in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.« less

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

Zheng, Tianyong; Olson, Daniel G.; Tian, Liang

Clostridium thermocellum and Thermoanaerobacterium saccharolyticumare thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticumproduce ethanol with a yield of 90% of the theoretical maximum, engineered strains ofC. thermocellumproduce ethanol at lower yields (~50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in theiradhEgenes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, theadhEgenes from six strains ofC. thermocellumandT. saccharolyticumwere cloned and expressed inEscherichia coli, the enzymesmore » produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains ofT. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain ofC. thermocellumhas acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced intoC. thermocellumandT. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. This work describes the characterization of the AdhE enzyme from different strains ofC. thermocellumandT. saccharolyticum.C. thermocellumandT. saccharolyticumare thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production byC. thermocellumandT. saccharolyticum.« less

Applying the design-build-test paradigm in microbiome engineering.

PubMed

Pham, Hoang Long; Ho, Chun Loong; Wong, Adison; Lee, Yung Seng; Chang, Matthew Wook

2017-12-01

The recently discovered roles of human microbiome in health and diseases have inspired research efforts across many disciplines to engineer microbiome for health benefits. In this review, we highlight recent progress in human microbiome research and how modifications to the microbiome could result in implications to human health. Furthermore, we discuss the application of a 'design-build-test' framework to expedite microbiome engineering efforts by reviewing current literature on three key aspects: design principles to engineer the human microbiome, methods to engineer microbiome with desired functions, and analytical techniques to examine complex microbiome samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aerospace engineering curriculum for the 21st century

NASA Technical Reports Server (NTRS)

Simitses, George J.

1995-01-01

The second year of the study was devoted to completing the information-gathering phase of this redesign effort, using the conclusions from that activity to prepare the initial structure for the new curriculum, publicizing activities to a wider engineering forum, and preparing the department faculty (Aerospace Engineering and Engineering Mechanics at University of Cincinnati) for the roles they will play in the curriculum redesign and implementation. These activities are summarized briefly in this progress report. Attached is a paper resulting from the data acquisition of this effort, 'Educating Aerospace Engineers for the Twenty-First Century: Results of a Survey.'

Strategies for design of improved biocatalysts for industrial applications.

PubMed

Madhavan, Aravind; Sindhu, Raveendran; Binod, Parameswaran; Sukumaran, Rajeev K; Pandey, Ashok

2017-12-01

Biocatalysts are creating increased interest among researchers due to their unique properties. Several enzymes are efficiently produced by microorganisms. However, the use of natural enzymes as biocatalysts is hindered by low catalytic efficiency and stability during various industrial processes. Many advanced enzyme technologies have been developed to reshape the existing natural enzymes to reduce these limitations and prospecting of novel enzymes. Frequently used enzyme technologies include protein engineering by directed evolution, immobilisation techniques, metagenomics etc. This review summarizes recent and emerging advancements in the area of enzyme technologies for the development of novel biocatalysts and further discusses the future directions in this field. Copyright © 2017 Elsevier Ltd. All rights reserved.

How Do Enzymes 'Meet' Nanoparticles and Nanomaterials?

PubMed

Chen, Ming; Zeng, Guangming; Xu, Piao; Lai, Cui; Tang, Lin

2017-11-01

Enzymes are fundamental biological catalysts responsible for biological regulation and metabolism. The opportunity for enzymes to 'meet' nanoparticles and nanomaterials is rapidly increasing due to growing demands for applications in nanomaterial design, environmental monitoring, biochemical engineering, and biomedicine. Therefore, understanding the nature of nanomaterial-enzyme interactions is becoming important. Since 2014, enzymes have been used to modify, degrade, or make nanoparticles/nanomaterials, while numerous nanoparticles/nanomaterials have been used as materials for enzymatic immobilization and biosensors and as enzyme mimicry. Among the various nanoparticles and nanomaterials, metal nanoparticles and carbon nanomaterials have received extensive attention due to their fascinating properties. This review provides an overview about how enzymes meet nanoparticles and nanomaterials. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Pushing the frontiers of first-principles based computer simulations of chemical and biological systems.

PubMed

Brunk, Elizabeth; Ashari, Negar; Athri, Prashanth; Campomanes, Pablo; de Carvalho, F Franco; Curchod, Basile F E; Diamantis, Polydefkis; Doemer, Manuel; Garrec, Julian; Laktionov, Andrey; Micciarelli, Marco; Neri, Marilisa; Palermo, Giulia; Penfold, Thomas J; Vanni, Stefano; Tavernelli, Ivano; Rothlisberger, Ursula

2011-01-01

The Laboratory of Computational Chemistry and Biochemistry is active in the development and application of first-principles based simulations of complex chemical and biochemical phenomena. Here, we review some of our recent efforts in extending these methods to larger systems, longer time scales and increased accuracies. Their versatility is illustrated with a diverse range of applications, ranging from the determination of the gas phase structure of the cyclic decapeptide gramicidin S, to the study of G protein coupled receptors, the interaction of transition metal based anti-cancer agents with protein targets, the mechanism of action of DNA repair enzymes, the role of metal ions in neurodegenerative diseases and the computational design of dye-sensitized solar cells. Many of these projects are done in collaboration with experimental groups from the Institute of Chemical Sciences and Engineering (ISIC) at the EPFL.

Biodegradation of Chlorinated Solvents: Reactions near DNAPL and Enzyme Function

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

McCarty, Perry L.; Spormann, Alfred M.; Criddle, Craig S.

The anaerobic biodegradation of chlorinated solvents is of great interest both for natural attenuation and for engineered remediation of these hazardous contaminants in groundwater. Compounds to be studied are carbon tetrachloride (CT) and the chlorinated ethenes, tetrachloroethene (PCE), trichloroethene (TCE) cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC). The chlorinated solvents often are present as dense non-aqueous-phase liquids (DNAPLs), which are difficult to remove. Biodegradation of DNAPLs was previously thought not possible because of toxicity, but recent evidence indicates that under the right conditions, biodegradation is possible. Anaerobic biodegradation of DNAPLs is the major subject of this research. The specific objectives ofmore » this multi-investigator effort are: (1) Evaluate the potential for chlorinated solvent biodegradation near DNAPLs, (2) Provide a molecular understanding of the biological mechanisms involved, (3) Determine cellular components involved in carbon tetrachloride transformation by Pseudomonas stutzeri strain KC without chloroform formation.« less

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B 2 Biosynthesis

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

Barajas, Jesus F.; Zargar, Amin; Pang, Bo

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. While β-amino acid loading in the fluvirucin B 2 polyketide pathway has been proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. In this paper, we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferasesmore » to selectively transfer β-amino acids onto a PKS loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.« less

Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort.

PubMed

Jeschek, Markus; Gerngross, Daniel; Panke, Sven

2016-03-31

Rational flux design in metabolic engineering approaches remains difficult since important pathway information is frequently not available. Therefore empirical methods are applied that randomly change absolute and relative pathway enzyme levels and subsequently screen for variants with improved performance. However, screening is often limited on the analytical side, generating a strong incentive to construct small but smart libraries. Here we introduce RedLibs (Reduced Libraries), an algorithm that allows for the rational design of smart combinatorial libraries for pathway optimization thereby minimizing the use of experimental resources. We demonstrate the utility of RedLibs for the design of ribosome-binding site libraries by in silico and in vivo screening with fluorescent proteins and perform a simple two-step optimization of the product selectivity in the branched multistep pathway for violacein biosynthesis, indicating a general applicability for the algorithm and the proposed heuristics. We expect that RedLibs will substantially simplify the refactoring of synthetic metabolic pathways.

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B 2 Biosynthesis

DOE PAGES

Barajas, Jesus F.; Zargar, Amin; Pang, Bo; ...

2018-03-30

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. While β-amino acid loading in the fluvirucin B 2 polyketide pathway has been proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. In this paper, we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferasesmore » to selectively transfer β-amino acids onto a PKS loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.« less

Dissecting the active site of a photoreceptor protein

NASA Astrophysics Data System (ADS)

Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

DOE PAGES

Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

2013-06-17

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

Continually emerging mechanistic complexity of the multi-enzyme cellulosome complex.

PubMed

Smith, Steven P; Bayer, Edward A; Czjzek, Mirjam

2017-06-01

The robust plant cell wall polysaccharide-degrading properties of anaerobic bacteria are harnessed within elegant, marcomolecular assemblages called cellulosomes, in which proteins of complementary activities amass on scaffold protein networks. Research efforts have focused and continue to focus on providing detailed mechanistic insights into cellulosomal complex assembly, topology, and function. The accumulated information is expanding our fundamental understanding of the lignocellulosic biomass decomposition process and enhancing the potential of engineered cellulosomal systems for biotechnological purposes. Ongoing biochemical studies continue to reveal unexpected functional diversity within traditional cellulase families. Genomic, proteomic, and functional analyses have uncovered unanticipated cellulosomal proteins that augment the function of the native and designer cellulosomes. In addition, complementary structural and computational methods are continuing to provide much needed insights on the influence of cellulosomal interdomain linker regions on cellulosomal assembly and activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Programming adaptive control to evolve increased metabolite production.

PubMed

Chou, Howard H; Keasling, Jay D

2013-01-01

The complexity inherent in biological systems challenges efforts to rationally engineer novel phenotypes, especially those not amenable to high-throughput screens and selections. In nature, increased mutation rates generate diversity in a population that can lead to the evolution of new phenotypes. Here we construct an adaptive control system that increases the mutation rate in order to generate diversity in the population, and decreases the mutation rate as the concentration of a target metabolite increases. This system is called feedback-regulated evolution of phenotype (FREP), and is implemented with a sensor to gauge the concentration of a metabolite and an actuator to alter the mutation rate. To evolve certain novel traits that have no known natural sensors, we develop a framework to assemble synthetic transcription factors using metabolic enzymes and construct four different sensors that recognize isopentenyl diphosphate in bacteria and yeast. We verify FREP by evolving increased tyrosine and isoprenoid production.

Insight into metabolic and cometabolic activities of autotrophic and heterotrophic microorganisms in the biodegradation of emerging trace organic contaminants.

PubMed

Tran, Ngoc Han; Urase, Taro; Ngo, Huu Hao; Hu, Jiangyong; Ong, Say Leong

2013-10-01

Many efforts have been made to understand the biodegradation of emerging trace organic contaminants (EOCs) in the natural and engineered systems. This review summarizes the current knowledge on the biodegradation of EOCs while having in-depth discussion on metabolism and cometabolism of EOCs. Biodegradation of EOCs is mainly attributed to cometabolic activities of both heterotrophic and autotrophic microorganisms. Metabolism of EOCs can only be observed by heterotrophic microbes. Autotrophic ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaeal (AOA) cometabolize a variety of EOCs via the non-specific enzymes, such as ammonia monooxygenase (AMO). Higher biodegradation of EOCs is often noted under nitrification at high ammonia loading rate. The presence of a growth substrate promotes cometabolic biodegradation of EOCs. Potential strategies for enhancing the biodegradation of EOCs were also proposed in this review. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Fully Non-metallic Gas Turbine Engine Enabled by Additive Manufacturing

NASA Technical Reports Server (NTRS)

Grady, Joseph E.

2014-01-01

The Non-Metallic Gas Turbine Engine project, funded by NASA Aeronautics Research Institute (NARI), represents the first comprehensive evaluation of emerging materials and manufacturing technologies that will enable fully nonmetallic gas turbine engines. This will be achieved by assessing the feasibility of using additive manufacturing technologies for fabricating polymer matrix composite (PMC) and ceramic matrix composite (CMC) gas turbine engine components. The benefits of the proposed effort include: 50 weight reduction compared to metallic parts, reduced manufacturing costs due to less machining and no tooling requirements, reduced part count due to net shape single component fabrication, and rapid design change and production iterations. Two high payoff metallic components have been identified for replacement with PMCs and will be fabricated using fused deposition modeling (FDM) with high temperature capable polymer filaments. The first component is an acoustic panel treatment with a honeycomb structure with an integrated back sheet and perforated front sheet. The second component is a compressor inlet guide vane. The CMC effort, which is starting at a lower technology readiness level, will use a binder jet process to fabricate silicon carbide test coupons and demonstration articles. The polymer and ceramic additive manufacturing efforts will advance from monolithic materials toward silicon carbide and carbon fiber reinforced composites for improved properties. Microstructural analysis and mechanical testing will be conducted on the PMC and CMC materials. System studies will assess the benefits of fully nonmetallic gas turbine engine in terms of fuel burn, emissions, reduction of part count, and cost. The proposed effort will be focused on a small 7000 lbf gas turbine engine. However, the concepts are equally applicable to large gas turbine engines. The proposed effort includes a multidisciplinary, multiorganization NASA - industry team that includes experts in ceramic materials and CMCs, polymers and PMCs, structural engineering, additive manufacturing, engine design and analysis, and system analysis.

Biosynthetic engineering of nonribosomal peptide synthetases.

PubMed

Kries, Hajo

2016-09-01

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Transporter engineering in biomass utilization by yeast.

PubMed

Hara, Kiyotaka Y; Kobayashi, Jyumpei; Yamada, Ryosuke; Sasaki, Daisuke; Kuriya, Yuki; Hirono-Hara, Yoko; Ishii, Jun; Araki, Michihiro; Kondo, Akihiko

2017-11-01

Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

Environmental and High-Strain Rate effects on composites for engine applications

NASA Technical Reports Server (NTRS)

Chamis, C. C.; Smith, G. T.

1982-01-01

The Lewis Research Center is conducting a series of programs intended to investigate and develop the application of composite materials to structural components for turbojet engines. A significant part of that effort is directed to establishing resistance, defect growth, and strain rate characteristics of composite materials over the wide range of environmental and load conditions found in commercial turbojet engine operations. Both analytical and experimental efforts are involved.

An overview of NASA research on positive displacement general-aviation engines

NASA Technical Reports Server (NTRS)

Kempke, E. E., Jr.

1980-01-01

The research and technology program related to improved and advanced general aviation engines is described. Current research is directed at the near-term improvement of conventional air-cooled spark-ignition piston engines and at future alternative engine systems based on all-new spark-ignition piston engines, lightweight diesels, and rotary combustion engines that show potential for meeting program goals in the midterm and long-term future. The conventional piston engine activities involve efforts on applying existing technology to improve fuel economy, investigation of key processes to permit leaner operation and reduce drag, and the development of cost effective technology to permit flight at high-altitudes where fuel economy and safety are improved. The advanced engine concepts activities include engine conceptual design studies and enabling technology efforts on the critical or key technology items.

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions.

PubMed

Beld, Joris; Blatti, Jillian L; Behnke, Craig; Mendez, Michael; Burkart, Michael D

2014-08-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

PubMed Central

Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

2014-01-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

Engineering bacteriophage for a pragmatic low-resource setting bacterial diagnostic platform.

PubMed

Talbert, Joey N; Alcaine, Samuel D; Nugen, Sam R

2016-04-01

Bacteriophages represent multifaceted building blocks that can be incorporated as substitutes for, or in unison with other detection methods, to create powerful new diagnostics for the detection of bacteria. The ease of phage manipulation, production, and detection speed clearly highlights that there remains unrealized opportunities to leverage these phage-based components in diagnostics amenable to resource-limited settings. The passage of regulations like the Food Safety Modernization act, and the ever increasing extent of global trade and travel, will create further demand for these types of diagnostics. While phage-based diagnostics have begun to entering the market place, further research is needed to ensure the potential benefits of phage-based technologies for public health are fully realized. We are just beginning to explore the possibilities that phage-based detection can offer us in the future. The combination of engineered phages as well as engineered enzymes could result in ultrasensitive detection systems for low-resource settings. Because the reporter enzyme is synthesized in vivo, we need to consider the options outside of normal enzyme reporters. In this case, common enzyme issues such as purification and long-term stability are less important. Phage-based diagnostics were conceptualized from out-of-the box thinking and the evolution of these systems should be as well.

JPRS Report, Science & Technology, Europe & Latin America

DTIC Science & Technology

1988-04-06

courses and in polytechnics a growing number of undergraduate research theses [ tesi di laurea] are increasingly coming to resemble authentic feasibility...Information Science Eleven Priorities Research Priority Actions — Microbiological engineering —Enzyme engineering —Biotechnological engineering —Food...Foodstuffs Medicine Human and social sciences Technology, computer-integrated manufacturing Electronics, data processing Microbiological

Rocketdyne PSAM: In-house enhancement/application

NASA Technical Reports Server (NTRS)

Newell, J. F.; Rajagopal, K. R.; Ohara, K.

1991-01-01

The development was initiated of the Probabilistic Design Analysis (PDA) Process for rocket engines. This will enable engineers a quantitative assessment of calculated reliability during the design process. The PDA will help choose better designs, make them more robust, and help decide on critical tests to help demonstrate key reliability issues to aid in improving the confidence of the engine capabilities. Rockedyne's involvement with the Composite Loads Spectra (CLS) and Probabilistic Structural Analysis Methodology (PSAM) contracts started this effort and are key elements in the on-going developments. Internal development efforts and hardware applications complement and extend the CLS and PSAM efforts. The completion of the CLS option work and the follow-on PSAM developments will also be integral parts of this methodology. A brief summary of these efforts is presented.

Fermentation of dilute acid pretreated Populus by Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis

DOE PAGES

Yee, Kelsey L.; Rodriguez, Jr., Miguel; Hamilton, Choo Yieng; ...

2015-07-25

Consolidated bioprocessing (CBP), which merges enzyme production, biomass hydrolysis, and fermentation into a single step, has the potential to become an efficient and economic strategy for the bioconversion of lignocellulosic feedstocks to transportation fuels or chemicals. In this study, we evaluated Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis, three , thermophilic,cellulolytic, mixed-acid fermenting candidate CBP microorganisms, for their fermentation capabilities using dilute acid pretreated Populus as a model biomass feedstock. Under pH controlled, anaerobic fermentation conditions, each candidate successfully digested a minimum of 75% of the cellulose from dilute acid pretreated Populus, as indicated by an increase in planktonic cellsmore » and end-product metabolites and a concurrent decrease in glucan content. C. thermocellum, which employs a cellulosomal approach to biomass degradation, required 120 hours to achieve 75% cellulose utilization. In contrast, the non-cellulosomal, secreted hydrolytic enzyme system of the Caldicellulosiruptor sp. required 300 hours to achieve similar results. End-point fermentation conversions for C. thermocellum, C. bescii, and C. obsidiansis were determined to be 0.29, 0.34, and 0.38 grams of total metabolites per gram of loaded glucan, respectively. This data provide a starting point for future strain engineering efforts that can serve to improve the biomass fermentation capabilities of these three promising candidate CBP platforms.« less

Fermentation of dilute acid pretreated Populus by Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis

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

Yee, Kelsey L.; Rodriguez, Jr., Miguel; Hamilton, Choo Yieng

Consolidated bioprocessing (CBP), which merges enzyme production, biomass hydrolysis, and fermentation into a single step, has the potential to become an efficient and economic strategy for the bioconversion of lignocellulosic feedstocks to transportation fuels or chemicals. In this study, we evaluated Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis, three , thermophilic,cellulolytic, mixed-acid fermenting candidate CBP microorganisms, for their fermentation capabilities using dilute acid pretreated Populus as a model biomass feedstock. Under pH controlled, anaerobic fermentation conditions, each candidate successfully digested a minimum of 75% of the cellulose from dilute acid pretreated Populus, as indicated by an increase in planktonic cellsmore » and end-product metabolites and a concurrent decrease in glucan content. C. thermocellum, which employs a cellulosomal approach to biomass degradation, required 120 hours to achieve 75% cellulose utilization. In contrast, the non-cellulosomal, secreted hydrolytic enzyme system of the Caldicellulosiruptor sp. required 300 hours to achieve similar results. End-point fermentation conversions for C. thermocellum, C. bescii, and C. obsidiansis were determined to be 0.29, 0.34, and 0.38 grams of total metabolites per gram of loaded glucan, respectively. This data provide a starting point for future strain engineering efforts that can serve to improve the biomass fermentation capabilities of these three promising candidate CBP platforms.« less

Vertical Integration of Biomass Saccharification of Enzymes for Sustainable Cellulosic Biofuel Production in a Biorefinery

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

Kumar, Manoj

2011-05-09

These are a set of slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Streptomyces spp. in the biocatalysis toolbox.

PubMed

Spasic, Jelena; Mandic, Mina; Djokic, Lidija; Nikodinovic-Runic, Jasmina

2018-04-01

About 20,100 research publications dated 2000-2017 were recovered searching the PubMed and Web of Science databases for Streptomyces, which are the richest known source of bioactive molecules. However, these bacteria with versatile metabolism are powerful suppliers of biocatalytic tools (enzymes) for advanced biotechnological applications such as green chemical transformations and biopharmaceutical and biofuel production. The recent technological advances, especially in DNA sequencing coupled with computational tools for protein functional and structural prediction, and the improved access to microbial diversity enabled the easier access to enzymes and the ability to engineer them to suit a wider range of biotechnological processes. The major driver behind a dramatic increase in the utilization of biocatalysis is sustainable development and the shift toward bioeconomy that will, in accordance to the UN policy agenda "Bioeconomy to 2030," become a global effort in the near future. Streptomyces spp. already play a significant role among industrial microorganisms. The intention of this minireview is to highlight the presence of Streptomyces in the toolbox of biocatalysis and to give an overview of the most important advances in novel biocatalyst discovery and applications. Judging by the steady increase in a number of recent references (228 for the 2000-2017 period), it is clear that biocatalysts from Streptomyces spp. hold promises in terms of valuable properties and applicative industrial potential.

Seonah Kim, Ph.D. | NREL

Science.gov Websites

lignocellulosic biomass enzymes Design of new catalysts for vapor phase upgrading of biomass pyrolysis Enzymatic Engineering for Continuous Hydrocarbon Fuel Production (PI) Computational Pyrolysis Consortium Zeolite design Celluase enzyme structure-function relationships to design enhanced cellulose systems Catalyst

Deactivating Chemical Agents Using Enzyme-Coated Nanofibers Formed by Electrospinning

DTIC Science & Technology

2011-11-16

method we have previously explored the formation of specialized fibers for several applications, including tissue engineering,28 superhydrophobic fab...dissolved during the first exposure and subsequent Figure 9. Multilayer fiber mat with outer superhydrophobic fibers and inner enzyme containing fibers

Tissue engineering, stem cells, and cloning for the regeneration of urologic organs.

PubMed

Atala, Anthony

2003-10-01

Tissue engineering efforts are currently being undertaken for every type of tissue and organ within the urinary system. Most of the effort expended to engineer genitourinary tissues has occurred within the last decade. Tissue engineering techniques require a cell culture facility designed for human application. Personnel who have mastered the techniques of cell harvest, culture, and expansion as well as polymer design are essential for the successful application of this technology. Various engineered genitourinary tissues are at different stages of development, with some already being used clinically, a few in preclinical trials, and some in the discovery stage. Recent progress suggests that engineered urologic tissues may have an expanded clinical applicability in the future.

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.

Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

PubMed Central

Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna

2013-01-01

Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. PMID:24038690

%22Trojan Horse%22 strategy for deconstruction of biomass for biofuels production.

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

Simmons, Blake Alexander; Sinclair, Michael B.; Yu, Eizadora

2011-02-01

Production of renewable biofuels to displace fossil fuels currently consumed in the transportation sector is a pressing multiagency national priority (DOE/USDA/EERE). Currently, nearly all fuel ethanol is produced from corn-derived starch. Dedicated 'energy crops' and agricultural waste are preferred long-term solutions for renewable, cheap, and globally available biofuels as they avoid some of the market pressures and secondary greenhouse gas emission challenges currently facing corn ethanol. These sources of lignocellulosic biomass are converted to fermentable sugars using a variety of chemical and thermochemical pretreatments, which disrupt cellulose and lignin cross-links, allowing exogenously added recombinant microbial enzymes to more efficiently hydrolyzemore » the cellulose for 'deconstruction' into glucose. This process is plagued with inefficiencies, primarily due to the recalcitrance of cellulosic biomass, mass transfer issues during deconstruction, and low activity of recombinant deconstruction enzymes. Costs are also high due to the requirement for enzymes and reagents, and energy-intensive cumbersome pretreatment steps. One potential solution to these problems is found in synthetic biology-engineered plants that self-produce a suite of cellulase enzymes. Deconstruction can then be integrated into a one-step process, thereby increasing efficiency (cellulose-cellulase mass-transfer rates) and reducing costs. The unique aspects of our approach are the rationally engineered enzymes which become Trojan horses during pretreatment conditions. During this study we rationally engineered Cazy enzymes and then integrated them into plant cells by multiple transformation techniques. The regenerated plants were assayed for first expression of these messages and then for the resulting proteins. The plants were then subjected to consolidated bioprocessing and characterized in detail. Our results and possible implications of this work on developing dedicated energy crops and their advantage in a consolidated bioprocessing system.« less

The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

PubMed

Marcella, Aaron M; Barb, Adam W

2017-12-01

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

Progress of Stirling cycle analysis and loss mechanism characterization

NASA Technical Reports Server (NTRS)

Tew, R. C., Jr.

1986-01-01

An assessment of Stirling engine thermodynamic modeling and design codes shows a general deficiency; this deficiency is due to poor understanding of the fluid flow and heat transfer phenomena that occur in the oscillating flow and pressure level environment within the engines. Stirling engine thermodynamic loss mechanisms are listed. Several experimental and computational research efforts now underway to characterize various loss mechanisms are reviewed. The need for additional experimental rigs and rig upgrades is discussed. Recent developments and current efforts in Stirling engine thermodynamic modeling are also reviewed.

Large Engine Technology (LET) Task XXXVII Low-Bypass Ratio Mixed Turbofan Engine Subsonic Jet Noise Reduction Program Test Report

NASA Technical Reports Server (NTRS)

Hauser, Joseph R.; Zysman, Steven H.; Barber, Thomas J.

2001-01-01

NASA Glenn Research Center supported a three year effort to develop the technology for reducing jet noise from low-bypass ratio engines. This effort concentrated on both analytical and experimental approaches using various mixer designs. CFD and MGB predictions are compared with LDV and noise data, respectively. While former predictions matched well with data, experiment shows a need for improving the latter predictions. Data also show that mixing noise can be sensitive to engine hardware upstream of the mixing exit plane.

Stable metal-organic frameworks containing single-molecule traps for enzyme encapsulation.

PubMed

Feng, Dawei; Liu, Tian-Fu; Su, Jie; Bosch, Mathieu; Wei, Zhangwen; Wan, Wei; Yuan, Daqiang; Chen, Ying-Pin; Wang, Xuan; Wang, Kecheng; Lian, Xizhen; Gu, Zhi-Yuan; Park, Jihye; Zou, Xiaodong; Zhou, Hong-Cai

2015-01-19

Enzymatic catalytic processes possess great potential in chemical manufacturing, including pharmaceuticals, fuel production and food processing. However, the engineering of enzymes is severely hampered due to their low operational stability and difficulty of reuse. Here, we develop a series of stable metal-organic frameworks with rationally designed ultra-large mesoporous cages as single-molecule traps (SMTs) for enzyme encapsulation. With a high concentration of mesoporous cages as SMTs, PCN-333(Al) encapsulates three enzymes with record-high loadings and recyclability. Immobilized enzymes that most likely undergo single-enzyme encapsulation (SEE) show smaller Km than free enzymes while maintaining comparable catalytic efficiency. Under harsh conditions, the enzyme in SEE exhibits better performance than free enzyme, showing the effectiveness of SEE in preventing enzyme aggregation or denaturation. With extraordinarily large pore size and excellent chemical stability, PCN-333 may be of interest not only for enzyme encapsulation, but also for entrapment of other nanoscaled functional moieties.

Stable metal-organic frameworks containing single-molecule traps for enzyme encapsulation

NASA Astrophysics Data System (ADS)

Feng, Dawei; Liu, Tian-Fu; Su, Jie; Bosch, Mathieu; Wei, Zhangwen; Wan, Wei; Yuan, Daqiang; Chen, Ying-Pin; Wang, Xuan; Wang, Kecheng; Lian, Xizhen; Gu, Zhi-Yuan; Park, Jihye; Zou, Xiaodong; Zhou, Hong-Cai

2015-01-01

Enzymatic catalytic processes possess great potential in chemical manufacturing, including pharmaceuticals, fuel production and food processing. However, the engineering of enzymes is severely hampered due to their low operational stability and difficulty of reuse. Here, we develop a series of stable metal-organic frameworks with rationally designed ultra-large mesoporous cages as single-molecule traps (SMTs) for enzyme encapsulation. With a high concentration of mesoporous cages as SMTs, PCN-333(Al) encapsulates three enzymes with record-high loadings and recyclability. Immobilized enzymes that most likely undergo single-enzyme encapsulation (SEE) show smaller Km than free enzymes while maintaining comparable catalytic efficiency. Under harsh conditions, the enzyme in SEE exhibits better performance than free enzyme, showing the effectiveness of SEE in preventing enzyme aggregation or denaturation. With extraordinarily large pore size and excellent chemical stability, PCN-333 may be of interest not only for enzyme encapsulation, but also for entrapment of other nanoscaled functional moieties.

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

PubMed Central

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao

2017-01-01

ABSTRACT To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (−)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (−)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (−)-Vince lactam. PMID:29054871

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one).

PubMed

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao; Zheng, Guojun

2018-01-01

To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (-)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (-)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (-)-Vince lactam. Copyright © 2017 American Society for Microbiology.

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

PubMed

Latimer, Luke N; Dueber, John E

2017-06-01

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ∼70% increase in biomass yield and ∼240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models

NASA Astrophysics Data System (ADS)

Dodani, Sheel C.; Kiss, Gert; Cahn, Jackson K. B.; Su, Ye; Pande, Vijay S.; Arnold, Frances H.

2016-05-01

The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.

Effect of ethyleneoxide groups of anionic surfactants on lipase activity.

PubMed

Magalhães, Solange S; Alves, Luís; Sebastião, Marco; Medronho, Bruno; Almeida, Zaida L; Faria, Tiago Q; Brito, Rui M M; Moreno, Maria J; Antunes, Filipe E

2016-09-01

The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant-enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE 0 S) and of sodium lauryl ether sulfate with two EO units (SLE 2 S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE 2 S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276-1282, 2016. © 2016 American Institute of Chemical Engineers.

Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

PubMed

Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

2015-04-21

Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

Novel Biocatalysts Combining the Special Assembly Properties of S-Layer Proteins and the Functionality of Enzymes of Extremophiles (BIOCAT)

DTIC Science & Technology

2010-04-14

THE FUNCTIONALITY OF ENZYMES OF EXTREMOPHILES 5a. CONTRACT NUMBER 5b. GRANT NUMBER FA9550-07-1-0313 5c. PROGRAM ELEMENT NUMBER 61102F 6. AUTHOR... Enzymes of Extremophiles (BIOCAT) Agreement Award No.: FA9550-07-1-0313 Reporting period: April 15th. 2008 to April 14th, 2010 3Lo\\*p<\\\\%\\%, Prof...Status of effort 4 4. Nanobiotechnologie of S-layers 4 5. Biocat accomplishments 6 5.1. The S-layer-based enzyme immobilization system 6 5.2. S

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases

PubMed Central

Lambert, Abigail R.; Kuhar, Ryan; Jarjour, Jordan; Kulshina, Nadia; Parmeggiani, Fabio; Danaher, Patrick; Gano, Jacob; Baker, David; Stoddard, Barry L.; Scharenberg, Andrew M.

2012-01-01

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40–70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ∼70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications. PMID:22684507

Inhibition of biofilm formation by T7 bacteriophages producing quorum-quenching enzymes.

PubMed

Pei, Ruoting; Lamas-Samanamud, Gisella R

2014-09-01

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes

PubMed Central

Lamas-Samanamud, Gisella R.

2014-01-01

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings. PMID:24951790

One more thing: Faculty response to increased emphasis on project teams in undergraduate engineering education

NASA Astrophysics Data System (ADS)

Hunter, Jane

Tenured and tenure-track faculty members at institutions of higher education, especially those at Research I institutions, are being asked to do more than ever before. With rapidly changing technology, significant decreases in public funding, the shift toward privately funded research, and the ever increasing expectations of students for an education that adequately prepares them for professional careers, engineering faculty are particularly challenged by the escalating demands on their time. In 1996, the primary accreditation organization for engineering programs (ABET) adopted new criteria that required, among other things, engineering programs to teach students to function on multidisciplinary teams and to communicate effectively. In response, most engineering programs utilize project teams as a strategy for teaching these skills. The purpose of this qualitative study of tenured and tenure track engineering faculty at a Research I institution in the southwestern United States was to explore the variety of ways in which the engineering faculty responded to the demands placed upon them as a result of the increased emphasis on project teams in undergraduate engineering education. Social role theory and organizational climate theory guided the study. Some faculty viewed project teams as an opportunity for students to learn important professional skills and to benefit from collaborative learning but many questioned the importance and feasibility of teaching teamwork skills and had concerns about taking time away from other essential fundamental material such as mathematics, basic sciences and engineering sciences. Although the administration of the College of Engineering articulated strong support for the use of project teams in undergraduate education, the prevailing climate did little to promote significant efforts related to effective utilization of project teams. Too often, faculty were unwilling to commit sufficient time or effort to make project teamwork a truly valuable learning opportunity because those efforts were not perceived to be valuable and were rarely rewarded. Few formal professional development opportunities were available and few incentives were in place to encourage other informal efforts to develop the necessary skills. Those who committed significant effort to project teams were challenged by concerns about team composition, student accountability and assigning individual grades for group teamwork.

Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems.

PubMed

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-02-28

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TK(SR39) mutants), yeast cytosine deaminase:uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs' bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. Copyright © 2015 Elsevier B.V. All rights reserved.

DragonflyTV: "Investigating the Nanoworld". Summative Evaluation Report

ERIC Educational Resources Information Center

Robles, Dawn; Helms, Jenifer; Phillips, Michelle

2009-01-01

In recent years academic, engineering, business, and other fields, have launched major research and development efforts into the study and application of nanoscale science, engineering, and technology. In spite of all these efforts and the investment of millions of dollars, the general public has had little access to research findings and…

Applications and Methods for Continuous Monitoring of Physiological Chemistry

DTIC Science & Technology

2016-02-04

product and test platform to verify the performance characteristics of the enzymes when used in diagnostic device fabrication. 1.3 Results This...project had three primary objectives: 1. Engineer a cortisol oxidase enzyme suitable for use in diagnostic devices 2. Large scale production and...for both animal and human use , and for direct sale to other entities to manufacture biosensors and other products for human monitoring. The enzymes

Annual Report to Congress - Fiscal Year 2002, from the Strategic Environmental Research and Development Program

DTIC Science & Technology

2003-03-01

and Catabolic Enzymes Involved in Phytoremediation of the Nitro-Substituted Explosives TNT, RDX, and HMX . . . . . A-48 CU-1318 – Engineering Transgenic...1317 Identification of Metabolic Routes and Catabolic Enzymes Involved in Phytoremediation of the Nitro-Substituted Explosives TNT, RDX, and HMX...A A-48 PROJECT SUMMARY PROJECT TITLE & ID: Identification of Metabolic Routes and Catabolic Enzymes Involved in Phytoremediation of the Nitro

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases

PubMed Central

Oh, Doo-Byoung

2015-01-01

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444] PMID:25999178

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

PubMed Central

Abi-Ghanem, Josephine; Chusainow, Janet; Karimova, Madina; Spiegel, Christopher; Hofmann-Sieber, Helga; Hauber, Joachim; Buchholz, Frank; Pisabarro, M. Teresa

2013-01-01

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine. PMID:23275541

The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization

PubMed Central

Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

2016-01-01

Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell’s capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified. PMID:27812109

Engineering yeast for the expression and secretion of cellulase cocktails

USDA-ARS?s Scientific Manuscript database

Enzyme systems that digest the cellulose in plant cell walls have potential value in the biorefining of renewable feedstocks such as crop residues, straws, and grasses to biofuels and other bioproducts. The bacterium Clostridium cellulovorans is a useful source of biomass-degrading enzymes because ...

Nuclear Engine System Simulation (NESS). Volume 1: Program user's guide

NASA Astrophysics Data System (ADS)

Pelaccio, Dennis G.; Scheil, Christine M.; Petrosky, Lyman J.

1993-03-01

A Nuclear Thermal Propulsion (NTP) engine system design analysis tool is required to support current and future Space Exploration Initiative (SEI) propulsion and vehicle design studies. Currently available NTP engine design models are those developed during the NERVA program in the 1960's and early 1970's and are highly unique to that design or are modifications of current liquid propulsion system design models. To date, NTP engine-based liquid design models lack integrated design of key NTP engine design features in the areas of reactor, shielding, multi-propellant capability, and multi-redundant pump feed fuel systems. Additionally, since the SEI effort is in the initial development stage, a robust, verified NTP analysis design tool could be of great use to the community. This effort developed an NTP engine system design analysis program (tool), known as the Nuclear Engine System Simulation (NESS) program, to support ongoing and future engine system and stage design study efforts. In this effort, Science Applications International Corporation's (SAIC) NTP version of the Expanded Liquid Engine Simulation (ELES) program was modified extensively to include Westinghouse Electric Corporation's near-term solid-core reactor design model. The ELES program has extensive capability to conduct preliminary system design analysis of liquid rocket systems and vehicles. The program is modular in nature and is versatile in terms of modeling state-of-the-art component and system options as discussed. The Westinghouse reactor design model, which was integrated in the NESS program, is based on the near-term solid-core ENABLER NTP reactor design concept. This program is now capable of accurately modeling (characterizing) a complete near-term solid-core NTP engine system in great detail, for a number of design options, in an efficient manner. The following discussion summarizes the overall analysis methodology, key assumptions, and capabilities associated with the NESS presents an example problem, and compares the results to related NTP engine system designs. Initial installation instructions and program disks are in Volume 2 of the NESS Program User's Guide.

Nuclear Engine System Simulation (NESS). Volume 1: Program user's guide

NASA Technical Reports Server (NTRS)

Pelaccio, Dennis G.; Scheil, Christine M.; Petrosky, Lyman J.

1993-01-01

A Nuclear Thermal Propulsion (NTP) engine system design analysis tool is required to support current and future Space Exploration Initiative (SEI) propulsion and vehicle design studies. Currently available NTP engine design models are those developed during the NERVA program in the 1960's and early 1970's and are highly unique to that design or are modifications of current liquid propulsion system design models. To date, NTP engine-based liquid design models lack integrated design of key NTP engine design features in the areas of reactor, shielding, multi-propellant capability, and multi-redundant pump feed fuel systems. Additionally, since the SEI effort is in the initial development stage, a robust, verified NTP analysis design tool could be of great use to the community. This effort developed an NTP engine system design analysis program (tool), known as the Nuclear Engine System Simulation (NESS) program, to support ongoing and future engine system and stage design study efforts. In this effort, Science Applications International Corporation's (SAIC) NTP version of the Expanded Liquid Engine Simulation (ELES) program was modified extensively to include Westinghouse Electric Corporation's near-term solid-core reactor design model. The ELES program has extensive capability to conduct preliminary system design analysis of liquid rocket systems and vehicles. The program is modular in nature and is versatile in terms of modeling state-of-the-art component and system options as discussed. The Westinghouse reactor design model, which was integrated in the NESS program, is based on the near-term solid-core ENABLER NTP reactor design concept. This program is now capable of accurately modeling (characterizing) a complete near-term solid-core NTP engine system in great detail, for a number of design options, in an efficient manner. The following discussion summarizes the overall analysis methodology, key assumptions, and capabilities associated with the NESS presents an example problem, and compares the results to related NTP engine system designs. Initial installation instructions and program disks are in Volume 2 of the NESS Program User's Guide.

Infusing Software Engineering Technology into Practice at NASA

NASA Technical Reports Server (NTRS)

Pressburger, Thomas; Feather, Martin S.; Hinchey, Michael; Markosia, Lawrence

2006-01-01

We present an ongoing effort of the NASA Software Engineering Initiative to encourage the use of advanced software engineering technology on NASA projects. Technology infusion is in general a difficult process yet this effort seems to have found a modest approach that is successful for some types of technologies. We outline the process and describe the experience of the technology infusions that occurred over a two year period. We also present some lessons from the experiences.

Cyanide bioremediation: the potential of engineered nitrilases.

PubMed

Park, Jason M; Trevor Sewell, B; Benedik, Michael J

2017-04-01

The cyanide-degrading nitrilases are of notable interest for their potential to remediate cyanide contaminated waste streams, especially as generated in the gold mining, pharmaceutical, and electroplating industries. This review provides a brief overview of cyanide remediation in general but with a particular focus on the cyanide-degrading nitrilases. These are of special interest as the hydrolysis reaction does not require secondary substrates or cofactors, making these enzymes particularly good candidates for industrial remediation processes. The genetic approaches that have been used to date for engineering improved enzymes are described; however, recent structural insights provide a promising new approach.

Engineered microorganisms capable of producing target compounds under anaerobic conditions

DOEpatents

Buelter, Thomas [Denver, CO; Meinhold, Peter [Denver, CO; Feldman, Reid M. Renny [San Francisco, CA; Hawkins, Andrew C [Parker, CO; Urano, Jun [Irvine, CA; Bastian, Sabine [Pasadena, CA; Arnold, Frances [La Canada, CA

2012-01-17

The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

Artificial concurrent catalytic processes involving enzymes.

PubMed

Köhler, Valentin; Turner, Nicholas J

2015-01-11

The concurrent operation of multiple catalysts can lead to enhanced reaction features including (i) simultaneous linear multi-step transformations in a single reaction flask (ii) the control of intermediate equilibria (iii) stereoconvergent transformations (iv) rapid processing of labile reaction products. Enzymes occupy a prominent position for the development of such processes, due to their high potential compatibility with other biocatalysts. Genes for different enzymes can be co-expressed to reconstruct natural or construct artificial pathways and applied in the form of engineered whole cell biocatalysts to carry out complex transformations or, alternatively, the enzymes can be combined in vitro after isolation. Moreover, enzyme variants provide a wider substrate scope for a given reaction and often display altered selectivities and specificities. Man-made transition metal catalysts and engineered or artificial metalloenzymes also widen the range of reactivities and catalysed reactions that are potentially employable. Cascades for simultaneous cofactor or co-substrate regeneration or co-product removal are now firmly established. Many applications of more ambitious concurrent cascade catalysis are only just beginning to appear in the literature. The current review presents some of the most recent examples, with an emphasis on the combination of transition metal with enzymatic catalysis and aims to encourage researchers to contribute to this emerging field.

Definition of the Engineering Method.

ERIC Educational Resources Information Center

Koen, Billy Vaughn

In an effort to more clearly define the engineering method, this document attempts to draw distinctions between engineering and science. Part I, "Some Thoughts on Engineering," discusses strategies that engineers employ to solve problems, and the characteristics of the types of engineering problems. Part II, "The Principal Rule of the Engineering…

Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes.

PubMed

Styles, Matthew Q; Nesbitt, Edward A; Marr, Scott; Hutchby, Marc; Leak, David J

2017-06-01

The terpenoid family of natural products is being targeted for heterologous microbial production as a cheaper and more reliable alternative to extraction from plants. The key enzyme responsible for diversification of terpene structure is the class-I terpene synthase (TS), and these often require engineering to improve properties such as thermostability, robustness and catalytic activity before they are suitable for industrial use. Improving thermostability typically relies on screening a large number of mutants, as there are no naturally thermostable TSs described upon which to base rational design decisions. We have characterized the first examples of natural TSs exhibiting thermostability, which catalyse the formation of the sesquiterpene τ-muurolol at temperatures up to 78 °C. We also report an enzyme with a k cat value of 0.95 s -1 at 65 °C, the highest k cat recorded for a bacterial sesquiterpene synthase. In turn, these thermostable enzymes were used as a model to inform the rational engineering of another TS, with the same specificity but low sequence identity to the model. The newly engineered variant displayed increased thermostability and turnover. Given the high structural homology of the class-I TS domain, this approach could be generally applicable to improving the properties of other enzymes in this class. Model data are available in the PMDB database under the accession number PM0080780. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

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.

Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco.

PubMed

Cai, Zhen; Liu, Guoxia; Zhang, Junli; Li, Yin

2014-07-01

Photosynthetic CO(2) fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of photosynthetic CO(2) fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving photosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an efficient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coli-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G substitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxylation activity and a 45% improvement in catalytic efficiency towards CO(2). The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4'-substituted acetophenones, and follows Swain-Lupton parameterisation.

PubMed

Cassimjee, Karim Engelmark; Humble, Maria Svedendahl; Land, Henrik; Abedi, Vahak; Berglund, Per

2012-07-28

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

Appreciating Formal Similarities in the Kinetics of Homogeneous, Heterogeneous, and Enzyme Catalysis

ERIC Educational Resources Information Center

Ashby, Michael T.

2007-01-01

Because interest in catalysts is widespread, the kinetics of catalytic reactions have been investigated by widely diverse groups of individuals, including chemists, engineers, and biologists. This has lead to redundancy in theories, particularly with regard to the topics of homogeneous, heterogeneous, and enzyme catalysis. From a pedagogical…

Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass

USDA-ARS?s Scientific Manuscript database

The Aspergillus niger ferulic acid esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interactio...

Australian engineering educators' attitudes towards Aboriginal cultures and perspectives

NASA Astrophysics Data System (ADS)

Goldfinch, Thomas; Prpic, Juliana Kaya; Jolly, Lesley; Leigh, Elyssebeth; Kennedy, Jade

2017-07-01

In Australia, representation of Aboriginal populations within the engineering profession is very low despite participation targets set by Government departments, professional bodies and Universities. Progressing the Aboriginal inclusion agenda within Australian Engineering Education requires a clearer understanding of engineering educators' preparedness for increased numbers of students from this non-traditional cohort. This research stems from a recently completed project that explored Aboriginal perspectives in engineering education and proposed a model for embedding perspectives in curricula. Nine engineering academics were interviewed to explore attitudes towards Aboriginal perspectives in engineering and the viability of the proposed model. Results of the interviews indicate efforts to embed Aboriginal perspectives are starting from a small base of knowledge and experience. Individuals' motivations and values indicate that there is significant support for improving this, but that efforts can be hampered by conceptions of Aboriginal perspectives that do not consider how Aboriginal knowledges may change engineering itself.

Establishing a K-12 Circuit Design Program

ERIC Educational Resources Information Center

Inceoglu, Mustafa M.

2010-01-01

Outreach, as defined by Wikipedia, is an effort by an organization or group to connect its ideas or practices to the efforts of other organizations, groups, specific audiences, or the general public. This paper describes a computer engineering outreach project of the Department of Computer Engineering at Ege University, Izmir, Turkey, to a local…

Embracing Scientific and Engineering Practices in 4-H

ERIC Educational Resources Information Center

Worker, Steven M.

2013-01-01

The 4-H Science Initiative has renewed efforts to strengthen 4-H programmatic and evaluation efforts in science and engineering education. A fundamental component of this initiative is to provide opportunities to youth to aid in their development of science process skills; however, emerging research stresses the importance of engaging youth in…

Engineering of Paraoxonases for Pre- and Post-treatment of Intoxication by a Broad Spectrum of Nerve Agents and Pesticides

DTIC Science & Technology

2014-08-01

chemical warfare nerve agents (CWNA). Enzymes identified in these screens should be capable of catalytically neutralizing the target agent under...soluble form. 4. Large-scale production of selected enzyme candidates, and their kinetic, structural and pharmacological evaluation 6...employed, with an enzyme protein concentration of 0.5-2 mM in the assay cuvette, the activity measured was indistinguishable from the rate of

Hyper-X Engine Design and Ground Test Program

NASA Technical Reports Server (NTRS)

Voland, R. T.; Rock, K. E.; Huebner, L. D.; Witte, D. W.; Fischer, K. E.; McClinton, C. R.

1998-01-01

The Hyper-X Program, NASA's focused hypersonic technology program jointly run by NASA Langley and Dryden, is designed to move hypersonic, air-breathing vehicle technology from the laboratory environment to the flight environment, the last stage preceding prototype development. The Hyper-X research vehicle will provide the first ever opportunity to obtain data on an airframe integrated supersonic combustion ramjet propulsion system in flight, providing the first flight validation of wind tunnel, numerical and analytical methods used for design of these vehicles. A substantial portion of the integrated vehicle/engine flowpath development, engine systems verification and validation and flight test risk reduction efforts are experimentally based, including vehicle aeropropulsive force and moment database generation for flight control law development, and integrated vehicle/engine performance validation. The Mach 7 engine flowpath development tests have been completed, and effort is now shifting to engine controls, systems and performance verification and validation tests, as well as, additional flight test risk reduction tests. The engine wind tunnel tests required for these efforts range from tests of partial width engines in both small and large scramjet test facilities, to tests of the full flight engine on a vehicle simulator and tests of a complete flight vehicle in the Langley 8-Ft. High Temperature Tunnel. These tests will begin in the summer of 1998 and continue through 1999. The first flight test is planned for early 2000.

In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose.

PubMed

Zhu, Zhiguang; Zhang, Y-H Percival

2017-01-01

The direct generation of electricity from the most abundant renewable sugar, glucose, is an appealing alternative to the production of liquid biofuels and biohydrogen. However, enzyme-catalyzed bioelectricity generation from glucose suffers from low yields due to the incomplete oxidation of the six-carbon compound glucose via one or few enzymes. Here, we demonstrate a synthetic ATP- and CoA-free 12-enzyme pathway to implement the complete oxidation of glucose in vitro. This pathway is comprised of glucose phosphorylation via polyphosphate glucokinase, NADH generation catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), electron transfer from NADH to the anode, and glucose 6-phosphate regeneration via the non-oxidative pentose phosphate pathway and gluconeogenesis. The faraday efficiency from glucose to electrons via this pathway was as high as 98.8%, suggesting the generation of nearly 24 electrons per molecule of glucose. The generated current density was greatly increased from 2.8 to 6.9mAcm -2 by replacing a low-activity G6PDH with a high-activity G6PDH and introducing a new enzyme, 6-phosphogluconolactonase, between G6PDH and 6PGDH. These results suggest the great potential of high-yield bioelectricity generation through in vitro metabolic engineering. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

PubMed Central

Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun

2013-01-01

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. PMID:23974145

Knowledge Integration and Wise Engineering

ERIC Educational Resources Information Center

Chiu, Jennifer L.; Linn, M. C.

2011-01-01

Recent efforts in engineering education focus on introducing engineering into secondary math and science courses to improve science, technology, engineering, and math (STEM) education (NAS, 2010). Infusing engineering into secondary classrooms can increase awareness of and interest in STEM careers, help students see the relevance of science and…

Philip T. Pienkos | NREL

Science.gov Websites

Valorization: Development of a Methane-to-Adipic Acid Bioprocess (consultant) Areas of Expertise Molecular engineering Enzyme and protein engineering Techno-economic analysis Education Ph.D., Molecular Biology Principal Research Supervisor, NREL, Aug. 2007-May 2011 Research Director, Molecular Logix, Inc., The

Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization.

PubMed

Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio

2011-04-01

Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

Research and application of microbial enzymes--India's contribution.

PubMed

Chand, Subhash; Mishra, Prashant

2003-01-01

Enzymes have attracted the attention of scientists world over due to their wide range of physiological, analytical and industrial applications. Although enzymes have been isolated, purified and studied from microbial, animal and plant sources, microorganisms represent the most common source of enzymes due to their broad biochemical diversity, feasibility of mass culture and ease of genetic manipulation. With the advent of molecular biology techniques, a number of genes of industrially important enzymes has been cloned and expressed in order to improve the production of enzymes, substrate utilization and other commercially useful properties. Special attention has been focused on enzymes isolated from thermophiles due to their inherent stability and industrial applications. In addition, a variety of methods have been employed to modify enzymes for their industrial usage including strain improvement, chemical modifications, modification of reaction environment, immobilization and protein engineering. A wide range of applications of enzymes in different bioprocess industries is discussed.

Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms.

PubMed

Thallinger, Barbara; Prasetyo, Endry N; Nyanhongo, Gibson S; Guebitz, Georg M

2013-01-01

With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme-based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi-enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum-sensing molecules. The inclusion of anti-quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti-biofilm enzymes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ExplorEnz: the primary source of the IUBMB enzyme list

PubMed Central

McDonald, Andrew G.; Boyce, Sinéad; Tipton, Keith F.

2009-01-01

ExplorEnz is the MySQL database that is used for the curation and dissemination of the International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature. A simple web-based query interface is provided, along with an advanced search engine for more complex Boolean queries. The WWW front-end is accessible at http://www.enzyme-database.org, from where downloads of the database as SQL and XML are also available. An associated form-based curatorial application has been developed to facilitate the curation of enzyme data as well as the internal and public review processes that occur before an enzyme entry is made official. Suggestions for new enzyme entries, or modifications to existing ones, can be made using the forms provided at http://www.enzyme-database.org/forms.php. PMID:18776214

Production of Enzymes from Marine Actinobacteria.

PubMed

Zhao, X Q; Xu, X N; Chen, L Y

Marine actinobacteria are well recognized for their capabilities to produce valuable natural products, which have great potential for applications in medical, agricultural, and fine chemical industries. In addition to producing unique enzymes responsible for biosynthesis of natural products, many marine actinobacteria also produce hydrolytic enzymes which are able to degrade various biopolymers, such as cellulose, xylan, and chitin. These enzymes are important to produce biofuels and biochemicals of interest from renewable biomass. In this chapter, the recent reports of novel enzymes produced by marine actinobacteria are reviewed, and advanced technologies that can be applied to search for novel marine enzymes as well as for improved enzyme production by marine actinobacteria are summarized, which include ribosome engineering, genome mining, as well as synthetic biology studies. © 2016 Elsevier Inc. All rights reserved.

Rapid purification of fluorescent enzymes by ultrafiltration

NASA Technical Reports Server (NTRS)

Benjaminson, M. A.; Satyanarayana, T.

1983-01-01

In order to expedite the preparation of fluorescently tagged enzymes for histo-cyctochemistry, a previously developed method employing gel column purification was compared with a more rapid modern technique using the Millipore Immersible CX-ultrafilter. Microscopic evaluation of the resulting conjugates showed comparable products. Much time and effort is saved using the new technique.

Rapid purification of fluorescent enzymes by ultrafiltration

NASA Technical Reports Server (NTRS)

Benjaminson, M. A.; Satyanarayana, T.

1983-01-01

In order to expedite the preparation of fluorescently tagged enzymes for histo/cytochemistry, a previously developed method employing gel column purification was compared with a more rapid modern technique using the Millipore Immersible CX-ultrafilter. Microscopic evaluation of the resulting conjugates showed comparable products. Much time and effort is saved using the new technique.

Engine environmental effects on composite behavior

NASA Technical Reports Server (NTRS)

Chamis, C. C.; Smith, G. T.

1980-01-01

A series of programs were conducted to investigate and develop the application of composite materials to turbojet engines. A significant part of that effort was directed to establishing the impact resistance and defect growth chracteristics of composite materials over the wide range of environmental conditions found in commercial turbojet engine operations. Both analytical and empirical efforts were involved. The experimental programs and the analytical methodology development as well as an evaluation program for the use of composite materials as fan exit guide vanes are summarized.

Rational design of engineered microbial cell surface multi-enzyme co-display system for sustainable NADH regeneration from low-cost biomass.

PubMed

Han, Lei; Liang, Bo; Song, Jianxia

2018-02-01

As an important cofactor, NADH is essential for most redox reactions and biofuel cells. However, supply of exogenous NADH is challenged, due to the low production efficiency and high cost of NADH regeneration system, as well as low stability of NADH. Here, we constructed a novel cell surface multi-enzyme co-display system with ratio- and space-controllable manner as exogenous NADH regeneration system for the sustainable NADH production from low-cost biomass. Dockerin-fused glucoamylase (GA) and glucose dehydrogenase (GDH) were expressed and assembled on the engineered bacterial surfaces, which displayed protein scaffolds with various combinations of different cohesins. When the ratio of GA and GDH was 3:1, the NADH production rate of the whole-cell biocatalyst reached the highest level using starch as substrate, which was three times higher than that of mixture of free enzymes, indicating that the highly ordered spatial organization of enzymes would promote reactions, due to the ratio of enzymes and proximity effect. To confirm performance of the established NADH regeneration system, the highly efficient synthesis of L-lactic acid (L-LA) was conducted by the system and the yield of L-LA (16 g/L) was twice higher than that of the mixture of free enzymes. The multi-enzyme co-display system showed good stability in the cyclic utilization. In conclusion, the novel sustainable NADH system would provide a cost-effective strategy to regenerate cofactor from low-cost biomass.

Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana

DOE PAGES

Smith, Sarah R.; Gle, Corine; Abbriano, Raffaela M.; ...

2016-02-04

Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity. To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterizedmore » alongside lipid accumulation. Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes. Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal-derived biofuels.« less

Enhanced pyruvate production in Candida glabrata by carrier engineering.

PubMed

Luo, Zhengshan; Liu, Song; Du, Guocheng; Xu, Sha; Zhou, Jingwen; Chen, Jian

2018-02-01

Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria, a high central carbon metabolism rate, specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate-limiting glycolytic enzymes. Moreover, pyruvate production of CGΔura3-Sp-MPC1, CGΔura3-Sp-MPC2, and CGΔura3-Sp-MPC1-Sp-MPC2 increased 134.4%, 120.3%, and 30.0%, respectively. In conclusion, lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms. © 2017 Wiley Periodicals, Inc.

A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.

PubMed

Hong, Jin-Bon; Chou, Fu-Ju; Ku, Amy T; Fan, Hsiang-Hsuan; Lee, Tung-Lung; Huang, Yung-Hsin; Yang, Tsung-Lin; Su, I-Chang; Yu, I-Shing; Lin, Shu-Wha; Chien, Chung-Liang; Ho, Hong-Nerng; Chen, You-Tzung

2014-01-01

PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

RetroPath2.0: A retrosynthesis workflow for metabolic engineers.

PubMed

Delépine, Baudoin; Duigou, Thomas; Carbonell, Pablo; Faulon, Jean-Loup

2018-01-01

Synthetic biology applied to industrial biotechnology is transforming the way we produce chemicals. However, despite advances in the scale and scope of metabolic engineering, the research and development process still remains costly. In order to expand the chemical repertoire for the production of next generation compounds, a major engineering biology effort is required in the development of novel design tools that target chemical diversity through rapid and predictable protocols. Addressing that goal involves retrosynthesis approaches that explore the chemical biosynthetic space. However, the complexity associated with the large combinatorial retrosynthesis design space has often been recognized as the main challenge hindering the approach. Here, we provide RetroPath2.0, an automated open source workflow for retrosynthesis based on generalized reaction rules that perform the retrosynthesis search from chassis to target through an efficient and well-controlled protocol. Its easiness of use and the versatility of its applications make this tool a valuable addition to the biological engineer bench desk. We show through several examples the application of the workflow to biotechnological relevant problems, including the identification of alternative biosynthetic routes through enzyme promiscuity or the development of biosensors. We demonstrate in that way the ability of the workflow to streamline retrosynthesis pathway design and its major role in reshaping the design, build, test and learn pipeline by driving the process toward the objective of optimizing bioproduction. The RetroPath2.0 workflow is built using tools developed by the bioinformatics and cheminformatics community, because it is open source we anticipate community contributions will likely expand further the features of the workflow. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Halophiles and their enzymes: negativity put to good use.

PubMed

DasSarma, Shiladitya; DasSarma, Priya

2015-06-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. Recent efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. Copyright © 2015 Elsevier Ltd. All rights reserved.

Halophiles and their enzymes: Negativity put to good use

PubMed Central

DasSarma, Shiladitya; DasSarma, Priya

2015-01-01

Halophilic microorganisms possess stable enzymes that function in very high salinity, an extreme condition that leads to denaturation, aggregation, and precipitation of most other proteins. Genomic and structural analyses have established that the enzymes of halophilic Archaea and many halophilic Bacteria are negatively charged due to an excess of acidic over basic residues, and altered hydrophobicity, which enhance solubility and promote function in low water activity conditions. Here, we provide an update on recent bioinformatic analysis of predicted halophilic proteomes as well as experimental molecular studies on individual halophilic enzymes. On-going efforts on discovery and utilization of halophiles and their enzymes for biotechnology, including biofuel applications are also considered. PMID:26066288

Emerging engineering principles for yield improvement in microbial cell design.

PubMed

Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo

2012-01-01

Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling.

Emerging engineering principles for yield improvement in microbial cell design

PubMed Central

Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo

2012-01-01

Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling. PMID:24688676

Activities in Support of Two-Year College Science, Engineering, Technology, and Mathematics Education, Fiscal Year 1993. Highlights.

ERIC Educational Resources Information Center

National Science Foundation, Washington, DC. Directorate for Education and Human Resources.

This report describes the efforts of the National Science Foundation (NSF) and its Division of Undergraduate Education (DUE) to provide educational support to two-year colleges to strengthen science, technology, engineering, and mathematics programs through grants, collaborative efforts, and support for curriculum materials and teacher activities.…

48 CFR 48.101 - General.

Code of Federal Regulations, 2013 CFR

2013-10-01

... engineering effort is applied to areas of the contract that offer opportunities for considerable savings... ENGINEERING Policies and Procedures 48.101 General. (a) Value engineering is the formal technique by which... performing more economically. Value engineering attempts to eliminate, without impairing essential functions...

48 CFR 48.101 - General.

Code of Federal Regulations, 2014 CFR

2014-10-01

... engineering effort is applied to areas of the contract that offer opportunities for considerable savings... ENGINEERING Policies and Procedures 48.101 General. (a) Value engineering is the formal technique by which... performing more economically. Value engineering attempts to eliminate, without impairing essential functions...

48 CFR 48.101 - General.

Code of Federal Regulations, 2012 CFR

2012-10-01

... engineering effort is applied to areas of the contract that offer opportunities for considerable savings... ENGINEERING Policies and Procedures 48.101 General. (a) Value engineering is the formal technique by which... performing more economically. Value engineering attempts to eliminate, without impairing essential functions...

Engineering--A Career for 330 Women at Boeing

ERIC Educational Resources Information Center

Minor, Barbara

1974-01-01

Describes the efforts of the Boeing Company to encourage and hire women for engineering jobs. Much of the article is devoted to women employed by Boeing in engineering jobs and their comments about engineering as a career for women. (BR)

Nanobiocatalyst advancements and bioprocessing applications

PubMed Central

Misson, Mailin; Zhang, Hu; Jin, Bo

2015-01-01

The nanobiocatalyst (NBC) is an emerging innovation that synergistically integrates advanced nanotechnology with biotechnology and promises exciting advantages for improving enzyme activity, stability, capability and engineering performances in bioprocessing applications. NBCs are fabricated by immobilizing enzymes with functional nanomaterials as enzyme carriers or containers. In this paper, we review the recent developments of novel nanocarriers/nanocontainers with advanced hierarchical porous structures for retaining enzymes, such as nanofibres (NFs), mesoporous nanocarriers and nanocages. Strategies for immobilizing enzymes onto nanocarriers made from polymers, silicas, carbons and metals by physical adsorption, covalent binding, cross-linking or specific ligand spacers are discussed. The resulting NBCs are critically evaluated in terms of their bioprocessing performances. Excellent performances are demonstrated through enhanced NBC catalytic activity and stability due to conformational changes upon immobilization and localized nanoenvironments, and NBC reutilization by assembling magnetic nanoparticles into NBCs to defray the high operational costs associated with enzyme production and nanocarrier synthesis. We also highlight several challenges associated with the NBC-driven bioprocess applications, including the maturation of large-scale nanocarrier synthesis, design and development of bioreactors to accommodate NBCs, and long-term operations of NBCs. We suggest these challenges are to be addressed through joint collaboration of chemists, engineers and material scientists. Finally, we have demonstrated the great potential of NBCs in manufacturing bioprocesses in the near future through successful laboratory trials of NBCs in carbohydrate hydrolysis, biofuel production and biotransformation. PMID:25392397

Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates.

PubMed

Aldor, Ilana S; Keasling, Jay D

2003-10-01

Implementing several metabolic engineering strategies, either individually or in combination, it is possible to construct microbial plastic factories to produce a variety of polyhydroxyalkanoate (PHA) biopolymers with desirable structures and material properties. Approaches include external substrate manipulation, inhibitor addition, recombinant gene expression, host cell genome manipulation and, most recently, protein engineering of PHA biosynthetic enzymes. In addition, mathematical models and molecular methods can be used to elucidate metabolically engineered systems and to identify targets for performance improvement.

Sensitising effects of genetically modified enzymes used in flavour, fragrance, detergence and pharmaceutical production: cross-sectional study.

PubMed

Budnik, Lygia T; Scheer, Edwin; Burge, P Sherwood; Baur, Xaver

2017-01-01

The use of genetically engineered enzymes in the synthesis of flavourings, fragrances and other applications has increased tremendously. There is, however, a paucity of data on sensitisation and/or allergy to the finished products. We aimed to review the use of genetically modified enzymes and the enormous challenges in human biomonitoring studies with suitable assays of specific IgE to a variety of modified enzyme proteins in occupational settings and measure specific IgE to modified enzymes in exposed workers. Specific IgE antibodies against workplace-specific individual enzymes were measured by the specific fluorescence enzyme-labelled immunoassay in 813 exposed workers seen in cross-sectional surveys. Twenty-three per cent of all exposed workers showed type I sensitisation with IgE antibodies directed against respective workplace-specific enzymes. The highest sensitisation frequencies observed were for workers exposed enzymes derived from α-amylase (44%), followed by stainzyme (41%), pancreatinin (35%), savinase (31%), papain (31%), ovozyme (28%), phytase (16%), trypsin (15%) and lipase (4%). The highest individual antibody levels (up to 110 kU/L) were detected in workers exposed to phytase, xylanase and glucanase. In a subgroup comprising 134 workers, detailed clinical diagnostics confirmed work-related symptoms. There was a strong correlation (r=0.75, p<0.0001) between the symptoms and antibody levels. Workers with work-related respiratory symptoms showed a higher prevalence for the presence of specific IgE antibodies against workplace-specific enzymes than asymptomatic exposed workers (likelihood ratio 2.32, sensitivity 0.92, specificity 0.6). Our data confirm the previous findings showing that genetically engineered enzymes are potent allergens eliciting immediate-type sensitisation. Owing to lack of commercial diagnostic tests, few of those exposed receive regular surveillance including biomonitoring with relevant specific IgE assays. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Engineering Background: Modern Formats and Challenges of Conceptual Engineering

NASA Astrophysics Data System (ADS)

Khamidullina, A. F.; Kuzmina, M. A.; Khusnutdinova, E. M.; Konakhina, I. A.

2017-09-01

This paper describes the analysis of problems and development perspectives of engineering education in our and other countries. Special attention is given to modern formats of education that motivate creative efforts of engineers-to-be as well as issues of conceptual engineering taking the challenges of modernity into account.

Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

DOE PAGES

Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong; ...

2014-12-09

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gainsmore » in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.« less

Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes.

PubMed

Loder, Andrew J; Zeldes, Benjamin M; Garrison, G Dale; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

2015-10-01

n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes

PubMed Central

Loder, Andrew J.; Zeldes, Benjamin M.; Garrison, G. Dale; Lipscomb, Gina L.; Adams, Michael W. W.

2015-01-01

n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. PMID:26253677

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.

System Engineering and Technical Challenges Overcome in the J-2X Rocket Engine Development Project

NASA Technical Reports Server (NTRS)

Ballard, Richard O.

2012-01-01

Beginning in 2006, NASA initiated the J-2X engine development effort to develop an upper stage propulsion system to enable the achievement of the primary objectives of the Constellation program (CxP): provide continued access to the International Space Station following the retirement of the Space Station and return humans to the moon. The J-2X system requirements identified to accomplish this were very challenging and the time expended over the five years following the beginning of the J- 2X effort have been noteworthy in the development of innovations in both the fields for liquid rocket propulsion and system engineering.

Exploration Medical Capability System Engineering Introduction and Vision

NASA Technical Reports Server (NTRS)

Mindock, J.; Reilly, J.

2017-01-01

Human exploration missions to beyond low Earth orbit destinations such as Mars will require more autonomous capability compared to current low Earth orbit operations. For the medical system, lack of consumable resupply, evacuation opportunities, and real-time ground support are key drivers toward greater autonomy. Recognition of the limited mission and vehicle resources available to carry out exploration missions motivates the Exploration Medical Capability (ExMC) Element's approach to enabling the necessary autonomy. The Element's work must integrate with the overall exploration mission and vehicle design efforts to successfully provide exploration medical capabilities. ExMC is applying systems engineering principles and practices to accomplish its integrative goals. This talk will briefly introduce the discipline of systems engineering and key points in its application to exploration medical capability development. It will elucidate technical medical system needs to be met by the systems engineering work, and the structured and integrative science and engineering approach to satisfying those needs, including the development of shared mental and qualitative models within and external to the human health and performance community. These efforts are underway to ensure relevancy to exploration system maturation and to establish medical system development that is collaborative with vehicle and mission design and engineering efforts.

An Account of Women's Progress in Engineering: a Social Cognitive Perspective

NASA Astrophysics Data System (ADS)

Vogt, Christina

Traditionally, women were not welcome in higher education, especially in male-dominated fields. Undoubtedly, women have dramatically increased their enrollments in many once male-only fields, such as law, medicine, and several of the sciences; nevertheless, engineering remains a field where women continue to be underrepresented. This has often been attributed to social barriers in engineering classrooms. However, a new turn of events has been reported: Young women entering engineering may receive higher grades and have a greater tendency to remain than men. To examine what has recently changed, the author applied Bandura's triadic model of reciprocity between environment, self, and behavior. The measured variables included academic integration or discrimination, self-measures of academic self-confidence, engineering self-efficacy, and behaviors taken to self-regulate learning: critical thinking, effort, peer learning, and help seeking. The data revealed that women apply slightly more effort and have slightly less self-efficacy than men. Their academic confidence is nearly equal in almost all areas. Most significantly, many previous gender biases appear diminished, and those that do exist are slight. However, it is recommended that continued efforts be undertaken to attract and retain women in engineering programs.

The limits to biocatalysis: pushing the envelope.

PubMed

Sheldon, Roger A; Brady, Dean

2018-06-12

In the period 1985 to 1995 applications of biocatalysis, driven by the need for more sustainable manufacture of chemicals and catalytic, (enantio)selective methods for the synthesis of pharmaceutical intermediates, largely involved the available hydrolases. This was followed, in the next two decades, by revolutionary developments in protein engineering and directed evolution for the optimisation of enzyme function and performance that totally changed the biocatalysis landscape. In the same period, metabolic engineering and synthetic biology revolutionised the use of whole cell biocatalysis in the synthesis of commodity chemicals by fermentation. In particular, developments in the enzymatic enantioselective synthesis of chiral alcohols and amines are highlighted. Progress in enzyme immobilisation facilitated applications under harsh industrial conditions, such as in organic solvents. The emergence of biocatalytic or chemoenzymatic cascade processes, often with co-immobilised enzymes, has enabled telescoping of multi-step processes. Discovering and inventing new biocatalytic processes, based on (meta)genomic sequencing, evolving enzyme promiscuity, chemomimetic biocatalysis, artificial metalloenzymes, and the introduction of non-canonical amino acids into proteins, are pushing back the limits of biocatalysis function. Finally, the integral role of biocatalysis in developing a biobased carbon-neutral economy is discussed.

The production, properties, and applications of thermostable steryl glucosidases.

PubMed

Aguirre, Andres; Eberhardt, Florencia; Hails, Guillermo; Cerminati, Sebastian; Castelli, María Eugenia; Rasia, Rodolfo M; Paoletti, Luciana; Menzella, Hugo G; Peiru, Salvador

2018-02-21

Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable β-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.

[Advances of consolidated bioprocessing based on recombinant strategy].

PubMed

Zheng, Zongbao; Zhao, Meina; Chen, Tao; Zhao, Xueming

2013-10-01

Lignocellulosic biomass represents an abundant, low-cost and renewable source of potentially fermentable sugars. It is acandidate besides petroleum as feedstock for fuel and chemical production. Recent researches on utilizing lignocellulosicsas feedstock boost development of numerous-promising processes for a variety of fuels and chemicals, such as biodiesel, biohydrogen and ethanol. However, high cost in depolymerization is a primary obstacle preventing the use of lignocellulosic biomass as feedstock. Consolidated bioprocessing (CBP), refers to the bioprocess without any exogenous cellulolyotic enzymes added, converting the lignocellulosic material into biochemicals directly, which could potentially avoid the cost of the dedicated enzyme generation step by incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single organism through genetic engineering. There are two CBP strategies, native strategy and recombinant strategy. We mainly introduce the recombinant strategy, including its principle, the two responding styles, the contributions of synthetic biology and metabolic engineering and the future challenges.

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis.

PubMed

Jacques, Philippe; Béchet, Max; Bigan, Muriel; Caly, Delphine; Chataigné, Gabrielle; Coutte, François; Flahaut, Christophe; Heuson, Egon; Leclère, Valérie; Lecouturier, Didier; Phalip, Vincent; Ravallec, Rozenn; Dhulster, Pascal; Froidevaux, Rénato

2017-02-01

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

Engineering the intracellular metabolism of Escherichia coli to produce gamma-aminobutyric acid by co-localization of GABA shunt enzymes.

PubMed

Pham, Van Dung; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

2016-02-01

To direct the carbon flux from Krebs cycle into the gamma-aminobutyric acid (GABA) shunt pathway for the production of GABA by protein scaffold introduction in Escherichia coli. Escherichia coli was engineered to produce GABA from glucose by the co-localization of enzymes succinate semialdehyde dehydrogenase (GadD), GABA aminotransferase (PuuE) and GABA transporter (GadC) by protein scaffold. 0.7 g GABA l(-1) was produced from 10 g glucose l(-1) while no GABA was produced in wild type E. coli. pH 6 and 30 °C were optimum for GABA production, and GABA concentration increased to 1.12 g GABA l(-1) when 20 g glucose l(-1) was used. When competing metabolic networks were inactivated, GABA increased by 24 % (0.87 g GABA l(-1)). The novel GABA production system was constructed by co-localization of GABA shunt enzymes.

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

Sunden, Fanny; Peck, Ariana; Salzman, Julia

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modesmore » of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.« less

Dietary incorporation of feedstuffs naturally high in organic selenium for racing pigeons (Columba livia): effects on plasma antioxidant markers after a standardised simulation of a flying effort.

PubMed

Schoonheere, N; Dotreppe, O; Pincemail, J; Istasse, L; Hornick, J L

2009-06-01

Selenium is a trace element of importance for animal health. It is essential for adequate functioning of many enzymes such as, the antioxidant enzyme, glutathione peroxidase, which protects the cell against free radicals. A muscular effort induces a rise in reactive oxygen species production which, in turn, can generate an oxidative stress. Two groups of eight racing pigeons were fed respectively with a diet containing 30.3 (control group) and 195.3 (selenium group) microg selenium/kg diet. The pigeons were submitted to a standardised simulation of a flying effort during 2 h. Blood was taken before and after the effort to measure antioxidant markers and blood parameters related to muscle metabolism. Plasma selenium concentration and glutathione peroxidase activity were significantly higher in the selenium group. There were no significant differences for the other measured parameters. As a consequence of the effort, the pigeons of the selenium group showed a higher increase of glutathione peroxidase activity and a smaller increase of plasma lactate concentration. Variations because of the effort in the other markers were not significantly different between the two groups. It is concluded that the selenium status was improved with the feeding of feedstuffs high in Selenium.

Engineering Isoprene Synthase Expression and Activity in Cyanobacteria.

PubMed

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

2017-12-15

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

Sensitivity and Antioxidant Response of Chlorella sp. MM3 to Used Engine Oil and Its Water Accommodated Fraction.

PubMed

Ramadass, Kavitha; Megharaj, Mallavarapu; Venkateswarlu, Kadiyala; Naidu, Ravi

2016-07-01

We exposed the microalgal strain, Chlorella sp. MM3, to unused or used engine oil, or their water accommodated fractions (WAFs) to determine growth inhibition and response of antioxidant enzymes. Oil type and oil concentration greatly affected the microalgal growth. Used oil at 0.04 % (0.4 g L(-1)) resulted in 50 % inhibition in algal growth, measured in terms of chlorophyll-a, while the corresponding concentration of unused oil was nontoxic. Similarly, used oil WAF showed significant toxicity to the algal growth at 10 % level, whereas WAF from unused oil was nontoxic even at 100 % concentration. Peroxidase enzyme in the microalga significantly increased with used oil at concentrations above 0.04 g L(-1) whereas the induction of superoxide dismutase and catalase was apparent only at 0.06 g L(-1). Activities of the antioxidant enzymes increased significantly when the microalga was exposed to 75 and 100 % WAF obtained from used oil. The used oil toxicity on microalga could be due to the presence of toxic soluble mono- and polyaromatic compounds, heavy metals, and other compounds attained by the oil during its use in the motor engines.

Min Zhang | NREL

Science.gov Websites

Min Zhang Photo of Min Zhang Min Zhang Researcher V-Molecular Biology Min.Zhang@nrel.gov | 303-384 -7753 Research Interests Using a systems biology approach to identify, analyze, and engineer pathways Metabolic engineering Molecular biology Microbial physiology Systems biology Fermentation development Enzyme

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

Spreitzer, Robert Joseph

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO 2 fixation in photosynthesis. However, it is a slow enzyme, and O 2 competes with CO 2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO 2. If carboxylation could be increased or oxygenation decreased, an increase in net CO 2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants,more » and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO 2/O 2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a possible structural pathway between the small-subunit βA-βB loop and alpha-helix 8 of the large-subunit α/β-barrel active site. Hybrid enzymes were also created comprised of plant small subunits and Chlamydomonas large subunits, and these enzymes have increases in CO 2/O 2 specificity, further indicating that small subunits may be the key for ultimately engineering an improved Rubisco enzyme.« less

ExplorEnz: a MySQL database of the IUBMB enzyme nomenclature.

PubMed

McDonald, Andrew G; Boyce, Sinéad; Moss, Gerard P; Dixon, Henry B F; Tipton, Keith F

2007-07-27

We describe the database ExplorEnz, which is the primary repository for EC numbers and enzyme data that are being curated on behalf of the IUBMB. The enzyme nomenclature is incorporated into many other resources, including the ExPASy-ENZYME, BRENDA and KEGG bioinformatics databases. The data, which are stored in a MySQL database, preserve the formatting of chemical and enzyme names. A simple, easy to use, web-based query interface is provided, along with an advanced search engine for more complex queries. The database is publicly available at http://www.enzyme-database.org. The data are available for download as SQL and XML files via FTP. ExplorEnz has powerful and flexible search capabilities and provides the scientific community with the most up-to-date version of the IUBMB Enzyme List.

Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement.

PubMed

Farnoosh, Gholamreza; Khajeh, Khosro; Latifi, Ali Mohammad; Aghamollaei, Hossein

2016-12-01

The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, delta G inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.

Modeling to Mars: a NASA Model Based Systems Engineering Pathfinder Effort

NASA Technical Reports Server (NTRS)

Phojanamongkolkij, Nipa; Lee, Kristopher A.; Miller, Scott T.; Vorndran, Kenneth A.; Vaden, Karl R.; Ross, Eric P.; Powell, Bobby C.; Moses, Robert W.

2017-01-01

The NASA Engineering Safety Center (NESC) Systems Engineering (SE) Technical Discipline Team (TDT) initiated the Model Based Systems Engineering (MBSE) Pathfinder effort in FY16. The goals and objectives of the MBSE Pathfinder include developing and advancing MBSE capability across NASA, applying MBSE to real NASA issues, and capturing issues and opportunities surrounding MBSE. The Pathfinder effort consisted of four teams, with each team addressing a particular focus area. This paper focuses on Pathfinder team 1 with the focus area of architectures and mission campaigns. These efforts covered the timeframe of February 2016 through September 2016. The team was comprised of eight team members from seven NASA Centers (Glenn Research Center, Langley Research Center, Ames Research Center, Goddard Space Flight Center IV&V Facility, Johnson Space Center, Marshall Space Flight Center, and Stennis Space Center). Collectively, the team had varying levels of knowledge, skills and expertise in systems engineering and MBSE. The team applied their existing and newly acquired system modeling knowledge and expertise to develop modeling products for a campaign (Program) of crew and cargo missions (Projects) to establish a human presence on Mars utilizing In-Situ Resource Utilization (ISRU). Pathfinder team 1 developed a subset of modeling products that are required for a Program System Requirement Review (SRR)/System Design Review (SDR) and Project Mission Concept Review (MCR)/SRR as defined in NASA Procedural Requirements. Additionally, Team 1 was able to perform and demonstrate some trades and constraint analyses. At the end of these efforts, over twenty lessons learned and recommended next steps have been identified.

Engineering a self-sufficient Mycobacterium tuberculosis CYP130 by gene fusion with the reductase-domain of CYP102A1 from Bacillus megaterium.

PubMed

Ortega Ugalde, Sandra; Luirink, Rosa A; Geerke, Daan P; Vermeulen, Nico P E; Bitter, Wilbert; Commandeur, Jan N M

2018-03-01

CYP130 belongs to the subset of cytochrome P450s from Mycobacterium tuberculosis (Mtb) that have been structurally characterized. Despite several efforts for its functional characterization, CYP130 is still considered an orphan enzyme for which no endogenous or exogenous substrate has been identified. In addition, functional redox-partners for CYP130 have not been clearly established yet, hampering the elucidation of its physiological role. In the present study, a catalytically active fusion protein involving CYP130 and the NADPH reductase-domain of CYP102A1 from Bacillus megaterium was created. By screening a panel of known substrates of human P450s, dextromethorphan N-demethylation was identified as a reaction catalyzed by CYP130. The fusion enzyme showed higher catalytic activity, when compared to CYP130 reconstituted with a selection of non-native redox-partners. Molecular dynamics simulation studies based on the crystal structure of CYP130 revealed two primary docking poses of dextromethorphan within the active site consistent with the experimentally observed N-demethylation reaction during the entire molecular dynamics simulation. The dextromethorphan N-demethylation reaction was strongly inhibited by azole-drugs and maybe applied to identify mechanism-based inhibitors of CYP130. Furthermore, the present active CYP130-fusion protein may facilitate the identification of endogenous substrates from Mtb. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic Engineering to Develop a Pathway for the Selective Cleavage of Carbon-Nitrogen Bonds

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

John J. Kilbane II

The objective of the project is to develop a biochemical pathway for the selective cleavage of C-N bonds in molecules found in petroleum. Specifically a novel biochemical pathway will be developed for the selective cleavage of C-N bonds in carbazole. The cleavage of the first C-N bond in carbazole is accomplished by the enzyme carbazole dioxygenase, that catalyzes the conversion of carbazole to 2-aminobiphenyl-2,3-diol. The genes encoding carbazole dioxygenase were cloned from Sphingomonas sp. GTIN11 and from Pseudomonas resinovorans CA10. The selective cleavage of the second C-N bond has been challenging, and efforts to overcome that challenge have been themore » focus of recent research in this project. Enrichment culture experiments succeeded in isolating bacterial cultures that can metabolize 2-aminobiphenyl, but no enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl has been identified. Aniline is very similar to the structure of 2-aminobiphenyl and aniline dioxygenase catalyzes the conversion of aniline to catechol and ammonia. For the remainder of the project the emphasis of research will be to simultaneously express the genes for carbazole dioxygenase and for aniline dioxygenase in the same bacterial host and then to select for derivative cultures capable of using carbazole as the sole source of nitrogen.« less

Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.

PubMed

Sun, Zhoutong; Lonsdale, Richard; Li, Guangyue; Reetz, Manfred T

2016-10-04

Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane-1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R- and S,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzyme-catalyzed crosslinkable hydrogels: emerging strategies for tissue engineering.

PubMed

Teixeira, Liliana S Moreira; Feijen, Jan; van Blitterswijk, Clemens A; Dijkstra, Pieter J; Karperien, Marcel

2012-02-01

State-of-the-art bioactive hydrogels can easily and efficiently be formed by enzyme-catalyzed mild-crosslinking reactions in situ. Yet this cell-friendly and substrate-specific method remains under explored. Hydrogels prepared by using enzyme systems like tyrosinases, transferases and lysyl oxidases show interesting characteristics as dynamic scaffolds and as systems for controlled release. Increased attention is currently paid to hydrogels obtained via crosslinking of precursors by transferases or peroxidases as catalysts. Enzyme-mediated crosslinking has proven its efficiency and attention has now shifted to the development of enzymatically crosslinked hydrogels with higher degrees of complexity, mimicking extracellular matrices. Moreover, bottom-up approaches combining biocatalysts and self-assembly are being explored for the development of complex nano-scale architectures. In this review, the use of enzymatic crosslinking for the preparation of hydrogels as an innovative alternative to other crosslinking methods, such as the commonly used UV-mediated photo-crosslinking or physical crosslinking, will be discussed. Photo-initiator-based crosslinking may induce cytotoxicity in the formed gels, whereas physical crosslinking may lead to gels which do not have sufficient mechanical strength and stability. These limitations can be overcome using enzymes to form covalently crosslinked hydrogels. Herewith, we report the mechanisms involved and current applications, focusing on emerging strategies for tissue engineering and regenerative medicine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

PubMed Central

Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2012-01-01

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges.

PubMed

Zhang, Yi-Heng Percival

2015-11-15

The largest obstacle to the cost-competitive production of low-value and high-impact biofuels and biochemicals (called biocommodities) is high production costs catalyzed by microbes due to their inherent weaknesses, such as low product yield, slow reaction rate, high separation cost, intolerance to toxic products, and so on. This predominant whole-cell platform suffers from a mismatch between the primary goal of living microbes - cell proliferation and the desired biomanufacturing goal - desired products (not cell mass most times). In vitro synthetic biosystems consist of numerous enzymes as building bricks, enzyme complexes as building modules, and/or (biomimetic) coenzymes, which are assembled into synthetic enzymatic pathways for implementing complicated bioreactions. They emerge as an alternative solution for accomplishing a desired biotransformation without concerns of cell proliferation, complicated cellular regulation, and side-product formation. In addition to the most important advantage - high product yield, in vitro synthetic biosystems feature several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this perspective review, the general design rules of in vitro synthetic pathways are presented with eight supporting examples: hydrogen, n-butanol, isobutanol, electricity, starch, lactate,1,3-propanediol, and poly-3-hydroxylbutyrate. Also, a detailed economic analysis for enzymatic hydrogen production from carbohydrates is presented to illustrate some advantages of this system and the remaining challenges. Great market potentials will motivate worldwide efforts from multiple disciplines (i.e., chemistry, biology and engineering) to address the remaining obstacles pertaining to cost and stability of enzymes and coenzymes, standardized building parts and modules, biomimetic coenzymes, biosystem optimization, and scale-up, soon. Copyright © 2014 Elsevier Inc. All rights reserved.

Emissions and new technology programs for conventional spark-ignition aircraft engines

NASA Technical Reports Server (NTRS)

Wintucky, W. T.

1976-01-01

A long-range technology plan in support of general aviation engines was formulated and is being implemented at the Lewis Research Center. The overall program was described, and that part of the program that represents the in-house effort at Lewis was presented in detail. Three areas of government and industry effort involving conventional general-aviation piston engines were part of a coordinated overall plan: (1) FAA/NASA joint program, (2) NASA contract exhaust emissions pollution reduction program, and (3) NASA in-house emissions reduction and new technology program.

Engineering Ultrastable Protein Filaments into 2D and 3D Templates for Advanced Nanomaterials: A New Dimension in Materials Design

DTIC Science & Technology

2013-08-16

Littlechild,   Thermophilic  archaeal   enzymes  and  applications  in  biocatalysis,  Biochem  Soc  Trans  39   (2011...spatially arrange thermostable enzymes for high- temperature biocatalysis[7][8] and as biotemplates for fabrication and patterning of inorganic...upon which to position hyperthermostable enzymes that have optimal activities at or above 100°C  [8], which are temperatures beyond the stability of

Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

PubMed

Collins, Kathleen; Nilsen, Timothy W

2013-08-01

Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in Escherichia coli for enhanced production of 1-butanol.

PubMed

Han, Gui Hwan; Seong, Wonjae; Fu, Yaoyao; Yoon, Paul K; Kim, Seong Keun; Yeom, Soo-Jin; Lee, Dae-Hee; Lee, Seung-Goo

2017-03-01

Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions onto inclusion body (IB) in Escherichia coli. The utility of this system was examined by co-localizing four heterologous enzymes of the 1-butanol pathway onto an IB that was formed in E. coli through overexpression of the cellulose binding domain (CBD) of Cellulomonas fimi exoglucanase. To target the 1-butanol pathway enzymes to the CBD IB, we utilized a peptide-peptide interaction between leucine zipper (LZ) peptides. We genetically fused the LZ peptide to the N-termini of four heterologous genes involved in the synthetic 1-butanol pathway, whereas an antiparallel LZ peptide was fused to the CBD gene. The in vivo activity of the CBD IB-based metabolon was examined through the determination of 1-butanol synthesis using E. coli transformed with two plasmids containing the LZ-fused CBD and LZ-fused 1-butanol pathway genes, respectively. In vivo synthesis of 1-butanol using the engineered E. coli yielded 1.98g/L of 1-butanol from glucose, representing a 1.5-fold increase over that obtained from E. coli expressing the LZ-fused 1-butanol pathway genes alone. In an attempt to examine the in vitro 1-butanol productivity, we reconstituted CBD IB-based metabolon using CBD IB and individual enzymes of 1-butanol pathway. The 1-butanol productivity of in vitro reconstituted CBD IB-based metabolon using acetoacetyl-CoA as the starting material was 2.29mg/L/h, 7.9-fold higher than that obtained from metabolon-free enzymes of 1-butanol pathway. Therefore, this novel CBD-based artificial metabolon may prove useful in metabolic engineering both in vivo and in vitro for the efficient production of desired products. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies.

PubMed

Zeitoun, Ramsey I; Pines, Gur; Grau, Willliam C; Gill, Ryan T

2017-04-21

Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites. This approach works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This approach, called barcoded-TRACE (bTRACE) was applied to assess E. coli populations engineered by recursive multiplex recombineering across both 6-target sites and 31-target sites. The 31-target library was then tracked throughout growth selections in the presence and absence of isopentenol (a potential next-generation biofuel). We also use the resolution of bTRACE to compare the influence of technical and biological noise on genome engineering efforts.

Organizational Influences on Interdisciplinary Interactions during Research and Design of Large-Scale Complex Engineered Systems

NASA Technical Reports Server (NTRS)

McGowan, Anna-Maria R.; Seifert, Colleen M.; Papalambros, Panos Y.

2012-01-01

The design of large-scale complex engineered systems (LaCES) such as an aircraft is inherently interdisciplinary. Multiple engineering disciplines, drawing from a team of hundreds to thousands of engineers and scientists, are woven together throughout the research, development, and systems engineering processes to realize one system. Though research and development (R&D) is typically focused in single disciplines, the interdependencies involved in LaCES require interdisciplinary R&D efforts. This study investigates the interdisciplinary interactions that take place during the R&D and early conceptual design phases in the design of LaCES. Our theoretical framework is informed by both engineering practices and social science research on complex organizations. This paper provides preliminary perspective on some of the organizational influences on interdisciplinary interactions based on organization theory (specifically sensemaking), data from a survey of LaCES experts, and the authors experience in the research and design. The analysis reveals couplings between the engineered system and the organization that creates it. Survey respondents noted the importance of interdisciplinary interactions and their significant benefit to the engineered system, such as innovation and problem mitigation. Substantial obstacles to interdisciplinarity are uncovered beyond engineering that include communication and organizational challenges. Addressing these challenges may ultimately foster greater efficiencies in the design and development of LaCES and improved system performance by assisting with the collective integration of interdependent knowledge bases early in the R&D effort. This research suggests that organizational and human dynamics heavily influence and even constrain the engineering effort for large-scale complex systems.

A Magnetically Separable, Highly Stable Enzyme System Based on Nanocomposites of Enzymes and Magnetic Nanoparticles Shipped in Hierarchically Ordered, Mesocellular, Mesoporous Silica

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

Kim, Jungbae; Lee, Jinwoo; Na, Hyon Bin

2005-12-01

Enzymes are versatile nanoscale biocatalysts, and find increasing applications in many areas, including organic synthesis[1-3] and bioremediation.[4-5] However, the application of enzymes is often hampered by the short catalytic lifetime of enzymes and by the difficulty in recovery and recycling. To solve these problems, there have been a lot of efforts to develop effective enzyme immobilization techniques. Recent advances in nanotechnology provide more diverse materials and approaches for enzyme immobilization. For example, mesoporous materials offer potential advantages as a host of enzymes due to their well-controlled porosity and large surface area for the immobilization of enzymes.[6,7] On the other hand,more » it has been demonstrated that enzymes attached on magnetic iron oxide nanoparticles can be easily recovered using a magnet and recycled for iterative uses.[8] In this paper, we report the development of magnetically-separable and highly-stable enzyme system by the combined use of two different kinds of nanostructured materials: magnetic nanoparticles and mesoporous silica.« less

Biomedical Engineering Bionanosystems Research at Louisiana Tech University

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

Palmer, James; Lvov, Yuri; Hegab, Hisham

2010-03-25

The nature of this project is to equip and support research in nanoengineered systems for biomedical, bioenvironmental, and bioenergy applications. Funds provided by the Department of Energy (DoE) under this Congressional Directive were used to support two ongoing research projects at Louisiana Tech University in biomedical, bioenvironmental, and bioenergy applications. Two major projects (Enzyme Immobilization for Large Scale Reactors to Reduce Cellulosic Ethanol Costs, and Nanocatalysts for Coal and Biomass Conversion to Diesel Fuel) and to fund three to five additional seed projects were funded using the project budget. The project funds also allowed the purchase and repair of sophisticatedmore » research equipment that will support continued research in these areas for many years to come. Project funds also supported faculty, graduate students, and undergraduate students, contributing to the development of a technically sophisticated work force in the region and the State. Descriptions of the technical accomplishments for each funded project are provided. Biofuels are an important part of the solution for sustainable transportation fuel and energy production for the future. Unfortunately, the country's appetite for fuel cannot be satisfied with traditional sugar crops such as sugar cane or corn. Emerging technologies are allowing cellulosic biomass (wood, grass, stalks, etc.) to also be converted into ethanol. Cellulosic ethanol does not compete with food production and it has the potential to decrease greenhouse gas (GHG) emissions by 86% versus current fossil fuels (current techniques for corn ethanol only reduce greenhouse gases by 19%). Because of these advantages, the federal government has made cellulosic ethanol a high priority. The Energy Independence and Security Act of 2007 (EISA) requires a minimum production of at least 16 billion gallons of cellulosic ethanol by 2022. Indeed, the Obama administration has signaled an ambitious commitment of achieving 2 billion gallons of cellulosic ethanol by 2013. Louisiana is well positioned to become a national contributor in cellulosic ethanol, with an excellent growing season, a strong pulp/paper industry, and one of the nation's first cellulosic ethanol demonstration plants. Dr. Palmer in Chemical Engineering at Louisiana Tech University is collaborating with Drs. Lvov and Snow in Chemistry and Dr. Hegab in Mechanical Engineering to capitalize on these advantages by applying nanotechnology to improve the cellulosic ethanol processes. In many of these processes, expensive enzymes are used to convert the cellulose to sugars. The nanotechnology processes developed at Louisiana Tech University can immobilize these enzymes and therefore significantly reduce the overall costs of the process. Estimates of savings range from approximately $32 million at each cellulosic ethanol plant, to $7.5 billion total if the 16 billion gallons of cellulosic ethanol is achieved. This process has the advantage of being easy to apply in a large-scale commercial environment and can immobilize a wide variety or mixture of enzymes for production. Two primary objectives with any immobilization technique are to demonstrate reusability and catalytic activity (both reuse of the immobilized enzyme and reuse of the polymer substrate). The scale-up of the layering-by-layering process has been a focus this past year as some interesting challenges in the surface chemistry have become evident. Catalytic activity of cellulase is highly dependent upon how the feed material is pretreated to enhance digestion. Therefore, efforts this year have been performed this year to characterize our process on a few of the more prevalent pretreatment methods.« less

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

PubMed

Vickery, Christopher R; Cardenas, Javier; Bowman, Marianne E; Burkart, Michael D; Da Silva, Nancy A; Noel, Joseph P

2018-06-01

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts. © 2018 Wiley Periodicals, Inc.

Converting Enzymes into Tools of Industrial Importance.

PubMed

Prasad, Shivcharan; Roy, Ipsita

2018-01-01

Enzymes have applications in numerous biotechnological products and processes that are commonly used in the production of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes, however, are optimized to function under physiological conditions. Any change in reaction conditions results in their activity as well as stability being compromised. Hence, most of the natural biomolecules are not suitable for industrial applications. Modifications are required to develop efficient and successful reagents as per demand. Protein engineering can be applied to cope up with these situations. This review describes some of the novel uses/unusual properties of enzymes as biological catalysts. It explains the different ways in which enzymes can be and have been used under non-native conditions. Different strategies have been discussed regarding stabilization of enzyme as well optimum conditions of its uses in different industries. The following patents databases were consulted: European Patent Office (EPO), the United States Patent and Trademark Office (USPTO), Patent scope Search International and National Patent Collections (WIPO) and Google Patents. The review illustrates the width of the umbrella of applications covered by biocatalysts. Employing the tools of solvent and protein engineering, viz. non-aqueous media, additives, immobilization, mutagenesis, to name a few; biotechnology has been able to make enzyme catalyzed processes an essential components of the industrialist's armoury. The article lists a number of successful examples, both of patented technology as well as biocatalysts which are currently being used in the industry, to highlight the accomplishments of technologies which have been adopted till now for making enzyme technology industrially viable. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves

USDA-ARS?s Scientific Manuscript database

The seeds of many non-domesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered ...

Large-Scale Digital Geologic Map Databases and Reports of the North Coal District in Afghanistan

USGS Publications Warehouse

Hare, Trent M.; Davis, Philip A.; Nigh, Devon; Skinner, James A.; SanFilipo, John R.; Bolm, Karen S.; Fortezzo, Corey M.; Galuszka, Donna; Stettner, William R.; Sultani, Shafiqullah; Nader, Billal

2008-01-01

This report describes the Afghanistan coal resource maps and associated databases that have been digitally captured and maps that have been thus far converted to GIS databases. Several maps by V/O Technoexport, USSR (VOTU) and Bundesanstalt fur Bodenforschung (BGR), Hannover, Germany, are captured here. Most of the historical coal exploration is concentrated in north-central Afghanistan, a region referred to as the 'North Coal District', and almost all of the coal-related maps found Afghanistan Geological Survey (AGS) archives to date cover various locations within that district as shown in the index map. Most of the maps included herein were originally scanned during U.S. Geological Survey (USGS) site visits to Kabul in November 2004 and February 2006. The scanning was performed using equipment purchased by U.S. Agency for International Development (USAID) and U.S. Trade and Development Agency (USTDA) and installed at the AGS by USGS. Many of these maps and associated reports exist as single unpublished copies in the AGS archives, so these efforts served not only to provide a basis for digital capturing, but also as a means for preserving these rare geologic maps and reports. The data included herein represent most of the coal-related reports and maps that are available in the AGS archives. This report excludes the limited cases when a significant portion of a report's text could not be located, but it does not exclude reports with missing plates. The vector files are released using the Environmental Systems Research Institute (ESRI) Personal Geodatabase, ESRI shapefile vector format, and the open Geography Markup Language (GML) format. Scanned images are available in JPEG and, when rectified, GeoTIFF format. The authors wish to acknowledge the contributions made by the staff of the AGS Records and Coal Departments whose valuable assistance made it possible to locate and catalogue the data provided herein. We especially acknowledge the efforts of particular members of the coal team: Engineer Saifuddin Aminy (Team Leader); Engineer Gul Pacha Azizi; Engineer Abdul Haq Barakati; Engineer Abdul Basir; Engineer Mohammad Daoud; Engineer Abdullah Ebadi; Engineer Abdul Ahad Omaid; Engineer Spozmy; and Engineer Shapary Tokhi. The ongoing efforts of Engineer Mir M. Atiq Kazimi (Team leader); Engineer M. Anwar Housinzada; and Engineer Shereen Agha of the AGS Records Department to organize and catalogue the AGS material were invaluable in locating and preserving these data. The efforts of the entire AGS staff to personally preserve these data during war time, in the absence of virtually any supporting infrastructure, was truly remarkable. The efforts by the British Geological Survey (BGS) to assist the AGS in archiving these data, and the personal assistance provided by BGS (notably Robert McIntosh), to the USGS teams were also appreciated. The logistical support provided by the U.S. Embassy in Kabul, particularly the Afghanistan Reconstruction Group, was critical to the success of the USGS teams while in Afghanistan. Finally, the efforts of the Minister of the Ministry of Mines and Industries (M. Ibrahim Adel) to support the USGS coal resource assessment in Afghanistan, in both his current and former role as President of the Mines Affairs Department was vital to this effort.

Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase

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

Carlin, DA; Bertolani, SJ; Siegel, JB

Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

ERIC Educational Resources Information Center

Ott, Laura E.; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase.

PubMed

Carlin, D A; Bertolani, S J; Siegel, J B

2015-02-11

Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

Inductive knowledge acquisition experience with commercial tools for space shuttle main engine testing

NASA Technical Reports Server (NTRS)

Modesitt, Kenneth L.

1990-01-01

Since 1984, an effort has been underway at Rocketdyne, manufacturer of the Space Shuttle Main Engine (SSME), to automate much of the analysis procedure conducted after engine test firings. Previously published articles at national and international conferences have contained the context of and justification for this effort. Here, progress is reported in building the full system, including the extensions of integrating large databases with the system, known as Scotty. Inductive knowledge acquisition has proven itself to be a key factor in the success of Scotty. The combination of a powerful inductive expert system building tool (ExTran), a relational data base management system (Reliance), and software engineering principles and Computer-Assisted Software Engineering (CASE) tools makes for a practical, useful and state-of-the-art application of an expert system.

A Theory of Information Quality and a Framework for its Implementation in the Requirements Engineering Process

NASA Astrophysics Data System (ADS)

Grenn, Michael W.

This dissertation introduces a theory of information quality to explain macroscopic behavior observed in the systems engineering process. The theory extends principles of Shannon's mathematical theory of communication [1948] and statistical mechanics to information development processes concerned with the flow, transformation, and meaning of information. The meaning of requirements information in the systems engineering context is estimated or measured in terms of the cumulative requirements quality Q which corresponds to the distribution of the requirements among the available quality levels. The requirements entropy framework (REF) implements the theory to address the requirements engineering problem. The REF defines the relationship between requirements changes, requirements volatility, requirements quality, requirements entropy and uncertainty, and engineering effort. The REF is evaluated via simulation experiments to assess its practical utility as a new method for measuring, monitoring and predicting requirements trends and engineering effort at any given time in the process. The REF treats the requirements engineering process as an open system in which the requirements are discrete information entities that transition from initial states of high entropy, disorder and uncertainty toward the desired state of minimum entropy as engineering effort is input and requirements increase in quality. The distribution of the total number of requirements R among the N discrete quality levels is determined by the number of defined quality attributes accumulated by R at any given time. Quantum statistics are used to estimate the number of possibilities P for arranging R among the available quality levels. The requirements entropy H R is estimated using R, N and P by extending principles of information theory and statistical mechanics to the requirements engineering process. The information I increases as HR and uncertainty decrease, and the change in information AI needed to reach the desired state of quality is estimated from the perspective of the receiver. The HR may increase, decrease or remain steady depending on the degree to which additions, deletions and revisions impact the distribution of R among the quality levels. Current requirements trend metrics generally treat additions, deletions and revisions the same and simply measure the quantity of these changes over time. The REF evaluates the quantity of requirements changes over time, distinguishes between their positive and negative effects by calculating their impact on HR, Q, and AI, and forecasts when the desired state will be reached, enabling more accurate assessment of the status and progress of the requirements engineering effort. Results from random variable simulations suggest the REF is an improved leading indicator of requirements trends that can be readily combined with current methods. The increase in I, or decrease in H R and uncertainty, is proportional to the engineering effort E input into the requirements engineering process. The REF estimates the AE needed to transition R from their current state of quality to the desired end state or some other interim state of interest. Simulation results are compared with measured engineering effort data for Department of Defense programs published in the SE literature, and the results suggest the REF is a promising new method for estimation of AE.

Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway.

PubMed

Mak, Wai Shun; Tran, Stephen; Marcheschi, Ryan; Bertolani, Steve; Thompson, James; Baker, David; Liao, James C; Siegel, Justin B

2015-11-24

The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. Here we utilize two approaches to discover enzymes that enable specific production of longer-chain (C5-C8) alcohols from sugar. The first approach combines bioinformatics and molecular modelling to mine sequence databases, resulting in a diverse panel of enzymes capable of catalysing the targeted reaction. The median catalytic efficiency of the computationally selected enzymes is 75-fold greater than a panel of naively selected homologues. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. The second approach uses computational enzyme design to reprogramme specificity. Both approaches result in enzymes with >100-fold increase in specificity for the targeted reaction. When enzymes from either approach are integrated in vivo, longer-chain alcohol production increases over 10-fold and represents >95% of the total alcohol products.

Dissecting enzyme function with microfluidic-based deep mutational scanning.

PubMed

Romero, Philip A; Tran, Tuan M; Abate, Adam R

2015-06-09

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.

Technological advances and applications of hydrolytic enzymes for valorization of lignocellulosic biomass.

PubMed

Manisha; Yadav, Sudesh Kumar

2017-12-01

Hydrolytic enzymes are indispensable tools in the production of various foodstuffs, drugs, and consumables owing to their applications in almost every industrial process nowadays. One of the foremost areas of interest involving the use of hydrolytic enzymes is in the transformation of lignocellulosic biomass into value added products. However, limitations of the processes due to inadequate enzyme activity and stability with a narrow range of pH and temperature optima often limit their effective usage. The innovative technologies, involving manipulation of enzyme activity and stability through mutagenesis, genetic engineering and metagenomics lead to a major leap in all the fields using hydrolytic enzymes. This article provides recent advancement towards the isolation and use of microbes for lignocellulosic biomass utilisation, microbes producing the hydrolytic enzymes, the modern age technologies used to manipulate and enhance the hydrolytic enzyme activity and the applications of such enzymes in value added products development from lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

48 CFR 31.205-25 - Manufacturing and production engineering costs.

Code of Federal Regulations, 2013 CFR

2013-10-01

... production engineering costs. 31.205-25 Section 31.205-25 Federal Acquisition Regulations System FEDERAL... Commercial Organizations 31.205-25 Manufacturing and production engineering costs. (a) The costs of manufacturing and production engineering effort as described in (1) through (4) below are all allowable: (1...

48 CFR 31.205-25 - Manufacturing and production engineering costs.

Code of Federal Regulations, 2014 CFR

2014-10-01

... production engineering costs. 31.205-25 Section 31.205-25 Federal Acquisition Regulations System FEDERAL... Commercial Organizations 31.205-25 Manufacturing and production engineering costs. (a) The costs of manufacturing and production engineering effort as described in (1) through (4) below are all allowable: (1...

48 CFR 31.205-25 - Manufacturing and production engineering costs.

Code of Federal Regulations, 2012 CFR

2012-10-01

... production engineering costs. 31.205-25 Section 31.205-25 Federal Acquisition Regulations System FEDERAL... Commercial Organizations 31.205-25 Manufacturing and production engineering costs. (a) The costs of manufacturing and production engineering effort as described in (1) through (4) below are all allowable: (1...

48 CFR 31.205-25 - Manufacturing and production engineering costs.

Code of Federal Regulations, 2011 CFR

2011-10-01

... production engineering costs. 31.205-25 Section 31.205-25 Federal Acquisition Regulations System FEDERAL... Commercial Organizations 31.205-25 Manufacturing and production engineering costs. (a) The costs of manufacturing and production engineering effort as described in (1) through (4) below are all allowable: (1...

48 CFR 31.205-25 - Manufacturing and production engineering costs.

Code of Federal Regulations, 2010 CFR

2010-10-01

... production engineering costs. 31.205-25 Section 31.205-25 Federal Acquisition Regulations System FEDERAL... Commercial Organizations 31.205-25 Manufacturing and production engineering costs. (a) The costs of manufacturing and production engineering effort as described in (1) through (4) below are all allowable: (1...

Cost evaluation of cellulase enzyme for industrial-scale cellulosic ethanol production based on rigorous Aspen Plus modeling.

PubMed

Liu, Gang; Zhang, Jian; Bao, Jie

2016-01-01

Cost reduction on cellulase enzyme usage has been the central effort in the commercialization of fuel ethanol production from lignocellulose biomass. Therefore, establishing an accurate evaluation method on cellulase enzyme cost is crucially important to support the health development of the future biorefinery industry. Currently, the cellulase cost evaluation methods were complicated and various controversial or even conflict results were presented. To give a reliable evaluation on this important topic, a rigorous analysis based on the Aspen Plus flowsheet simulation in the commercial scale ethanol plant was proposed in this study. The minimum ethanol selling price (MESP) was used as the indicator to show the impacts of varying enzyme supply modes, enzyme prices, process parameters, as well as enzyme loading on the enzyme cost. The results reveal that the enzyme cost drives the cellulosic ethanol price below the minimum profit point when the enzyme is purchased from the current industrial enzyme market. An innovative production of cellulase enzyme such as on-site enzyme production should be explored and tested in the industrial scale to yield an economically sound enzyme supply for the future cellulosic ethanol production.

NASA's PEM Fuel Cell Power Plant Development Program for Space Applications

NASA Technical Reports Server (NTRS)

Hoberecht, Mark

2006-01-01

NASA embarked on a PEM fuel cell power plant development program beginning in 2001. This five-year program was conducted by a three-center NASA team of Glenn Research Center (lead), Johnson Space Center, and Kennedy Space Center. The program initially was aimed at developing hardware for a Reusable Launch Vehicle (RLV) application, but more recently had shifted to applications supporting the NASA Exploration Program. The first phase of the development effort, to develop breadboard hardware in the 1-5 kW power range, was conducted by two competing vendors. The second phase of the effort, to develop Engineering Model hardware at the 10 kW power level, was conducted by the winning vendor from the first phase of the effort. Both breadboard units and the single engineering model power plant were delivered to NASA for independent testing. This poster presentation will present a summary of both phases of the development effort, along with a discussion of test results of the PEM fuel cell engineering model under simulated mission conditions.

Compatibility of alternative fuels with advanced automotive gas turbine and stirling engines. A literature survey

NASA Technical Reports Server (NTRS)

Cairelli, J.; Horvath, D.

1981-01-01

The application of alternative fuels in advanced automotive gas turbine and Stirling engines is discussed on the basis of a literature survey. These alternative engines are briefly described, and the aspects that will influence fuel selection are identified. Fuel properties and combustion properties are discussed, with consideration given to advanced materials and components. Alternative fuels from petroleum, coal, oil shale, alcohol, and hydrogen are discussed, and some background is given about the origin and production of these fuels. Fuel requirements for automotive gas turbine and Stirling engines are developed, and the need for certain reseach efforts is discussed. Future research efforts planned at Lewis are described.

STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C.

PubMed

Gaßmeyer, Sarah Katharina; Yoshikawa, Hiroyuki; Enoki, Junichi; Hülsemann, Nadine; Stoll, Raphael; Miyamoto, Kenji; Kourist, Robert

2015-06-23

Structure-guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation-transfer-difference NMR (STD-NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functionalized nanocompartments (Synthosomes): limitations and prospective applications in industrial biotechnology.

PubMed

Onaca, Ozana; Nallani, Madhavan; Ihle, Saskia; Schenk, Alexander; Schwaneberg, Ulrich

2006-01-01

Synthosomes are mechanically stable vesicles with a block copolymer membrane and an engineered transmembrane protein acting as selective gate. The polymer vesicles are nanometer-sized (50-1000 nm) and functionalized by loading them with enzymes for bioconversions or encapsulating charged macromolecules for selective compound recovery/release. The Synthosome system might become a novel technology platform for biocatalysis and selective product recovery. Progress in Synthosome research comprises employed block copolymers, transmembrane channel engineering, and functionalizations, which are discussed here in detail. The challenges in transmembrane protein engineering, as well as cost-effective production, in block copolymer design and the state of the art in Synthosome characterization comprising quantification of encapsulated protein, translocation efficiency, number of transmembrane channels per vesicle, and enzyme kinetics are also presented and discussed. An assessment of the Synthosome technology platform for prospective applications in industrial (white) biotechnology concludes this review.

Overview of free-piston Stirling technology at the NASA Lewis Research Center

NASA Technical Reports Server (NTRS)

Slaby, J. G.

1985-01-01

An overview of the National Aeronautics and Space Administration (NASA) Lewis Research Center (Lewis) free-piston Stirling engine activities is presented. These activities include: (1) a generic free-piston Stirling technology project being conducted to develop technologies synergistic to both space power and terrestrial heat pump applications in a cooperative, cost-shared effort with the Department of Energy (DOE/Oak Ridge National Laboratory (ONRL)), and (2) a free-piston Stirling space-power technology demonstration project as part of the SP-100 program being conducted in support of the Department of Defense (DOD), DOE, and NASA/Lewis. The generic technology effort includes extensive parametric testing of a 1 kw free-piston Stirling engine (RE-1000), development and validation of a free-piston Stirling performance computer code, and fabrication and initial testing of an hydraulic output modification for the RE-1000 engine. The space power technology effort, under SP-100, addresses the status of the 25 kWe Space Power Demonstrator Engine (SPDE) including early test results.

Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

PubMed

Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, Veeranki V

2017-01-01

Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

From Planetary Imaging to Enzyme Screening

NASA Technical Reports Server (NTRS)

2006-01-01

Based in San Diego, KAIROS Scientific develops molecular biology methods, instrumentation, and computer algorithms to create solutions to challenging problems in the medical and chemical industries. company s pioneering efforts in digital imaging spectroscopy (DIS) enable researchers to obtain spectral and/or time-dependent information for each pixel or group of pixels in a two-dimensional scene. In addition to having Yang s NASA experience at its foundation, KAIROS Scientific was established with the support of many government grants and contracts. Its first was a NASA Small Business Innovation Research (SBIR) grant, from Ames Research Center, to develop HIRIM, a high-resolution imaging microscope embodying both novel hardware and software that can be used to simultaneously acquire hundreds of individual absorbance spectra from microscopic features. Using HIRIM s graphical user interface, MicroDIS, scientists and engineers are presented with a revolutionary new tool which enables them to point to a feature in an image and recall its associated spectrum in real time.

From chaperonins to Rubisco assembly and metabolic repair.

PubMed

Hayer-Hartl, Manajit

2017-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO 2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Despite its pivotal role, Rubisco is an inefficient enzyme and thus has been a key target for bioengineering. However, efforts to increase crop yields by Rubisco engineering remain unsuccessful, due in part to the complex machinery of molecular chaperones required for Rubisco biogenesis and metabolic repair. While the large subunit of Rubisco generally requires the chaperonin system for folding, the evolution of the hexadecameric Rubisco from its dimeric precursor resulted in the dependence on an array of additional factors required for assembly. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands. Metabolic repair of Rubisco depends on remodeling by the ATP-dependent Rubisco activase and hydrolysis of inhibitors by specific phosphatases. This review highlights our work toward understanding the structure and mechanism of these auxiliary machineries. © 2017 The Protein Society.

Nano-Engineered Biomimetic Optical Sensors for Glucose Monitoring in Diabetes.

PubMed

Rauf, Sajid; Hayat Nawaz, Muhammad Azhar; Badea, Mihaela; Marty, Jean Louis; Hayat, Akhtar

2016-11-17

Diabetes is a rapidly growing disease that can be monitored at an individual level by controlling the blood glucose level, hence minimizing the negative impact of the disease. Significant research efforts have been focused on the design of novel and improved technologies to overcome the limitations of existing glucose analysis methods. In this context, nanotechnology has enabled the diagnosis at the single cell and molecular level with the possibility of incorporation in advanced molecular diagnostic biochips. Recent years have witnessed the exploration and synthesis of various types of nanomaterials with enzyme-like properties, with their subsequent integration into the design of biomimetic optical sensors for glucose monitoring. This review paper will provide insights on the type, nature and synthesis of different biomimetic nanomaterials. Moreover, recent developments in the integration of these nanomaterials for optical glucose biosensing will be highlighted, with a final discussion on the challenges that must be addressed for successful implementation of these nano-devices in the clinical applications is presented.

Nano-Engineered Biomimetic Optical Sensors for Glucose Monitoring in Diabetes

PubMed Central

Rauf, Sajid; Hayat Nawaz, Muhammad Azhar; Badea, Mihaela; Marty, Jean Louis; Hayat, Akhtar

2016-01-01

Diabetes is a rapidly growing disease that can be monitored at an individual level by controlling the blood glucose level, hence minimizing the negative impact of the disease. Significant research efforts have been focused on the design of novel and improved technologies to overcome the limitations of existing glucose analysis methods. In this context, nanotechnology has enabled the diagnosis at the single cell and molecular level with the possibility of incorporation in advanced molecular diagnostic biochips. Recent years have witnessed the exploration and synthesis of various types of nanomaterials with enzyme-like properties, with their subsequent integration into the design of biomimetic optical sensors for glucose monitoring. This review paper will provide insights on the type, nature and synthesis of different biomimetic nanomaterials. Moreover, recent developments in the integration of these nanomaterials for optical glucose biosensing will be highlighted, with a final discussion on the challenges that must be addressed for successful implementation of these nano-devices in the clinical applications is presented. PMID:27869658

RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

PubMed

Gasiunas, Giedrius; Siksnys, Virginijus

2013-11-01

Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology.

PubMed

Lenz, Robert W; Marchessault, Robert H

2005-01-01

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years.

Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production

PubMed Central

Jin, Lu; Bhuiya, Mohammad Wadud; Li, Mengmeng; Liu, XiangQi; Han, Jixiang; Deng, WeiWei; Wang, Min; Yu, Oliver; Zhang, Zhengzhu

2014-01-01

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g. tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed. PMID:25133732

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects.

PubMed

Wang, Yan; Song, Qinghao; Zhang, Xiao-Hua

2016-09-22

Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes.

Industrial Fungal Enzymes: An Occupational Allergen Perspective

PubMed Central

Green, Brett J.; Beezhold, Donald H.

2011-01-01

Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes. PMID:21747869

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation.

PubMed

Sinha, Rajeshwari; Khare, Sunil K

2014-01-01

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants.

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

PubMed Central

Sinha, Rajeshwari; Khare, Sunil K.

2014-01-01

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants. PMID:24782853

African American Adolescent Female Identification with Engineering and Participation in Engineering Education

ERIC Educational Resources Information Center

Cornick, Shayla L.

2012-01-01

Experiences that females have during middle and high school have been found to influence the perceptions that they have of their ability to be successful as an engineer and the value that they place on participating in engineering education. Engineering education continues to suffer from a lack of female participation. Several efforts have been…

Designing for Success: Developing Engineers Who Consider Universal Design Principles

ERIC Educational Resources Information Center

Bigelow, Kimberly Edginton

2012-01-01

Engineers must design for a diverse group of potential users of their products; however, engineering curricula rarely include an emphasis on universal design principles. This research article details the effectiveness of a design project implemented in a first-year engineering course in an effort to raise awareness of the need for engineers to be…

Thermostable Carbonic Anhydrases in Biotechnological Applications

PubMed Central

Di Fiore, Anna; Alterio, Vincenzo; Monti, Simona M.; De Simone, Giuseppina; D’Ambrosio, Katia

2015-01-01

Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application. PMID:26184158

Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

PubMed

Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

2014-02-01

To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Positive dermal hypersensitivity and specific antibodies in workers exposed to bio-engineered enzymes

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

Biagini, R.E.; Henningsen, G.M.; Driscoll, R.

1991-03-15

Thirty-six employees who produced industrial enzymes from bio-engineered strains of bacteria and fungi were evaluated by skin prick testing and enzyme linked immunosorbent assays for specific IgE and IgG antibodies. The workers complained of asthma- and flu-like' symptoms which generally lessened away from work. The enzymes evaluated were {alpha}-amylase from A. niger (ind-AAN), B. licheniformis (ind-AAL) and B. subtilis (ind-AAS); purified {alpha}-amylase from B. subtilis (AAS) and A. niger (AAN); alkaline protease from B. licheniformis (ind-APL) and purified alkaline protease (APL); amylase glucosidase from A. niger (ind-AGN) and purified amylase glucosidase (AGN). Significantly positive skin tests were found for APL,more » AGN and ind-AAN. Significantly elevated specific IgE results were observed for AAN, AGN, and ind-AAN; elevated specific IgGs were observed for AAN, ind-AAN, ind-AAS, ind-AAL and ind-AGN. Radioimmunoassays of air filter samples (using sera with high Ab titers) for 4 of the ind-enzymes showed only ind-AAN at extremely high environmental levels. These results indicate that occupational exposure to some ind-enzymes causes immediate onset dermal hypersensitivity reactions. The results are equivocal as to whether these reactions are IgE mediated, as IgE titers were low. Contrary to this, IgG titers were extremely high and suggest that these biomarkers can be used as indicators of both individual exposure and environmental analyses.« less

The structure of Plasmodium falciparum serine hydroxymethyltransferase reveals a novel redox switch that regulates its activities

PubMed Central

Chitnumsub, Penchit; Ittarat, Wanwipa; Jaruwat, Aritsara; Noytanom, Krittikar; Amornwatcharapong, Watcharee; Pornthanakasem, Wichai; Chaiyen, Pimchai; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

2014-01-01

Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Å resolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similar to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme. PMID:24914963

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.

Energetic Combustion Devices for Aerospace Propulsion and Power

NASA Technical Reports Server (NTRS)

Litchford, Ron J.

2000-01-01

Chemical reactions have long been the mainstay thermal energy source for aerospace propulsion and power. Although it is widely recognized that the intrinsic energy density limitations of chemical bonds place severe constraints on maximum realizable performance, it will likely be several years before systems based on high energy density nuclear fuels can be placed into routine service. In the mean time, efforts to develop high energy density chemicals and advanced combustion devices which can utilize such energetic fuels may yield worthwhile returns in overall system performance and cost. Current efforts in this vein are being carried out at NASA MSFC under the direction of the author in the areas of pulse detonation engine technology development and light metals combustion devices. Pulse detonation engines are touted as a low cost alternative to gas turbine engines and to conventional rocket engines, but actual performance and cost benefits have yet to be convincingly demonstrated. Light metal fueled engines also offer potential benefits in certain niche applications such as aluminum/CO2 fueled engines for endo-atmospheric Martian propulsion. Light metal fueled MHD generators also present promising opportunities with respect to electric power generation for electromagnetic launch assist. This presentation will discuss the applications potential of these concepts with respect to aero ace propulsion and power and will review the current status of the development efforts.

Space Transportation Engine Program (STEP), phase B

NASA Technical Reports Server (NTRS)

1990-01-01

The Space Transportation Engine Program (STEP) Phase 2 effort includes preliminary design and activities plan preparation that will allow smooth and time transition into a Prototype Phase and then into Phases 3, 4, and 5. A Concurrent Engineering approach using Total Quality Management (TQM) techniques, is being applied to define an oxygen-hydrogen engine. The baseline from Phase 1/1' studies was used as a point of departure for trade studies and analyses. Existing STME system models are being enhanced as more detailed module/component characteristics are determined. Preliminary designs for the open expander, closed expander, and gas generator cycles were prepared, and recommendations for cycle selection made at the Design Concept Review (DCR). As a result of July '90 DCR, and information subsequently supplied to the Technical Review Team, a gas generator cycle was selected. Results of the various Advanced Development Programs (ADP's) for the Advanced Launch Systems (ALS) were contributive to this effort. An active vehicle integration effort is supplying the NASA, Air Force, and vehicle contractors with engine parameters and data, and flowing down appropriate vehicle requirements. Engine design and analysis trade studies are being documented in a data base that was developed and is being used to organize information. To date, seventy four trade studies were input to the data base.

Development and Testing of Assessment Instruments for Multidisciplinary Engineering Capstone Design Courses

ERIC Educational Resources Information Center

Gerlick, Robert Edward

2010-01-01

The research presented in this manuscript was focused on the development of assessments for engineering design outcomes. The primary goal was to support efforts by the Transferrable Integrated Design Engineering Education (TIDEE) consortium in developing assessment instruments for multidisciplinary engineering capstone courses. Research conducted…

Delivering Core Engineering Concepts to Secondary Level Students

ERIC Educational Resources Information Center

Merrill, Chris; Custer, Rodney L.; Daugherty, Jenny; Westrick, Martin; Zeng, Yong

2008-01-01

Through the efforts of National Center for Engineering and Technology Education (NCETE), three core engineering concepts within the realm of engineering design have emerged as crucial areas of need within secondary level technology education. These concepts are constraints, optimization, and predictive analysis (COPA). COPA appears to be at the…

The Application of Tissue Engineering Procedures to Repair the Larynx

ERIC Educational Resources Information Center

Ringel, Robert L.; Kahane, Joel C.; Hillsamer, Peter J.; Lee, Annie S.; Badylak, Stephen F.

2006-01-01

The field of tissue engineering/regenerative medicine combines the quantitative principles of engineering with the principles of the life sciences toward the goal of reconstituting structurally and functionally normal tissues and organs. There has been relatively little application of tissue engineering efforts toward the organs of speech, voice,…

Future of Chemical Engineering: Integrating Biology into the Undergraduate ChE Curriculum

ERIC Educational Resources Information Center

Mosto, Patricia; Savelski, Mariano; Farrell, Stephanie H.; Hecht, Gregory B.

2007-01-01

Integrating biology in the chemical engineering curriculum seems to be the future for chemical engineering programs nation and worldwide. Rowan University's efforts to address this need include a unique chemical engineering curriculum with an intensive biology component integrated throughout from freshman to senior years. Freshman and Sophomore…

Building Inclusive Engineering Identities: Implications for Changing Engineering Culture

ERIC Educational Resources Information Center

Atadero, Rebecca A.; Paguyo, Christina H.; Rambo-Hernandez, Karen E.; Henderson, Heather L.

2018-01-01

Ongoing efforts to broaden the participation of women and people of colour in engineering degree programmes and careers have had limited success. This paper describes a different approach to broadening participation that seeks to work with all students and develop inclusive engineering identities. Researchers worked with the instructors of two…

A design-build-test cycle using modeling and experiments reveals interdependencies between upper glycolysis and xylose uptake in recombinant S. cerevisiae and improves predictive capabilities of large-scale kinetic models.

PubMed

Miskovic, Ljubisa; Alff-Tuomala, Susanne; Soh, Keng Cher; Barth, Dorothee; Salusjärvi, Laura; Pitkänen, Juha-Pekka; Ruohonen, Laura; Penttilä, Merja; Hatzimanikatis, Vassily

2017-01-01

Recent advancements in omics measurement technologies have led to an ever-increasing amount of available experimental data that necessitate systems-oriented methodologies for efficient and systematic integration of data into consistent large-scale kinetic models. These models can help us to uncover new insights into cellular physiology and also to assist in the rational design of bioreactor or fermentation processes. Optimization and Risk Analysis of Complex Living Entities (ORACLE) framework for the construction of large-scale kinetic models can be used as guidance for formulating alternative metabolic engineering strategies. We used ORACLE in a metabolic engineering problem: improvement of the xylose uptake rate during mixed glucose-xylose consumption in a recombinant Saccharomyces cerevisiae strain. Using the data from bioreactor fermentations, we characterized network flux and concentration profiles representing possible physiological states of the analyzed strain. We then identified enzymes that could lead to improved flux through xylose transporters (XTR). For some of the identified enzymes, including hexokinase (HXK), we could not deduce if their control over XTR was positive or negative. We thus performed a follow-up experiment, and we found out that HXK2 deletion improves xylose uptake rate. The data from the performed experiments were then used to prune the kinetic models, and the predictions of the pruned population of kinetic models were in agreement with the experimental data collected on the HXK2 -deficient S. cerevisiae strain. We present a design-build-test cycle composed of modeling efforts and experiments with a glucose-xylose co-utilizing recombinant S. cerevisiae and its HXK2 -deficient mutant that allowed us to uncover interdependencies between upper glycolysis and xylose uptake pathway. Through this cycle, we also obtained kinetic models with improved prediction capabilities. The present study demonstrates the potential of integrated "modeling and experiments" systems biology approaches that can be applied for diverse applications ranging from biotechnology to drug discovery.

Controlling cell-free metabolism through physiochemical perturbations.

PubMed

Karim, Ashty S; Heggestad, Jacob T; Crowe, Samantha A; Jewett, Michael C

2018-01-01

Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore physiochemical perturbations and suggests the need for a more controllable, multi-step, separated cell-free framework for future pathway prototyping and enzyme discovery efforts. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Overview of thermal barrier coatings in diesel engines

NASA Technical Reports Server (NTRS)

Yonushonis, Thomas M.

1995-01-01

An understanding of delamination mechanisms in thermal barrier coatings has been developed for diesel engine applications through rig tests, structural analysis modeling, nondestructive evaluation, and engine evaluation of various thermal barrier coatings. This knowledge has resulted in improved thermal barrier coatings which survive abusive cyclic fatigue tests in high output diesel engines. Although much conflicting literature now exists regarding the impact of thermal barrier coatings on engine performance and fuel consumption, the changes in fuel consumption appear to be less than a few percent and can be negative for state-of-the-art diesel engines. The ability of the thermal barrier coating to improve fuel economy tends to be dependent on a number of factors including the fuel injection system, combustion chamber design, and the initial engine fuel economy. Limited investigations on state-of-the-art diesel engines have indicated that the surface connected porosity and coating surface roughness may influence engine fuel economy. Current research efforts on thermal barrier coatings are primarily directed at reducing in-cylinder heat rejection, thermal fatigue protection of underlying metal surfaces and a possible reduction in diesel engine emissions. Significant efforts are still required to improve the plasma spray processing capability and the economics for complex geometry diesel engine components.

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.

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

Synthetic biology for manufacturing chemicals: constraints drive the use of non-conventional microbial platforms.

PubMed

Czajka, Jeffrey; Wang, Qinhong; Wang, Yechun; Tang, Yinjie J

2017-10-01

Genetically modified microbes have had much industrial success producing protein-based products (such as antibodies and enzymes). However, engineering microbial workhorses for biomanufacturing of commodity compounds remains challenging. First, microbes cannot afford burdens with both overexpression of multiple enzymes and metabolite drainage for product synthesis. Second, synthetic circuits and introduced heterologous pathways are not yet as "robust and reliable" as native pathways due to hosts' innate regulations, especially under suboptimal fermentation conditions. Third, engineered enzymes may lack channeling capabilities for cascade-like transport of metabolites to overcome diffusion barriers or to avoid intermediate toxicity in the cytoplasmic environment. Fourth, moving engineered hosts from laboratory to industry is unreliable because genetic mutations and non-genetic cell-to-cell variations impair the large-scale fermentation outcomes. Therefore, synthetic biology strains often have unsatisfactory industrial performance (titer/yield/productivity). To overcome these problems, many different species are being explored for their metabolic strengths that can be leveraged to synthesize specific compounds. Here, we provide examples of non-conventional and genetically amenable species for industrial manufacturing, including the following: Corynebacterium glutamicum for its TCA cycle-derived biosynthesis, Yarrowia lipolytica for its biosynthesis of fatty acids and carotenoids, cyanobacteria for photosynthetic production from its sugar phosphate pathways, and Rhodococcus for its ability to biotransform recalcitrant feedstock. Finally, we discuss emerging technologies (e.g., genome-to-phenome mapping, single cell methods, and knowledge engineering) that may facilitate the development of novel cell factories.

Enzymes, trophoblasts, and cancer: the afterlife of an idea (1924-2008).

PubMed

Moss, Ralph W

2008-12-01

In the early 20th century, advocacy of the enzyme therapy of cancer was primarily the work of one man, John Beard, DSc (1858-1924). He and his collaborators made a determined effort to establish this mode of therapy, especially in the years 1905 to 1911. Despite a brief flowering of international interest, Beard's efforts came to naught. During the 20th century, there was a succession of American researchers who continued to investigate this topic. This included Marshall William McDuffie, MD (1882-1945), Frank LeForest Morse, MD (1876-1953), Franklin Lloyd Shively, MD (1887-1971), and William Donald Kelley (1926-2005). In central Europe, India, and other parts of the globe, the use of pancreatic enzymes as an adjuvant treatment for cancer has become a fairly routine practice, at least among those doctors who utilize complementary and alternative medicine (CAM). It is also a well-established method for reducing inflammation and mitigating the adverse effects of cytotoxic treatment.

Transgenic plants that exhibit enhanced nitrogen assimilation

DOEpatents

Coruzzi, Gloria M.; Brears, Timothy

2005-03-08

The present invention relates to a method for producing plants with improved agronomic and nutritional traits. Such traits include enhanced nitrogen assimilatory and utilization capacities, faster and more vigorous growth, greater vegetative and reproductive yields, and enriched or altered nitrogen content in vegetative and reproductive parts. More particularly, the invention relates to the engineering of plants modified to have altered expression of key enzymes in the nitrogen assimilation and utilization pathways. In one embodiment of the present invention, the desired altered expression is accomplished by engineering the plant for ectopic overexpression of one of more the native or modified nitrogen assimilatory enzymes. The invention also has a number of other embodiments, all of which are disclosed herein.

Transgenic plants that exhibit enhanced nitrogen assimilation

DOEpatents

Coruzzi, Gloria M.; Brears, Timothy

1999-01-01

The present invention relates to a method for producing plants with improved agronomic and nutritional traits. Such traits include enhanced nitrogen assimilatory and utilization capacities, faster and more vigorous growth, greater vegetative and reproductive yields, and enriched or altered nitrogen content in vegetative and reproductive parts. More particularly, the invention relates to the engineering of plants modified to have altered expression of key enzymes in the nitrogen assimilation and utilization pathways. In one embodiment of the present invention, the desired altered expression is accomplished by engineering the plant for ectopic overexpression of one of more the native or modified nitrogen assimilatory enzymes. The invention also has a number of other embodiments, all of which are disclosed herein.

Transgenic plants that exhibit enhanced nitrogen assimilation

DOEpatents

Coruzzi, Gloria M.; Brears, Timothy

2000-01-01

The present invention relates to a method for producing plants with improved agronomic and nutritional traits. Such traits include enhanced nitrogen assimilatory and utilization capacities, faster and more vigorous growth, greater vegetative and reproductive yields, and enriched or altered nitrogen content in vegetative and reproductive parts. More particularly, the invention relates to the engineering of plants modified to have altered expression of key enzymes in the nitrogen assimilation and utilization pathways. In one embodiment of the present invention, the desired altered expression is accomplished by engineering the plant for ectopic overexpression of one of more the native or modified nitrogen assimilatory enzymes. The invention also has a number of other embodiments, all of which are disclosed herein.

THE EMERGENCE OF ECOLOGICAL ENGINEERING AS A DISCIPLINE

EPA Science Inventory

Pioneering efforts in the field of ecological engineering research and practice have proven to be tremendous strides toward establishing a new engineering discipline with a science base in ecology. Case studies, demonstrations and applications pertaining to restoration, rehabili...