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Author : Sang-Weon Bang
Publisher :
ISBN 13 :
Total Pages : 234 pages
Book Rating : 4.:/5 (262 download)
Download or read book Ethanol Production by Zymomonas Mobilis Strains written by Sang-Weon Bang and published by . This book was released on 1991 with total page 234 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Patrick M. Weir
Publisher :
ISBN 13 :
Total Pages : 714 pages
Book Rating : 4.:/5 (551 download)
Download or read book Ethanol Production by Zymomonas Mobilis written by Patrick M. Weir and published by . This book was released on 1989 with total page 714 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Author : Manoj Agrawal
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (81 download)
Download or read book Metabolic Engineering of Zymomonas Mobilis for Improved Production of Ethanol from Lignocelluloses written by Manoj Agrawal and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Ethanol from lignocellulosic biomass is a promising alternative to rapidly depleting oil reserves. However, natural recalcitrance of lignocelluloses to biological and chemical treatments presents major engineering challenges in designing an ethanol conversion process. Current methods for pretreatment and hydrolysis of lignocelluloses generate a mixture of pentose (C5) and hexose (C6) sugars, and several microbial growth inhibitors such as acetic acid and phenolic compounds. Hence, an efficient ethanol production process requires a fermenting microorganism not only capable of converting mixed sugars to ethanol with high yield and productivity, but also having high tolerance to inhibitors. Although recombinant bacteria and yeast strains have been developed, ethanol yield and productivity from C5 sugars in the presence of inhibitors remain low and need to be further improved for a commercial ethanol production. The overarching objective of this work is to transform Zymomonas mobilis into an efficient whole-cell biocatalyst for ethanol production from lignocelluloses. Z. mobilis, a natural ethanologen, is ideal for this application but xylose (a C5 sugar) is not its 'natural' substrate. Back in 1995, researches at National Renewable Energy Laboratory (NREL) had managed to overcome this obstacle by metabolically engineering Z. mobilis to utilize xylose. However, even after more than a decade of research, xylose fermentation by Z. mobilis is still inefficient compared to that of glucose. For example, volumetric productivity of ethanol from xylose fermentation is 3- to 4- fold lower than that from glucose fermentation. Further reduction or complete inhibition of xylose fermentation occurs under adverse conditions. Also, high concentrations of xylose do not get metabolized completely. Thus, improvement in xylose fermentation by Z. mobilis is required. In this work, xylose fermentation in a metabolically engineered Z. mobilis was markedly improved by applying the technique of adaptive mutation. The adapted strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, the strain produced a total of 9% (w/v) ethanol from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Investigation was done to identify the molecular basis for efficient biocatalysis. An altered xylitol metabolism with reduced xylitol formation, increased xylitol tolerance and higher xylose isomerase activity were found to contribute towards improvement in xylose fermentation. Lower xylitol production in adapted strain was due to a single mutation in ZMO0976 gene, which drastically lowered the reductase activity of ZMO0976 protein. ZMO0976 was characterized as a novel aldo-keto reductase capable of reducing xylose, xylulose, benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, but not glucose or fructose. It exhibited nearly 150-times higher affinity with benzaldehyde than xylose. Knockout of ZMO0976 was found to facilitate the establishment of xylose fermentation in Z. mobilis ZM4. Equipped with molecular level understanding of the biocatalytic process and insight into Z. mobilis central carbon metabolism, further genetic engineering of Z. mobilis was undertaken to improve the fermentation of sugars and lignocellulosic hydrolysates. These efforts culminated in construction of a strain capable of fermenting glucose-xylose mixture in presence of high concentration of acetic acid and another strain with a partially operational EMP pathway.
Author : Yun Wang
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (52 download)
Download or read book Development of Acetic-acid Tolerant Zymomonas Mobilis Strains Through Adaptation written by Yun Wang and published by . This book was released on 2008 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Zymomonas mobilis is one of the most promising microorganisms for bioethanol production. However, its practical use on industrial scale is impeded by its high sensitivity to acetate, which is present in high concentration in pretreated biomass. This research develops an adaptive mutation method for generating acetate-tolerant strains for bioethanol production. The goal is to obtain Zymomonas mobilis strain capable of growing and producing ethanol in the presence of acetate at a concentration typical of a pretreated biomass (2-3%). The interplay between the ability of fermentative production of ethanol and acetate tolerance will be investigated through careful fermentation studies. The potential cross-tolerance to other inhibitors, commonly present in pretreated biomass will be evaluated. A preliminary study on the mechanism of acetate tolerance at the cell membrane level will be conducted. The strain developed through this research will be useful in bioethanol production from biomass. The insights into tolerance mechanisms gained through this study will allow a more rational approach to further engineer a better producing strain.
Author : H. W. Doelle
Publisher : Academic Press
ISBN 13 : 1483272745
Total Pages : 501 pages
Book Rating : 4.4/5 (832 download)
Download or read book Bacterial Metabolism written by H. W. Doelle and published by Academic Press. This book was released on 2014-06-28 with total page 501 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacterial Metabolism focuses on metabolic events that occur in microorganisms, as well as photosynthesis, oxidation, polysaccharide formation, and homofermentation. The book first discusses the thermodynamics of biological reactions, photosynthesis and photometabolism, and chemosynthesis. Free energy, photosynthesis, enzymes, and terminology in bacterial metabolism are elaborated. The manuscript then examines acetic acid bacteria and lactic acid bacteria. Discussions focus on lactate, ethanol, glucose, and glycerol metabolism, glycol oxidation, homofermentation, polysaccharide formation, and electron transport systems. The publication takes a look at pseudomonadaceae and nitrogen metabolism as an energy source for anaerobic microorganisms. Topics include metabolism of pairs of amino acids, single amino acid metabolism, oxidation of glycolate and malonate, and oxygenases. The book is a dependable source of information for readers interested in bacterial metabolism.
Author : Antonio Morata
Publisher : BoD – Books on Demand
ISBN 13 : 9535135996
Total Pages : 302 pages
Book Rating : 4.5/5 (351 download)
Download or read book Yeast written by Antonio Morata and published by BoD – Books on Demand. This book was released on 2017-11-08 with total page 302 pages. Available in PDF, EPUB and Kindle. Book excerpt: Yeast - Industrial Applications is a book that covers applications and utilities of yeasts in food, chemical, energy, and environmental industries collected in 12 chapters. The use of yeasts in the production of metabolites, enzymatic applications, fermented foods, microorganism controls, bioethanol production, and bioremediation of contaminated environments is covered showing results, methodologies, and processes and describing the specific role of yeasts in them. The traditional yeast Saccharomyces cerevisiae is complemented in many applications with the use of less known non-Saccharomyces yeasts that now are being used extensively in industry. This book compiles the experience and know-how of researchers and professors from international universities and research centers.
Download or read book Ethanol Production by Escherichia Coli Strains Co-expressing Zymomonas PDC and ADH Genes written by and published by . This book was released on 1991 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: A novel operon and plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase activities of Zymomonas mobilis are described. Also disclosed are methods for increasing the growth of microorganisms or eukaryotic cells and methods for reducing the accumulation of undesirable metabolic products in the growth medium of microorganisms or cells.
Author : Jung Woo Yang
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (879 download)
Download or read book Enhanced Bioethanol Production by Zymomonas Mobilis in Response to the Quorum Sensing Molecules AI-2 written by Jung Woo Yang and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The depletion of non-renewable energy resources, the environmental concern over the burning of fossil fuels, and the recent price rises and instability in the international oil markets have all combined to stimulate interest in the use of fermentation processes for the production of alternative bio-fuels. As a fuel, ethanol is mainly of interest as a petrol additive, or substrate, because ethanol-blended fuel produces a cleaner, more complete combustion that reduces greenhouse gas and toxic emissions. As a consequence of the surge in demand for biofuels, ethanol producing microorganisms, such as the bacterium Zymomonas mobilis, are of considerable interest due to their potential for industrial-scale bioethanol production. Although bioethanol has traditionally been produced in batch fermentation with the yeast Saccharomyces cerevisiae, there are advantages in using Z. mobilis as an alternative for bioethanol production. In comparison to yeast, Z. mobilis grows and ferments rapidly, without the requirement for the controlled supply of oxygen during fermentation, and has a significantly higher ethanol product rate and yield. Most importantly, it has a high tolerance for ethanol. Bacteria communicate with one another using chemical signalling molecules. In general, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules termed autoinducers (AI). This process allows bacteria to monitor the environment for other bacteria and to alter behaviour on a population-wide scale in response to changes in the number and/or species present in a community. Currently, there are three well-defined classes of molecules that serve as the paradigms for chemical signaling in bacteria: oligopeptides, AI-1 (AHLs) and AI-2. Oligopeptide signalling is the predominant signal used by Gram-positive bacteria, and AHLs (acyl-homoserine lactones) are for species-specific communication in Gram-negative bacteria. Finally, the LuxS/AI-2 pathway is generally considered as involved in interspecies communication because the luxS gene, which is responsible for AI-2 production, is found in various bacteria. Many physiological functions in bacteria such as toxin, virulence factor and bacteriocin production, biofilm formation, bioluminescence, type III secretion, have been shown to be under the control of AI-2 quorum sensing. In Z. mobilis, in vitro synthesized and in vivo produced AI-2 treatment enhanced ethanol production by this bacterium up to a maximum of 50% in comparison with untreated control cells. This appears attributable to the overproduction of the glycolytic enzymes, enolase and pyruvate carboxylase, which are only rarely found in bacteria and the key enzymes for ethanol production. From the perspective of interspecies communication, enhanced ethanol production in Z. mobilis, under the control of the AI-2 signalling molecules, could represent a good example of a bacterium that does not produce AI-2, but responds to it. Another interesting finding is that two extracellular proteins from Z. mobilis, ZMO0994 and ZMO0134 which were originally induced by AI-2, were secreted when they were cloned, transformed and expressed in E. coli strain BL21 DE3; since it is generally accepted that nonpathogenic strains of E. coli, particularly derivatives of K12, do not secrete proteins under routine growth conditions. Presumably, these proteins possess signal sequences for secretion that could be used to provide a strategy for their use as carriers of recombinant proteins produced in E. coli K12. The merit of this system is that there would be few contaminant cytoplasmic proteins, and could possibly solve problems in protein purification, such as protease activity, protein misfolding and inclusion body formation. Finally, the discovery that the metabolic pathway leading to ethanol production is regulated by AI-2 is of considerable biotechnological importance because it will provide a basis for further engineering of strains for more efficient ethanol production. Indeed, engineering Z. mobilis by introducing the genes that encode Pfs and LuxS to produce AI-2, would be a means to stimulate increased ethanol production.
Download or read book Xylose Utilizing Zymomonas Mobilis with Improved Ethanol Production in Biomass Hydrolysate Medium written by and published by . This book was released on 2013 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Xylose-utilizing, ethanol producing strains of Zymomonas mobilis with improved performance in medium comprising biomass hydrolysate were isolated using an adaptation process. Independently isolated strains were found to have independent mutations in the same coding region. Mutation in this coding may be engineered to confer the improved phenotype.
Author : Charles Wyman
Publisher : Routledge
ISBN 13 : 1351441760
Total Pages : 289 pages
Book Rating : 4.3/5 (514 download)
Download or read book Handbook on Bioethanol written by Charles Wyman and published by Routledge. This book was released on 2018-05-02 with total page 289 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bioethanol is a versatile transportation fuel and fuel additive that offers excellent performance and reduced air pollution compared to conventional fuels. Its production and use adds little, if any, net release of carbon dioxide to the atmosphere, dramatically reducing the potential for global climate change. Through a sustained research program and an emerging economic competitiveness, the technology for bioethanol production is poised for immediate widespread commercial applications. Written by engineers and scientists providing a technical focus, this handbook provides the up-to-date information needed by managers, engineers, and scientists to evaluate the technology, market, and economics of this fuel, while examining the development of production required to support its commercial use.
Author : Alain A. Vertes
Publisher : John Wiley & Sons
ISBN 13 : 111915202X
Total Pages : 702 pages
Book Rating : 4.1/5 (191 download)
Download or read book Green Energy to Sustainability: Strategies for Global Industries written by Alain A. Vertes and published by John Wiley & Sons. This book was released on 2020-06-10 with total page 702 pages. Available in PDF, EPUB and Kindle. Book excerpt: Reviews the latest advances in biofuel manufacturing technologies and discusses the deployment of other renewable energy for transportation Aimed at providing an interface useful to business and scientific managers, this book focuses on the key challenges that still impede the realization of the billion-ton renewable fuels vision. It places great emphasis on a global view of the topic, reviewing deployment and green energy technology in different countries across Africa, Asia, South America, the EU, and the USA. It also integrates scientific, technological, and business development perspectives to highlight the key developments that are necessary for the global replacement of fossil fuels with green energy solutions. Green Energy to Sustainability: Strategies for Global Industries examines the most recent developments in biofuel manufacturing technologies in light of business, financial, value chain, and supply chain concerns. It also covers the use of other renewable energy sources like solar energy for transportation and proposes a view of the challenges over the next two to five decades, and how these will deeply modify the industrial world in the third millennium. The coming of age of electric vehicles is also looked at, as is the impact of their deployment on the biomass to biofuels value chain. Offers extensive updates on the field of green energy for global industries Covers the structure of the energy business; chemicals and diesel from biomass; ethanol and butanol; hydrogen and methane; and more Provides an expanded focus on the next generation of energy technologies Reviews the latest advances in biofuel manufacturing technologies Integrates scientific, technological and business perspectives Highlights important developments needed for replacing fossil fuels with green energy Green Energy to Sustainability: Strategies for Global Industries will appeal to academic researchers working on the production of fuels from renewable feedstocks and those working in green and sustainable chemistry, and chemical/process engineering. It is also an excellent textbook for courses in bioprocessing technology, renewable resources, green energy, and sustainable chemistry.
Author : Lok-Yan So
Publisher :
ISBN 13 : 9781361279274
Total Pages : pages
Book Rating : 4.2/5 (792 download)
Download or read book The Development of New Methodologies and Genetic Tools for Proteomic and Metabolic Engineering Applications Within the Ethanol-Producing Bacterium Zymomonas Mobilis written by Lok-Yan So and published by . This book was released on 2017-01-26 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation, "The Development of New Methodologies and Genetic "tools" for Proteomic and "metabolic Engineering" Applications Within the Ethanol-producing Bacterium Zymomonas Mobilis" by Lok-yan, So, 蘇樂欣, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Zymomonas mobilis is a non-pathogenic, facultatively-anaerobic Gram-negative bacterium, which has historically been used for the fermentation of alcoholic beverages in many tropical/sub-tropical countries. Due to its excellent ethanol-producing capabilities, significant effort has been undertaken over recent years to utilize it for industrial ''bioethanol'' production. Its physiological and metabolic properties indicate that it may also be an excellent organism for the bio-production of many different types of organic molecules. Consequently, the aim of my thesis was to develop new molecular methodologies that would enable Z. mobilis to be ''engineered'' for use in future ''bioproduction'' endeavours. In the first part of my study, I analyzed the native (cryptic) plasmids present within a variety of Z. mobilis strains, including two poorly-studied Z. mobilis strains: NCIMB 11163 and NCIMB 8227. Several plasmid libraries containing restriction-digested fragments of Z. mobilis cryptic plasmid DNA were prepared, and their inserts were sequenced. This enabled the complete DNA sequences of three small (non-integrating, double-stranded DNA) cryptic plasmids to be determined: pZMO1A and pZMO7 from NCIMB 11163, and pZMO1B from NCIMB 8227. Their DNA sequences were analyzed using bioinformatic approaches, to identify open reading frames, and regions of DNA that were putatively involved in transcription or DNA replication. In the second part of this thesis, the minimally-replicating region from plasmid pZMO7 was used to construct a series of Escherichia coli-Z. mobilis shuttle vectors. These vectors were found to be stable within several Z. mobilis strains for over 60 generations without antibiotic selective pressure. A reliable and reproducible method based on quantitative real time PCR (Q-RT-PCR) was developed to accurately determine the copy number of cryptic plasmids and shuttle-vectors present in Z. mobilis cultures. The pZMO7-based shuttle vectors exhibited good compatibility with cryptic plasmids as well as the widely-used pZM2-based shuttle vectors. Genes encoding glutathione S-transferase (GST) as well as green and red fluorescent protein (GFP and RFP) reporters were cloned into various shuttle vector constructs; placing them under the control of endogenous (Ppdc) or exogenous (Plac and Ptac) promoters. Promoter strength was evaluated by quantifying the reporter gene expression. The plasmid-based expression of GFP and RFP was visualized within planktonic and biofilm cultures using confocal laser scanning microscopy (CLSM). Shuttle vector-based GST pull-down experiments were used to study intracellular protein-protein binding interactions. In the third part of my thesis, I explored the potential use of Z. mobilis for the bioproduction of isoprenoid (terpenoid) compounds. Five predicted sesquiterpene synthases (terpene cyclases) of unknown function from the dimorphic fungus Penicillium marneffei, and several terpene cyclases from several other bacteria, fungi and plants were initially functionally-analyzed in E. coli. Several cyclase genes were cloned into E. coli-Z. mobilis shuttle vectors for expression trials within Z. mobilis cells. In summary, this thesis describes the development of a variety of novel methodologies and genetic ''tools'' that may be used to express heterolog
Author : Lisbeth Olsson
Publisher : Springer
ISBN 13 : 3540736514
Total Pages : 372 pages
Book Rating : 4.5/5 (47 download)
Download or read book Biofuels written by Lisbeth Olsson and published by Springer. This book was released on 2007-09-11 with total page 372 pages. Available in PDF, EPUB and Kindle. Book excerpt: With contributions by numerous experts
Author : Seung Hee Cho
Publisher :
ISBN 13 :
Total Pages : 306 pages
Book Rating : 4.:/5 (127 download)
Download or read book Exploring Novel Regulatory Noncoding RNAs Associated with Ethanol Tolerance in Zymomonas Mobilis for Strain Engineering written by Seung Hee Cho and published by . This book was released on 2016 with total page 306 pages. Available in PDF, EPUB and Kindle. Book excerpt: Zymomonas mobilis has been identified as a promising cellular factory for biofuels due to its efficient, natural production of and tolerance to ethanol. With rising demands of efficiency and sustainability, the use of microbes as chemical factories is increasingly attractive. In bacteria, small noncoding RNAs have been highlighted as powerful tools due to their regulatory roles in cellular pathways and as such are being increasingly exploited for engineering purposes. As global controllers of gene expression, noncoding regulatory RNAs represent powerful tools for engineering complex phenotypes. However, mechanistic analysis of these regulators in bacteria lags far behind their high-throughput search and discovery; this makes it difficult to understand how to efficiently identify noncoding RNAs that could be used to engineer a phenotype of interest. This dissertation describes discovery of novel global regulatory small noncoding RNAs that impact ethanol tolerance in Z. mobilis using a forward systems approach. The effect of the bioinformatically predicted candidates were experimentally characterized by building overexpression and deletion strains. This led to the successful uncovering of several sRNAs that could be manipulated to enhance ethanol tolerance as well as ethanol production in Z. mobilis. Using these ethanol-related sRNAs, we then performed traditional genetic and biochemical approaches as well as transcriptomics and proteomics methods to identify a pool of mRNA targets and pathways that were being regulated by these sRNAs under conditions of enhanced ethanol tolerance. Additionally, control elements (especially untranslated regions (UTRs)) that regulate gene expression at the local level associated with ethanol as well as other metabolic stresses in Z. mobilis were searched and exploited. Specifically, ethanol responsive-UTR element identified in this work regulates the expression of Hfq and affects cell growth under ethanol stress condition. This work represents the first application of a de novo sRNA engineering strategy in a non-model organism, Z. mobilis, which is of relevance to biofuel technologies. Furthermore, our development and application of a novel bioinformatics pipeline for this work has demonstrated the ability to use formal computational approaches (in conjuction with systems-level methods) to accelerate the discovery of specific pathways that could be further optimized to enhance a given complex phenotype
Download or read book Ethanol Production from Rice-Straw Hydrolysate Using Zymomonas Mobilis in a Continuous Fluidized-Bed Reactor (FBR). written by and published by . This book was released on 2001 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Rice-straw hydrolysate obtained by the Arkenol's concentrated acid hydrolysis process was fermented to ethanol using a recombinant Zymomonas mobilis strain capable of utilizing both glucose and xylose in a continuous fluidized-bed reactor (FBR). The parameters studied included biocatalyst stability with and without antibiotic, feed composition, and retention time. Xylose utilization in the presence of tetracycline remained stable for at least 17 days. This was a significant improvement over the old strain, Z. mobilis CP4 (pZB5), which started to lose xylose utilization capability after seven days. In the absence of tetracycline, the xylose utilization rate started to decrease almost immediately. With tetracycline in the feed for the first six days, stability of xylose utilization was maintained for four days after the antibiotic was removed from the feed. The xylose utilization rate started to decrease on day 11. In the presence of tetracycline using the Arkenol's hydrolysate diluted to 48 g/L glucose and 13 g/L xylose at a retention time of 4.5 h, 95% xylose conversion and complete glucose conversion occurred. The ethanol concentration was 29 g/L, which gave a yield of 0.48 g/g sugar consumed or 94% of the theoretical yield. Using the Arkenol's hydrolysate diluted to 83 g/L glucose and 28 g/L xylose, 92% xylose conversion and complete glucose conversion were obtained. The ethanol concentration was 48 g/L, which gave a yield of 0.45 g/ g sugar consumed or 88% of the theoretical yield. Maximum productivity of 25.5 g/L-h was obtained at a retention time of 1.9 h. In this case, 84% xylose conversion was obtained.
Author : Yang Liu
Publisher :
ISBN 13 :
Total Pages : 247 pages
Book Rating : 4.:/5 (129 download)
Download or read book Regulatory Circuits to Program Metabolism of Zymomonas Mobilis for Advanced Cellulosic Biofuels Production written by Yang Liu and published by . This book was released on 2021 with total page 247 pages. Available in PDF, EPUB and Kindle. Book excerpt: Zymomonas mobilis is a potential organism for microbial biofuel production with attractive properties such as extraordinarily fast glycolysis and ethanol production. Production of advanced biofuels, such as butanol and isobutanol, is envisioned with Z. mobilis as a promising host. Z. mobilis grows exclusively through its efficient ethanol fermentation, which makes it difficult to achieve high flux into desired engineered pathways. In order to understand the physiological roles of ethanol fermentation and create strategies to divert carbon flux into production of chemicals and advanced biofuels, a Z. mobilis strain with regulated pyruvate decarboxylase (Pdc) expression was constructed. Growth, biochemical, and metabolomic experiments indicated that Pdc is essential for ethanologenic growth of Z. mobilis for redox balance and maintenance of fast glycolysis. For central carbon flux redirection, all flux can be redirected to a 2,3-butanediol pathway for which respiration maintains redox balance and the engineered 2,3-butanediol pathway allows growth without Pdc. Anaerobically, flux can be redirected to redox-balanced lactate or isobutanol pathways with ≥65% overall yield from glucose, where initial isobutanol titers reached 5 g/L. Subsequent improvements in expression of isobutanologenic enzymes allowed production up to 17 g isobutanol/L in Z. mobilis in 24 h. However, the current pathway can not support IBA-dependent growth in Z. moblis. Future optimization of the isobutanol pathway is still required. Biosensor regulatory systems, such as quorum sensing and metabolite sensing, offer practical approaches to control large-scale production of chemicals and biofuels. A xylose biosensor based on Bacillus subtlis XylR was engineered in Z. mobilis, capable of inducing gene expression at low xylose concentration. A xylose-sensing dual metabolic control circuit was constructed in Z. mobilis. Using this circuit, isobutanol pathway expression in Z. mobilis can be induced in the presence of xylose and pdc expression can be partially repressed by CRISPRi to achieve carbon flux redirection to isobutanol production. The partial pdc knockdown allowed growth and ethanol production at reduced rates and improved isobutanol production up to 3 g/L (~36% of theoretical yield from glucose) in rich medium. The circuit was also function in dilute switchgrass hydrolysate, where the engineered strain produced ~1.3 g IBA/L. Hundreds of biochemical reactions require NADH and NAD+ cofactors and their ratio is a key metabolic marker of cellular state. A genetically-encoded ratiometric biosensor for NADH/NAD+ based on redox-responsive bacterial transcription factor Rex was engineered in E. coli, which enabled non-invasive measurement of cellular redox state. The redox sensor was used to investigate the effect of removing respiratory chain enzymes on NADH/NAD+ ratio during aerobiosis. We found that the NADH/NAD+ ratio increased in five of the nine mutants by over three-fold compared to wild type, including an NADH dehydrogenase double mutant with six-fold elevation. We also found that among several common carbon sources, E. coli grown on acetate exhibited higher NADH/NAD+ ratios compared to E. coli grown on glucose. As a proof-of-concept for high-throughput redox screening, we were able to enrich high NADH mutants present at 1 in 10,000 among wild-type cells in a biosensor-guided, pooled screen. Thus, our Rex biosensor-reporter enabled facile, non-invasive, high-throughput redox measurement to understand and engineer redox metabolism.
Download or read book Final Technical Report written by and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Between 2007 and 2010 DuPont conducted a program under DOE award DE-FC36-07GO17056 to develop and improve Zymomonas mobilis as an ethanologen for commercial use in biorefineries to produce cellulosic ethanol. This program followed upon an earlier DOE funded program in which DuPont, in collaboration with the National Renewable Energy Laboratory (NREL) had developed a Zymomonas strain in conjunction with the development of an integrated cellulosic ethanol process. In the current project, we sought to maximize the utility of Zymomonas by adding the pathway to allow fermentation of the minor sugar arabinose, improve the utilization of xylose, improve tolerance to process hydrolysate and reduce the cost of producing the ethanologen. We undertook four major work streams to address these tasks, employing a range of approaches including genetic engineering, adaptation, metabolite and pathway analysis and fermentation process development. Through this project, we have developed a series of strains with improved characteristics versus the starting strain, and demonstrated robust scalability to at least the 200L scale. By a combination of improved ethanol fermentation yield and titer as well as reduced seed train costs, we have been able to reduce the capital investment and minimum ethanol selling price (MESP) by approximately 8.5% and 11% respectively vs. our starting point. Furthermore, the new strains we have developed, coupled with the learnings of this program, provide a platform for further strain improvements and advancement of cellulosic ethanol technology.
