Alain A. Vertès

Metabolic Analysis of Corynebacterium glutamicum during Lactate and Succinate Productions under Oxygen Deprivation Conditions

Lactate and succinate were produced from glucose by Corynebacterium glutamicum under oxygen deprivation conditions without growth. Addition of bicarbonate to the reaction mixture led not only to a 3.6-fold increase in succinate production rate, but also to a 2.3- and 2.5-fold increase, respectively, of the rates of lactate production and glucose consumption, compared to the control. Furthermore, when small amounts of pyruvate were added to the reaction mixture, acid production rates and the glucose consumption rate were multiplied by a factor ranging from 2 to 3. These phenomena were paralleled by an increase in the NAD+/NADH ratio, thus corroborating the view that the efficient regeneration of NAD+ could be triggered by the addition of either bicarbonate or pyruvate. To investigate the global metabolism of corynebacteria under oxygen deprivation conditions, we engineered several strains where the genes coding for key metabolic enzymes had been inactivated by gene disruption and replacement. A lactate dehydrogenase (LDH)-deficient mutant was not able to produce lactate, suggesting this enzyme has no other isozyme. Although a pyruvate carboxylase (pyc) mutant exhibited similar behavior to that of the wild type, phosphoenolpyruvate carboxylase (ppc) mutants were characterized by a dramatic decrease in succinate production, which was concomitant to decreased lactate production and glucose consumption rates. This set of observations corroborates the view that in coryneform bacteria under oxygen deprivation conditions the major anaplerotic reaction is driven by the ppc gene product rather than by the pyc gene product. Moreover, intracellular NADH concentrations in C. glutamicum were observed to correlate to oxygen-deprived metabolic flows.

Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions.

The central metabolic pathway of Corynebacterium glutamicum was engineered to produce ethanol. A recombinant strain which expressed the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) was constructed. Both genes placed under the control of the C. glutamicum ldhA promoter were expressed at high levels in C. glutamicum, resulting, under oxygen-deprivation conditions, in a significant yield ofethanol from glucose in a process characterized by the absence of cellular growth. Addition of pyruvate in trace amounts to the reaction mixture induced a 2-fold increase in the ethanol production rate. A similar effect was observed when acetaldehyde was added. Disruption of the lactate dehydrogenase (ldhA) gene led to a 3-fold higher ethanol yield than wild type, with no lactate production. Moreover, inactivation of the phosphoenolpyruvate carboxylase (ppc) and ldhA genes revealed a significant amount of ethanol production and a dramatic decrease in succinate without any lactate production, when pyruvate was added. Since the reaction occurred in the absence of cell growth, the ethanol volumetric productivity increased in proportion to cell density of ethanologenic C. glutamicum in a process under oxygen-deprivation conditions. These observations corroborate the view that intracellular NADH concentrations in C. glutamicum are correlated to oxygen-deprived metabolic flows.

Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

ABSTRACT The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

Biomass to biofuels : strategies for global industries

Foreword. Preface. Contributors. PART I STRUCTURE OF THE BIOEVERGY BUSINESS. 1 Characteristics of Biofuels and Renewable Fuel Standards ( Alan C. Hansen, Dimitrios C. Kyritsis, and Chia fon F. Lee ). 1.1 Introduction. 1.2 Molecular Structure. 1.3 Physical Properties. 1.4 Chemical Properties. 1.5 Biofuel Standards. 1.6 Perspective. References. 2 The Global Demand for Biofuels: Technologies, Markets and Policies (Jurgen Scheffran). 2.1 Introduction. 2.2 Motivation and Potential of Renewable Fuels. 2.3 Renewable Fuels in the Transportation Sector. 2.4 Status and Potential of Major Biofuels. 2.5 Biofuel Policies and Markets in Selected Countries. 2.6 Perspective. References. 3 Biofuel Demand Realization ( Stephen R. Hughes and Nasib Qureshi ). 3.1 Introduction. 3.2 Availability of Renewable Resources to Realize Biofuel Demand. 3.3 Technology Improvements to Enhance Biofuel Production Economics. 3.4 US Regulatory Requirements for Organisms Engineered to Meet Biofuel Demand. 3.5 Perspective. Acknowledgments. References. 4 Advanced Biorefineries for the Production of Fuel Ethanol ( Stephen R. Hughes, William Gibbons, and Scott Kohl ). 4.1 Introduction. 4.2 Ethanol Production Plants Using Sugar Feedstocks. 4.3 Dedicated Dry-Grind and Dry-Mill Starch Ethanol Production Plants. 4.4 Dedicated Wet-Mill Starch Ethanol Production Plants. 4.5 Dedicated Cellulosic Ethanol Production Plants. 4.6 Advanced Combined Biorefineries. 4.7 Perspective. Acknowledgments. References. PART II DIESEL FROM BIOMASS. 5 Biomass Liquefaction and Gasification ( Nicolaus Dahmen, Edmund Henrich, Andrea Kruse, and Klaus Raffelt ). 5.1 Introduction. 5.2 Direct Liquefaction. 5.3 Biosynfuels from Biosyngas. 5.4 Perspective. References. 6 Diesel from Syngas ( Yong-Wang Li, Jian Xu, and Yong Yang ). 6.1 Introduction. 6.2 Overview of Fischer-Tropsch Synthesis. 6.3 Historical Development of the Fischer-Tropsch Synthesis Process. 6.4 Modern Fischer-Tropsch Synthesis Processes. 6.5 Economics. 6.6 Perspective. Acknowledgements. References. 7 Biodiesel from Vegetable Oils ( Jon Van Gerpen ). 7.1 Introduction. 7.2 Use of Vegetable Oils as Diesel Fuels. 7.3 Renewable Diesel. 7.4 Properties. 7.5 Biodiesel Production. 7.6 Transesteritication. 7.7 Biodiesel Purification. 7.8 Perspective. References. 8 Biofuels from Microalgae and Seaweeds ( Michael Huesemann, G. Roesjadi, John Benemann, and F. Blaine Metting ). 8.1 Introduction. 8.2 Biofuels from Microalgae: Products, Processes, and Limitations. 8.3 Biofuels from Seaweeds: Products, Processes, and Limitations. 8.4 Perspective. References. PART III ETHANOL AND BUTANOL. 9 Improvements in Corn to Ethanol Production Technology Using Saccharomyces cerevisiae ( Vijay Singh, David B. Johnston, Kent D. Rausch, and M.E. Tumbleson ). 9.1 Introduction. 9.2 Current Industrial Ethanol Production Technology. 9.3 Granular Starch Hydrolysis. 9.4 Corn Fractionation. 9.5 Simultaneous SSF and Distillation. 9.6 Dynamic Control of SSF Processes. 9.7 Cost of Ethanol. 9.8 Perspective. References. 10 Advanced Technologies for Biomass Hydrolysis and Saccharification Using Novel Enzymes ( Margret E. Berg Miller, Jennifer M. Brulc, Edward A. Bayer, Raphael Lamed, Harry J. Flint, and Bryan A. White ). 10.1 Introduction. 10.2 The Substrate. 10.3 Glycosyl Hydrolases. 10.4 The Cellulosome Concept. 10.5 New Approaches for the Identification of Novel Glycoside Hydrolases. 10.6 Perspective. References. 11 Mass Balances and Analytical Methods for Biomass Pretreatment Experiments ( Bruce S. Dien ). 11.1 Introduction. 11.2 Analysis of Feedstocks for Composition and Potential Ethanol Yield. 11.3 Pretreatment. 11.4 Enzymatic Extraction of Sugars. 11.5 Fermentation of Pretreated Hydrolysates to Ethanol. 11.6 Feedstock and Process Integration. 11.7 Perspective. Acknowledgments. References. 12 Biomass Conversion Inhibitors and In Situ Detoxification ( Z. Lewis Liu and Hans P. Blaschek ). 12.1 Introduction. 12.2 Inhibitory Compounds Derived from Biomass Pretreatment. 12.3 Inhibitory Effects. 12.4 Removal of Inhibitors. 12.5 Inhibitor-Tolerant Strain Development. 12.6 Inhibitor Conversion Pathways. 12.7 Molecular Mechanisms of In Situ Detoxification. 12.8 Perspective. Acknowledgments. References. 13 Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts ( Grant Stanley and Barbel Hahn-Hagerdal ). 13.1 Introduction. 13.2 Consolidated Bioprocessing and Ethanol Production. 13.3 Pentose-Fermenting S. cerevisiae Strains. 13.4 Lignocellulose Fermentation and Ethanol Inhibition. 13.5 Perspective. Acknowledgments. References. 14 Conversion of Biomass to Ethanol by Other Organisms ( Siqing Liu ). 14.1 Introduction. 14.2 Desired Biocatalysts for Biomass to Bioethanol. 14.3 Gram-Negative Bacteria. 14.4 Gram-Positive Bacteria. 14.5 Perspective. Acknowledgments. References. 15 Advanced Fermentation Technologies ( Masayuki Inui, Alain A. Vertes and Hideaki Yukawa ). 15.1 Introduction. 15.2 Batch Processes. 15.3 Fed-Batch Processes. 15.4 Continuous Processes. 15.5 Immobilized Cell Systems. 15.6 Growth-Arrested Process. 15.7 Integrated Bioprocesses. 15.8 Consolidated Bioprocessing (CBP). 15.9 Perspective. References. 16 Advanced Product Recovery Technologies ( Thaddeus C Ezeji and Yebo Li ). 16.1 Introduction. 16.2 Membrane Separation. 16.3 Advanced Technologies for Biofuel Recovery: Industrially Relevant Processes. 16.4 Perspective. Acknowledgments. References. 17 Clostridia and Process Engineering for Energy Generation ( Nasib Qureshi and Hans P. Blaschek ). 17.1 Introduction. 17.2 Substrates, Cultures, and Traditional Technologies. 17.3 Agricultural Residues as Substrates for the Future. 17.4 Butanol-Producing Microbial Cultures. 17.5 Regulation of Butanol Production and Microbial Genetics. 17.6 Novel Fermentation Technologies. 17.7 Novel Product Recovery Technologies. 17.8 Fermentation of Lignocellulosic Substrates in Integrated Systems. 17.9 Integrated or Consolidated Processes. 17.10 Perspective. Acknowledgments. References. PART IV: HYDROGEN, METHANE, AND METHANOL. 18 Hydrogen Generation by Microbial Cultures ( Anja Hemschemeier, Katrin Mullner, Thilo Ruhle, and Thomas Happe ). 18.1. Introduction: Why Biological Hydrogen Production? 18.2. Biological Hydrogen Production. 18.3. Metabolic Basics for Hydrogen Production: Fermentation and Photosynthesis. 18.4. H 2 Production in Application: Cases in Point. 18.5. Perspective. References. 19 Engineering Photosynthesis for H 2 Production from H 2 O: Cyanobacteria as Design Organisms ( Nadine Waschewski, Gabor Bernat, and Matthias Rogner ). 19.1 The Basic Idea: Why Hydrogen from Water? 19.2 Realization: Three Mutually Supporting Strategies. 19.3 The Biological Strategy: How to Design a Hydrogen-Producing (Cyano-) Bacterial Cell. 19.4 Engineering the Environment of the Cells: Reactor Design. 19.5 How Much Can We Expect? The Limit of Natural Systems. 19.6 Perspective. Acknowledgments. References. 20 Production and Utilization of Methane Biogas as Renewable Fuel ( Zhongtang Yu, Mark Morrison, and Floyd L. Schanbacher ). 20.1 Introduction. 20.2 The Microbes and Metabolisms Underpinning Biomethanation. 20.3 Potential Feedstocks Used for Methane Biogas Production. 20.4 Biomethanation Technologies for Production of Methane Biogas. 20.5 Utilization of Methane Biogas as a Fuel. 20.6 Perspective. 20.7 Concluding Remarks. 20.8 Disclaimer. References. 21 Methanol Production and Utilization ( Gregory A. Dolan ). 21.1 Introduction. 21.2 Biomass Gasification: Mature and Immature. 21.3 Feedstocks: Diverse and Plentiful. 21.4 Biomethanol: ICEs, FFVs, and FCVs. 21.5 Case Study: Waste Wood Biorefinery. 21.6 Case Study: Two-Step Thermochemical Conversion Process. 21.7 Case Study: Mobile Methanol Machine. 21.8 Case Study: Scandinavia Leading the Way with Black Liquor Methanol Production. 21.9 Case Study: Methanol Fermentation through Anaerobic Digestion. References. PART V PERSPECTIVES. 22 Enhancing Primary Raw Materials for Biofuels ( Takahisa Hayashi, Rumi Kaida, Nobutaka Mitsuda, Masaru Ohme-Takagi, Nobuyuki Nishikuba, Shin-ichiro Kidou, and Kouki Yoshida ). 22.1 Introduction. 22.2 In-Fibril Modification. 22.3 In-Wall Modifications. 22.4 In-Planta Modifications. 22.5 In-CRES-T Modification. 22.6 A Catalogue of Gene Families for Glycan Synthases and Hydrolases. 22.7 Perspective. Acknowledgments. References. 23 Axes of Development in Chemical and Process Engineering for Converting Biomass to Energy ( Alain A. Vertes ). 23.1 Global Outlook. 23.2 Enhancement of Raw Material Biomass. 23.3 Conversion of Biomass to Fuels and Chemicals. 23.4 Chemical Engineering Development. 23.5 Perspective. References. 24 Financing Strategies for Industrial-Scale Biofuel Production and Technology Development Start-Ups ( Alain A. Vertes and Sarit Soccary Ben Yochanan ). 24.1 Background: The Financial Environment. 24.2 Biofuels Project: Steps in Value Creation and Required Funding at Each Stage. 24.3 Governmental Incentives to Support the Nascent Biofuel and Biomaterial Industry. 24.4 Perspective: What is the Best Funding Source for Each Step in a Companys Development? References. Index.

Engineering of an l-arabinose metabolic pathway in Corynebacterium glutamicum

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.

Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminal electron acceptor

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia colinarK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicumnarG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.

Identification and Functional Analysis of the Gene Cluster for l-Arabinose Utilization in Corynebacterium glutamicum

ABSTRACT Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (l-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on l-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l-arabinose (3.6 g liter−1) but not at a higher concentration of l-arabinose (40 g liter−1). The expression of the araBDA operon and the araE gene was l-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l-arabinose. Expression of the regulon was not repressed by d-glucose, and simultaneous utilization of l-arabinose and d-glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.

Manipulating corynebacteria, from individual genes to chromosomes

Corynebacterium glutamicum is an industrial organism with a long history of use for the production of various fine chemicals. The C. glutamicum -mediated production of l-glutamic acid by fermentation was one of the very first industrial processes of the biotechnology era. This fermentation

Technological options for biological fuel ethanol.

The current paradigm to produce biotechnological ethanol is to use the yeast Saccharomyces cerevisiae to ferment sugars derived from starch or sugar crops such as maize, sugar cane or sugar beet. Despite its current success, the global impact of this manufacturing model is restricted on the one hand by limits on the availability of these primary raw materials, and on the other hand by the maturity of baker’s yeast fermentation technologies. Revisiting the technical, economic, and value chain aspects of the biotechnological ethanol industry points to the need for radical innovation to complement the current manufacturing model. Implementation of lignocellulosic materials is clearly a key enabler to the billion-ton biofuel vision. However, realization of the full market potential of biofuels will be facilitated by the availability of an array of innovative technological options, as the flexibility generated by these alternative processes will not only enable the exploitation of heretofore untapped local market opportunities, but also it will confer to large biorefinery structures numerous opportunities for increased process integration as well as optimum reactivity to logistic and manufacturing challenges. In turn, all these factors will interplay in synergy to contribute in shifting the economic balance in favor of the global implementation of biotechnological ethanol.

Implementing biofuels on a global scale

Is the introduction of renewable biofuels a simple problem of technology development and diffusion or does it require an industrial revolution?