Engineering Central Metabolism for Production of Higher Alcohol-based Biofuels

Abstract Widespread concerns have been raised regarding the need to develop sustainable processes for the production of fuels from renewable resources. While bioethanol production processes have been studied and successfully developed at industrial scales, its inferior fuel properties (e.g., lower energy density and higher hygroscopicity) relative to higher chain alcohols have directed recent interest towards producing C3-C10 alcohols, a more challenging prospect than bioethanol production. Metabolic engineering enabled by systems biology and synthetic biology is an enabling technology in such efforts, and many research examples show great promise for addressing current issues, including engineering enzymes and pathway flux, enhancing cofactor and precursor availability, and improving hosts’ tolerance to toxic biofuel products. In this chapter, we discuss the challenges and research efforts towards engineering microbes for optimized production of alcohol-based biofuels, with an emphasis on C3-C10 alcohols produced via central metabolism. Section 1.1 begins with a general introduction and compares relevant properties of different fuels. Section 1.2 discusses two categories of alcohol-producing pathways in detail, including both fermentative (i.e., acetone-butanol-ethanol pathway called the ABE pathway) and non-fermentative (i.e., Ehrlich pathway linked with amino acid metabolism; and reverse β-oxidation pathway). In Section 1.3 , engineering strategies to improve higher alcohol production are reviewed, which include (1) enhancing the function of enzymes and pathways as well as cofactor and precursor availability, and (2) addressing product toxicity. Section 1.4 covers successes and challenges towards commercialization of higher alcohol-based biofuels, giving some examples of successful commercialization and current issues and topics such as product separation, host choice, and alternative feedstocks. Section 1.5 concludes this chapter with an outlook on the future of higher alcohol biofuel production.

[1]  Y.‐H.P. Zhang,et al.  Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: challenges and opportunities. , 2010, Biotechnology and bioengineering.

[2]  L. Ingram,et al.  Mechanism of ethanol inhibition of fermentation in Zymomonas mobilis CP4 , 1985, Journal of bacteriology.

[3]  E. Green.  Fermentative production of butanol--the industrial perspective. , 2011, Current opinion in biotechnology.

[4]  Katsuyuki Takahashi,et al.  Screened Butanol-Tolerant Enterococcus faecium Capable of Butanol Production , 2012, Applied Biochemistry and Biotechnology.

[5]  Y. Jang,et al.  Metabolic engineering of Clostridium acetobutylicum M 5 for highly selective butanol production , 2009 .

[6]  Michael Taylor,et al.  An overview of second generation biofuel technologies. , 2010, Bioresource technology.

[7]  Sang Yup Lee,et al.  Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation , 2011, Applied and Environmental Microbiology.

[8]  X. Jia,et al.  Engineering a Metabolic Pathway for Isobutanol Biosynthesis in Bacillus subtilis , 2012, Applied Biochemistry and Biotechnology.

[9]  S. Atsumi,et al.  Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity , 2012, Microbial Cell Factories.

[10]  L. Ingram.  Adaptation of membrane lipids to alcohols , 1976, Journal of bacteriology.

[11]  Kechun Zhang,et al.  A synthetic recursive "+1" pathway for carbon chain elongation. , 2012, ACS chemical biology.

[12]  Si-Yu Li,et al.  Process parameters for operating 1-butanol gas stripping in a fermentor. , 2014, Journal of bioscience and bioengineering.

[13]  Yanping Zhang,et al.  Engineering cellular robustness of microbes by introducing the GroESL chaperonins from extremophilic bacteria. , 2014, Journal of biotechnology.

[14]  N. Stoynova,et al.  Use of the valine biosynthetic pathway to convert glucose into isobutanol , 2011, Journal of Industrial Microbiology & Biotechnology.

[15]  S. Junne,et al.  Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic Clostridium acetobutylicum Fermentations , 2003, Journal of bacteriology.

[16]  P. Dürre,et al.  Clostridium ljungdahlii represents a microbial production platform based on syngas , 2010, Proceedings of the National Academy of Sciences.

[17]  C. Grisham,et al.  The effects of long-chain alcohols on membrane lipids and the (Na++K+)-ATPase. , 1973, Biochimica et biophysica acta.

[18]  E. Papoutsakis,et al.  Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance , 2012, mBio.

[19]  Shang-Tian Yang,et al.  Bioprocessing technologies in biorefinery for sustainable production of fuels, chemicals, and polymers , 2013 .

[20]  E. Papoutsakis,et al.  Aldehyde–alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations , 2009, Biotechnology and bioengineering.

[21]  V. Zverlov,et al.  Biofuels from microbes , 2007, Applied Microbiology and Biotechnology.

[22]  S. Ehrlich,et al.  Essential Bacillus subtilis genes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  M. Inui,et al.  Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli , 2008, Applied Microbiology and Biotechnology.

[24]  G. Church,et al.  A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion , 2010, Molecular systems biology.

[25]  Kevin M. Smith,et al.  Metabolic engineering of Escherichia coli for 1-butanol production. , 2008, Metabolic engineering.

[26]  J. Liao,et al.  Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli , 2011, Applied and Environmental Microbiology.

[27]  D. T. Jones,et al.  Acetone-butanol fermentation revisited. , 1986, Microbiological reviews.

[28]  A. Schmid,et al.  Metabolic engineering of Pseudomonas sp. strain VLB120 as platform biocatalyst for the production of isobutyric acid and other secondary metabolites , 2014, Microbial Cell Factories.

[29]  L. Nielsen,et al.  Fermentative butanol production by clostridia , 2008, Biotechnology and bioengineering.

[30]  Y. Jang,et al.  Enhanced Butanol Production Obtained by Reinforcing the Direct Butanol-Forming Route in Clostridium acetobutylicum , 2012, mBio.

[31]  Irina Borodina,et al.  Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism , 2011, Biotechnology for biofuels.

[32]  Kwang Myung Cho,et al.  Extending carbon chain length of 1-butanol pathway for 1-hexanol synthesis from glucose by engineered Escherichia coli. , 2011, Journal of the American Chemical Society.

[33]  Michelle C. Y. Chang,et al.  Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. , 2011, Nature chemical biology.

[34]  J. Gapes,et al.  The acetone-butanol fermentation in pilot plant and pre-industrial scale. , 2000, Journal of molecular microbiology and biotechnology.

[35]  G. Stephanopoulos,et al.  Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes , 2009, Applied and Environmental Microbiology.

[36]  Dorota Grabowska,et al.  The ALD6 Gene Product Is Indispensable for Providing NADPH in Yeast Cells Lacking Glucose-6-phosphate Dehydrogenase Activity* , 2003, The Journal of Biological Chemistry.

[37]  Luis H. Reyes,et al.  Visualizing evolution in real time to determine the molecular mechanisms of n-butanol tolerance in Escherichia coli. , 2012, Metabolic engineering.

[38]  James C. Liao,et al.  Production of 2-methyl-1-butanol in engineered Escherichia coli , 2008, Applied Microbiology and Biotechnology.

[39]  Frances H Arnold,et al.  Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli. , 2011, Metabolic engineering.

[40]  D. Clarke,et al.  Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli. , 1985, European journal of biochemistry.

[41]  Kwang Myung Cho,et al.  Integrated Electromicrobial Conversion of CO2 to Higher Alcohols , 2012, Science.

[42]  James C Liao,et al.  Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde , 2009, Nature Biotechnology.

[43]  Rongrong Jiang,et al.  Enhancing E. coli isobutanol tolerance through engineering its global transcription factor cAMP receptor protein (CRP) , 2014, Biotechnology and bioengineering.

[44]  Masayuki Inui,et al.  Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation , 2013, Biotechnology and bioengineering.

[45]  Yong-Su Jin,et al.  Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering. , 2010, Journal of biotechnology.

[46]  F. Hui,et al.  Mesomorphic behaviour of some phospholipids with aliphatic alcohols and other non-ionic substances. , 1973, Biochimica et biophysica acta.

[47]  Blake A. Simmons,et al.  Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli , 2011, Proceedings of the National Academy of Sciences.

[48]  Xiaoxu Tian,et al.  Integrated OMICS guided engineering of biofuel butanol-tolerance in photosynthetic Synechocystis sp. PCC 6803 , 2013, Biotechnology for Biofuels.

[49]  E. Papoutsakis,et al.  Dissecting the assays to assess microbial tolerance to toxic chemicals in bioprocessing. , 2013, Trends in biotechnology.

[50]  T. Tajima,et al.  Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host , 2011, AMB Express.

[51]  Haishan Qi,et al.  Model-Driven Redox Pathway Manipulation for Improved Isobutanol Production in Bacillus subtilis Complemented with Experimental Validation and Metabolic Profiling Analysis , 2014, PloS one.

[52]  Jee Loon Foo,et al.  Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules , 2013, Biotechnology for Biofuels.

[53]  J. Weiner,et al.  Comparative proteomic and metabolomic analysis of Staphylococcus warneri SG1 cultured in the presence and absence of butanol. , 2013, Journal of proteome research.

[54]  Ramon Gonzalez,et al.  Engineered Respiro-Fermentative Metabolism for the Production of Biofuels and Biochemicals from Fatty Acid-Rich Feedstocks , 2010, Applied and Environmental Microbiology.

[55]  Yanhe Ma,et al.  Activating transhydrogenase and NAD kinase in combination for improving isobutanol production. , 2013, Metabolic engineering.

[56]  Laura R. Jarboe,et al.  Furfural Inhibits Growth by Limiting Sulfur Assimilation in Ethanologenic Escherichia coli Strain LY180 , 2009, Applied and Environmental Microbiology.

[57]  Shoshana D. Brown,et al.  Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily , 2012, Proceedings of the National Academy of Sciences.

[58]  J. Heap,et al.  The ClosTron: a universal gene knock-out system for the genus Clostridium. , 2007, Journal of microbiological methods.

[59]  S. Kashket,et al.  Maintenance of DpH by a butanol-tolerant mutant of Clostridium beijerinckii , 2004 .

[60]  J C Liao,et al.  A synthetic iterative pathway for ketoacid elongation. , 2011, Methods in enzymology.

[61]  M. Studer,et al.  Consolidated bioprocessing of lignocellulose by a microbial consortium , 2014 .

[62]  Harvey W Blanch,et al.  Bioprocessing for biofuels. , 2012, Current opinion in biotechnology.

[63]  Xueli Zhang,et al.  Metabolic Engineering for Production of Biorenewable Fuels and Chemicals: Contributions of Synthetic Biology , 2010, Journal of biomedicine & biotechnology.

[64]  Leland M. Vane,et al.  Hybrid vapor stripping–vapor permeation process for recovery and dehydration of 1‐butanol and acetone/butanol/ethanol from dilute aqueous solutions. Part 2. Experimental validation with simple mixtures and actual fermentation broth , 2013 .

[65]  Yinjie J. Tang,et al.  Metabolic Engineering of Synechocystis sp. Strain PCC 6803 for Isobutanol Production , 2012, Applied and Environmental Microbiology.

[66]  James C Liao,et al.  Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass , 2013, Proceedings of the National Academy of Sciences.

[67]  M. Dunlop.  Engineering microbes for tolerance to next-generation biofuels , 2011, Biotechnology for biofuels.

[68]  Y.‐H.P. Zhang,et al.  Analysis of biofuels production from sugar based on three criteria: Thermodynamics, bioenergetics, and product separation , 2011 .

[69]  James C. Liao,et al.  Engineering Corynebacterium glutamicum for isobutanol production , 2010, Applied Microbiology and Biotechnology.

[70]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[71]  James C. Liao,et al.  Expanding metabolism for biosynthesis of nonnatural alcohols , 2008, Proceedings of the National Academy of Sciences.

[72]  I. Yumoto,et al.  Isolation of Butanol- and Isobutanol-Tolerant Bacteria and Physiological Characterization of Their Butanol Tolerance , 2013, Applied and Environmental Microbiology.

[73]  William L. Luyben,et al.  Energy-saving distillation designs in ethanol production , 1988 .

[74]  D. Porro,et al.  A novel pathway to produce butanol and isobutanol in Saccharomyces cerevisiae , 2013, Biotechnology for Biofuels.

[75]  James C. Liao,et al.  Engineering of an Escherichia coli Strain for the Production of 3-Methyl-1-Butanol , 2008, Applied and Environmental Microbiology.

[76]  N. Qureshi,et al.  How microbes tolerate ethanol and butanol. , 2009, New biotechnology.

[77]  C. Tomas,et al.  Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum , 2004, Journal of bacteriology.

[78]  James C. Liao,et al.  ATP drives direct photosynthetic production of 1-butanol in cyanobacteria , 2012, Proceedings of the National Academy of Sciences.

[79]  L. Ingram,et al.  Alcohol-induced changes in the phospholipid molecular species of Escherichia coli , 1980, Journal of bacteriology.

[80]  James C. Liao,et al.  Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose , 2011, Applied and Environmental Microbiology.

[81]  Cong T. Trinh,et al.  Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol , 2011, Applied and Environmental Microbiology.

[82]  Costas D Maranas,et al.  Analysis of NADPH supply during xylitol production by engineered Escherichia coli , 2009, Biotechnology and bioengineering.

[83]  J. Pronk,et al.  Genome-scale analyses of butanol tolerance in Saccharomyces cerevisiae reveal an essential role of protein degradation , 2013, Biotechnology for Biofuels.

[84]  Volker F. Wendisch,et al.  Corynebacterium glutamicum Tailored for Efficient Isobutanol Production , 2011, Applied and Environmental Microbiology.

[85]  E. Papoutsakis,et al.  A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: From biofuels and chemicals, to biocatalysis and bioremediation. , 2010, Metabolic engineering.

[86]  J. Keasling,et al.  Engineering microbial biofuel tolerance and export using efflux pumps , 2011, Molecular systems biology.

[87]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[88]  Isobutanol production as an alternative metabolic sink to rescue the growth deficiency of the glycogen mutant of Synechococcus elongatus PCC 7942 , 2014, Photosynthesis Research.

[89]  Dong Gu Choi,et al.  Coordinated EV adoption: double-digit reductions in emissions and fuel use for $40/vehicle-year. , 2013, Environmental science & technology.

[90]  Michael C. Flickinger,et al.  Encyclopedia of bioprocess technology : fermentation, biocatalysis, and bioseparation , 1999 .

[91]  W. M. Ingledew,et al.  The acetone-butanol-ethanol fermentation. , 1988, Critical reviews in microbiology.

[92]  Patrick J. Evans,et al.  Enhancement of Butanol Formation by Clostridium acetobutylicum in the Presence of Decanol-Oleyl Alcohol Mixed Extractants , 1988, Applied and environmental microbiology.

[93]  G. Stephanopoulos,et al.  Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? , 2009, Nature Reviews Microbiology.

[94]  K. Trenberth,et al.  Modern Global Climate Change , 2003, Science.

[95]  J. Liao,et al.  High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal , 2011, Applied Microbiology and Biotechnology.

[96]  J. D. de Bont,et al.  Bacteria tolerant to organic solvents , 1998, Extremophiles.

[97]  Nasib Qureshi,et al.  Improving performance of a gas stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation , 2005, Bioprocess and biosystems engineering.

[98]  J. Liao,et al.  Pathway analysis, engineering, and physiological considerations for redirecting central metabolism. , 1996, Biotechnology and bioengineering.

[99]  Wolfgang Arlt,et al.  Separation of Azeotropic Mixtures Using Hyperbranched Polymers or Ionic Liquids , 2004 .

[100]  William L. Luyben,et al.  Control of the Heterogeneous Azeotropic n-Butanol/Water Distillation System , 2008 .

[101]  Jun Ishii,et al.  Genetic engineering to enhance the Ehrlich pathway and alter carbon flux for increased isobutanol production from glucose by Saccharomyces cerevisiae. , 2012, Journal of biotechnology.

[102]  P. Aakko-Saksa,et al.  Ethanol, isobutanol, and biohydrocarbons as gasoline components in relation to gaseous emissions and particulate matter. , 2014, Environmental science & technology.

[103]  Chengyan Zhang,et al.  Performance evaluation of acetone–butanol continuous flash extractive fermentation process , 2005, Bioprocess and biosystems engineering.

[104]  James C. Liao,et al.  Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes , 2009, Applied Microbiology and Biotechnology.

[105]  P. Gervais,et al.  Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses , 2001, Applied Microbiology and Biotechnology.

[106]  J. Houghton,et al.  Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda , 2006 .

[107]  Pao-Yang Chen,et al.  Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli , 2010, Molecular systems biology.

[108]  E. Papoutsakis,et al.  Metabolite stress and tolerance in the production of biofuels and chemicals: Gene‐expression‐based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum , 2010, Biotechnology and bioengineering.

[109]  U. Sauer,et al.  The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli* , 2004, Journal of Biological Chemistry.

[110]  Z. Dragovic,et al.  Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress , 2012, Biotechnology Letters.

[111]  James M Clomburg,et al.  Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals , 2011, Nature.

[112]  1-Butanol synthesis by Escherichia coli cells through butyryl-CoA formation by heterologous enzymes of clostridia and native enzymes of fatty acid β-oxidation , 2012, Applied Biochemistry and Microbiology.

[113]  L. Blank,et al.  Selected Pseudomonas putida Strains Able To Grow in the Presence of High Butanol Concentrations , 2009, Applied and Environmental Microbiology.

[114]  V. Zverlov,et al.  Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis , 2010, Applied Microbiology and Biotechnology.

[115]  Anthony J. Sinskey,et al.  Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha , 2012, Applied Microbiology and Biotechnology.

[116]  Christopher A. Voigt,et al.  Synthesis of methyl halides from biomass using engineered microbes. , 2009, Journal of the American Chemical Society.

[117]  Jean-Marie Rouillard,et al.  Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli , 2011, Microbial cell factories.

[118]  J. Nielsen,et al.  Transcriptional control of metabolic fluxes , 2011, Molecular systems biology.

[119]  R. Srivastava,et al.  Performance of batch, fed-batch, and continuous A-B-E fermentation with pH-control. , 2011, Bioresource technology.

[120]  R. Datta,et al.  Production of butanol and ethanol from synthesis gas via fermentation , 1991 .

[121]  Christer Larsson,et al.  Physiological adaptations of Saccharomyces cerevisiae evolved for improved butanol tolerance , 2013, Biotechnology for Biofuels.

[122]  James C. Liao,et al.  Directed Evolution of Methanococcus jannaschii Citramalate Synthase for Biosynthesis of 1-Propanol and 1-Butanol by Escherichia coli , 2008, Applied and Environmental Microbiology.

[123]  G. Stephanopoulos,et al.  Engineering Yeast Transcription Machinery for Improved Ethanol Tolerance and Production , 2006, Science.

[124]  Jens Nielsen,et al.  Improving biobutanol production in engineered Saccharomyces cerevisiae by manipulation of acetyl-CoA metabolism , 2013, Journal of Industrial Microbiology & Biotechnology.

[125]  G. Unden,et al.  Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. , 1997, Biochimica et biophysica acta.

[126]  Xiaoxu Tian,et al.  Global metabolomic and network analysis of Escherichia coli responses to exogenous biofuels. , 2013, Journal of proteome research.

[127]  Danielle Tullman-Ercek,et al.  Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol. , 2014, ACS synthetic biology.

[128]  Alyssa M. Redding,et al.  Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol , 2008, Microbial cell factories.

[129]  Thomas Wallner,et al.  Analytical Assessment of C2–C8 Alcohols as Spark-Ignition Engine Fuels , 2013 .

[130]  Sang Woo Seo,et al.  Model-driven rebalancing of the intracellular redox state for optimization of a heterologous n-butanol pathway in Escherichia coli. , 2013, Metabolic engineering.

[131]  Weihong Jiang,et al.  Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio. , 2009, Metabolic engineering.

[132]  Luis H. Reyes,et al.  Genomic Library Screens for Genes Involved in n-Butanol Tolerance in Escherichia coli , 2011, PloS one.

[133]  J. Nielsen,et al.  Advanced biofuel production by the yeast Saccharomyces cerevisiae. , 2013, Current opinion in chemical biology.

[134]  Min Lin,et al.  Laboratory-Evolved Mutants of an Exogenous Global Regulator, IrrE from Deinococcus radiodurans, Enhance Stress Tolerances of Escherichia coli , 2011, PloS one.

[135]  E. Papoutsakis,et al.  Dynamics of Genomic-Library Enrichment and Identification of Solvent Tolerance Genes for Clostridium acetobutylicum , 2007, Applied and Environmental Microbiology.

[136]  A. C. O'sullivan.  Cellulose: the structure slowly unravels , 1997, Cellulose.

[137]  P. Huang,et al.  The effects of alcohols on lipid bilayers: a spin label study. , 1972, Biochimica et biophysica acta.

[138]  Ye Sun,et al.  Hydrolysis of lignocellulosic materials for ethanol production: a review. , 2002, Bioresource technology.

[139]  L. Lynd,et al.  Metabolic engineering of Thermoanaerobacterium saccharolyticum for n-butanol production. , 2014, Metabolic engineering.

[140]  C. Tomas,et al.  Overexpression of groESL in Clostridium acetobutylicum Results in Increased Solvent Production and Tolerance, Prolonged Metabolism, and Changes in the Cell's Transcriptional Program , 2003, Applied and Environmental Microbiology.

[141]  Rubens Maciel Filho,et al.  Butanol production in a first-generation Brazilian sugarcane biorefinery: technical aspects and economics of greenfield projects. , 2013, Bioresource technology.

[142]  Peter Dürre,et al.  Fermentative production of butanol--the academic perspective. , 2011, Current opinion in biotechnology.

[143]  Jay D. Keasling,et al.  Functional Genomic Study of Exogenous n-Butanol Stress in Escherichia coli , 2010, Applied and Environmental Microbiology.

[144]  Xiaoqiang Jia,et al.  Engineering Bacillus subtilis for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression , 2011, Applied Microbiology and Biotechnology.

[145]  Diego Barba,et al.  Hyperazeotropic ethanol salted-out by extractive distillation. Theoretical evaluation and experimental check , 1985 .

[146]  Jun Ishii,et al.  Increased isobutanol production in Saccharomyces cerevisiae by eliminating competing pathways and resolving cofactor imbalance , 2013, Microbial Cell Factories.

[147]  R. P. Ross,et al.  Improved Stress Tolerance of GroESL-Overproducing Lactococcus lactis and Probiotic Lactobacillus paracasei NFBC 338 , 2004, Applied and Environmental Microbiology.

[148]  J. Liao,et al.  Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels , 2008, Nature.

[149]  Leland M. Vane,et al.  Separation technologies for the recovery and dehydration of alcohols from fermentation broths , 2008 .

[150]  Luis H. Reyes,et al.  Genetic Determinants for n-Butanol Tolerance in Evolved Escherichia coli Mutants: Cross Adaptation and Antagonistic Pleiotropy between n-Butanol and Other Stressors , 2013, Applied and Environmental Microbiology.

[151]  Christian Weber,et al.  Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae , 2012, Biotechnology for Biofuels.

[152]  M. Tyurin,et al.  Genome tailoring powered production of isobutanol in continuous CO2/H2 blend fermentation using engineered acetogen biocatalyst , 2014, Journal of Industrial Microbiology & Biotechnology.

[153]  Ana Segura,et al.  Mechanisms of solvent tolerance in gram-negative bacteria. , 2002, Annual review of microbiology.

[154]  G. Stephanopoulos,et al.  Compartmentalization of metabolic pathways in yeast mitochondria improves production of branched chain alcohols , 2013, Nature Biotechnology.

[155]  J. Liao,et al.  Microbial synthesis of n-butanol, isobutanol, and other higher alcohols from diverse resources. , 2013, Bioresource technology.

[156]  Hans P. Blaschek,et al.  Effect of Butanol Challenge and Temperature on Lipid Composition and Membrane Fluidity of Butanol-Tolerant Clostridium acetobutylicum , 1987, Applied and environmental microbiology.

[157]  M. Turner,et al.  Correlation of biocatalytic activity in an organic-aqueous two-liquid phase system with solvent concentration in the cell membrane. , 1990, Enzyme and microbial technology.

[158]  Ryan T Gill,et al.  Strategy for directing combinatorial genome engineering in Escherichia coli , 2012, Proceedings of the National Academy of Sciences.

[159]  J. Liao,et al.  Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways. , 2008, Metabolic engineering.

[160]  Heikki Ojamo,et al.  Biobutanol: the outlook of an academic and industrialist , 2013 .

[161]  M. Seeger,et al.  Site-Directed Mutagenesis Reveals Putative Substrate Binding Residues in the Escherichia coli RND Efflux Pump AcrB , 2008, Journal of bacteriology.

[162]  K. Ch. A. M. Luyben,et al.  In situ product recovery by adsorption in the butanol/isopropanol batch fermentation , 1986, Applied Microbiology and Biotechnology.

[163]  D. Das,et al.  Development of suitable photobioreactors for CO2 sequestration addressing global warming using green algae and cyanobacteria. , 2011, Bioresource technology.

[164]  Yajun Yan,et al.  Conversion of proteins into biofuels by engineering nitrogen flux , 2011, Nature Biotechnology.

[165]  H. Blaschek,et al.  Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101 , 1999, Applied Microbiology and Biotechnology.

[166]  Alexander J. Ninfa,et al.  Activation of the glnA, glnK, and nac Promoters as Escherichia coli Undergoes the Transition from Nitrogen Excess Growth to Nitrogen Starvation , 2002, Journal of bacteriology.

[167]  K. Prather,et al.  Engineering alternative butanol production platforms in heterologous bacteria. , 2009, Metabolic Engineering.

[168]  C. Lange,et al.  Ploidy in cyanobacteria. , 2011, FEMS microbiology letters.

[169]  Hans P. Blaschek,et al.  Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth , 1983, Applied and environmental microbiology.

[170]  Xiaobin Yu,et al.  Repetitive domestication to enhance butanol tolerance and production in Clostridium acetobutylicum through artificial simulation of bio-evolution. , 2013, Bioresource technology.

[171]  J. Liao,et al.  An integrated network approach identifies the isobutanol response network of Escherichia coli , 2009, Molecular systems biology.

[172]  J. Liao,et al.  Improvement of isopropanol production by metabolically engineered Escherichia coli using gas stripping. , 2010, Journal of bioscience and bioengineering.

[173]  Huimin Zhao,et al.  Utilizing an endogenous pathway for 1-butanol production in Saccharomyces cerevisiae. , 2014, Metabolic engineering.

[174]  Tina Lütke-Eversloh,et al.  Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum , 2013, Biotechnology and bioengineering.

[175]  Yong-Su Jin,et al.  Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes , 2012, Bioprocess and Biosystems Engineering.

[176]  Jean-Michel Lavoie,et al.  From first- to third-generation biofuels: Challenges of producing a commodity from a biomass of increasing complexity , 2013 .

[177]  J. Li,et al.  Screening and characterization of butanol‐tolerant micro‐organisms , 2010, Letters in applied microbiology.

[178]  J. Liao,et al.  A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols. , 2012, Metabolic engineering.

[179]  T. Hermann.  Industrial production of amino acids by coryneform bacteria. , 2003, Journal of biotechnology.

[180]  L. T. Angenent,et al.  Biocatalytic reduction of short‐chain carboxylic acids into their corresponding alcohols with syngas fermentation , 2013, Biotechnology and bioengineering.

[181]  A. Wolfe,et al.  Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids , 1994, Journal of bacteriology.

[182]  L. Ingram.  Changes in lipid composition of Escherichia coli resulting from growth with organic solvents and with food additives , 1977, Applied and environmental microbiology.

[183]  Yanning Zheng,et al.  Problems with the microbial production of butanol , 2009, Journal of Industrial Microbiology & Biotechnology.

[184]  J. C. Liao,et al.  Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli , 2007, Applied and Environmental Microbiology.

[185]  Min Zhang,et al.  Butanol Tolerance in a Selection of Microorganisms , 2009, Applied biochemistry and biotechnology.

[186]  Philippe Soucaille,et al.  Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture. , 2013, Metabolic engineering.

[187]  H. Bahl,et al.  Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production. , 2011, Current opinion in biotechnology.