Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

[1]  Herbert J. Strobel,et al.  Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum , 2007, Applied Microbiology and Biotechnology.

[2]  Sue E. Nokes,et al.  Analysis of composition and structure of Clostridium thermocellum membranes from wild-type and ethanol-adapted strains , 2009, Applied Microbiology and Biotechnology.

[3]  Johannes Söding,et al.  The HHpred interactive server for protein homology detection and structure prediction , 2005, Nucleic Acids Res..

[4]  M. Parrinello,et al.  Polymorphic transitions in single crystals: A new molecular dynamics method , 1981 .

[5]  H. Berendsen,et al.  Molecular dynamics with coupling to an external bath , 1984 .

[6]  Hoover,et al.  Canonical dynamics: Equilibrium phase-space distributions. , 1985, Physical review. A, General physics.

[7]  Gwo-Liang Chen,et al.  Improved genome annotation for Zymomonas mobilis , 2009, Nature Biotechnology.

[8]  Carsten Kutzner,et al.  GROMACS 4:  Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. , 2008, Journal of chemical theory and computation.

[9]  J. Zeikus,et al.  Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum , 1984, Applied and environmental microbiology.

[10]  Ying Xu,et al.  DOOR: a database for prokaryotic operons , 2008, Nucleic Acids Res..

[11]  L. Lynd,et al.  End-product pathways in the xylose fermenting bacterium, Thermoanaerobacterium saccharolyticum , 2008 .

[12]  SödingJohannes.  Protein homology detection by HMM--HMM comparison , 2005 .

[13]  Richard Sparling,et al.  Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405. , 2009, Journal of biotechnology.

[14]  L. Lynd,et al.  Consolidated bioprocessing of cellulosic biomass: an update. , 2005, Current opinion in biotechnology.

[15]  W. H. Zyl,et al.  Engineering cellulolytic ability into bioprocessing organisms , 2010, Applied Microbiology and Biotechnology.

[16]  Lee R. Lynd,et al.  Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant , 2010, Applied and Environmental Microbiology.

[17]  Jack Snoeyink,et al.  Nucleic Acids Research Advance Access published April 22, 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids , 2007 .

[18]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[19]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[20]  J. Zeikus,et al.  Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase. , 1994, The Biochemical journal.

[21]  Yang Zhang,et al.  I-TASSER: a unified platform for automated protein structure and function prediction , 2010, Nature Protocols.

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

[23]  Jianpeng Ma,et al.  CHARMM: The biomolecular simulation program , 2009, J. Comput. Chem..

[24]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[25]  Ben M. Webb,et al.  Comparative Protein Structure Modeling Using MODELLER , 2007, Current protocols in protein science.

[26]  A. Aubry,et al.  A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+. , 1997, Journal of molecular biology.

[27]  A. Sali,et al.  Modeling of loops in protein structures , 2000, Protein science : a publication of the Protein Society.

[28]  P Argos,et al.  NADP‐Dependent enzymes. I: Conserved stereochemistry of cofactor binding , 1997, Proteins.

[29]  I. S. Pretorius,et al.  Microbial Cellulose Utilization: Fundamentals and Biotechnology , 2002, Microbiology and Molecular Biology Reviews.

[30]  A. Herrero,et al.  Development of ethanol tolerance in Clostridium thermocellum: effect of growth temperature , 1980, Applied and environmental microbiology.

[31]  Slawomir K. Grzechnik,et al.  Crystal structure of an iron‐containing 1,3‐propanediol dehydrogenase (TM0920) from Thermotoga maritima at 1.3 Å resolution , 2003, Proteins.

[32]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

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

[34]  Dale A Pelletier,et al.  Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae , 2010, Proceedings of the National Academy of Sciences.

[35]  Genia Dubrovsky,et al.  Deletion of the Cel48S cellulase from Clostridium thermocellum , 2010, Proceedings of the National Academy of Sciences.

[36]  Francisco J. Enguita,et al.  1,3-Propanediol Dehydrogenase from Klebsiella pneumoniae: Decameric Quaternary Structure and Possible Subunit Cooperativity , 2008, Journal of bacteriology.

[37]  Johannes Söding,et al.  The MPI Bioinformatics Toolkit for protein sequence analysis , 2006, Nucleic Acids Res..

[38]  A. Sali,et al.  Statistical potential for assessment and prediction of protein structures , 2006, Protein science : a publication of the Protein Society.

[39]  Brian G. Fox,et al.  Global Gene Expression Patterns in Clostridium thermocellum as Determined by Microarray Analysis of Chemostat Cultures on Cellulose or Cellobiose , 2010, Applied and Environmental Microbiology.

[40]  P. Dürre,et al.  Enzymatic investigations on butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum , 1987, Applied Microbiology and Biotechnology.

[41]  S. Nosé,et al.  Constant pressure molecular dynamics for molecular systems , 1983 .

[42]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[43]  G. Seenayya,et al.  Improved ethanol tolerance and production in strains of Clostridium thermocellum , 1996, World journal of microbiology & biotechnology.

[44]  J. Zeikus,et al.  Ethanol Production by Thermophilic Bacteria: Relationship Between Fermentation Product Yields of and Catabolic Enzyme Activities in Clostridium thermocellum and Thermoanaerobium brockii , 1980, Journal of bacteriology.

[45]  G. Stephanopoulos.  Challenges in Engineering Microbes for Biofuels Production , 2007, Science.

[46]  L. Ingram,et al.  Effects of alcohols on micro-organisms. , 1984, Advances in microbial physiology.

[47]  J. Zeikus,et al.  Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum , 1988, Journal of bacteriology.

[48]  A. Sali,et al.  Comparative protein structure modeling of genes and genomes. , 2000, Annual review of biophysics and biomolecular structure.

[49]  T. Darden,et al.  A smooth particle mesh Ewald method , 1995 .

[50]  M. Tomaa,et al.  Respiratory behaviour of a Zymomonas mobilis adhB::kan mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions , 2006 .

[51]  Ignacio Fita,et al.  Complete Reversal of Coenzyme Specificity by Concerted Mutation of Three Consecutive Residues in Alcohol Dehydrogenase* , 2003, Journal of Biological Chemistry.

[52]  C. Lim,et al.  Empirical force fields for biologically active divalent metal cations in water. , 2006, The journal of physical chemistry. A.

[53]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

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

[55]  R. Poole,et al.  Respiratory behaviour of a Zymomonas mobilis adhB::kan r mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions , 2006, FEBS letters.

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

[57]  S. Nosé.  A molecular dynamics method for simulations in the canonical ensemble , 1984 .