Engineering Clostridial Aldehyde/Alcohol Dehydrogenase for Selective Butanol Production

Renewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineered Clostridium acetobutylicum strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity. ABSTRACT Butanol production by Clostridium acetobutylicum is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased butanol selectivity by a series of design and analysis procedures, including random mutagenesis, substrate specificity feature analysis, and structure-based butanol selectivity design. The butanol/ethanol ratios (B/E ratios) were dramatically increased to 17.47 and 15.91 g butanol/g ethanol for AADF716L and AADN655H, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD. The much-increased B/E ratio obtained was due to the dramatic reduction in ethanol production (0.59 ± 0.01 g/liter) that resulted from engineering the substrate binding chamber and the active site of AAD. This protein design strategy can be applied generally for engineering enzymes to alter substrate selectivity. IMPORTANCE Renewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineered Clostridium acetobutylicum strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity.

[1]  D. R. Woods,et al.  Acetone-butanol fermentation revisited , 1986 .

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

[3]  E. Papoutsakis,et al.  Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824 , 1994, Journal of bacteriology.

[4]  P. Rellos,et al.  Alteration of substrate specificity of Zymomonas mobilis alcohol dehydrogenase-2 using in vitro random mutagenesis. , 1997, Protein expression and purification.

[5]  P. Soucaille,et al.  Molecular Characterization and Transcriptional Analysis of adhE2, the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824 , 2002, Journal of bacteriology.

[6]  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.

[7]  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.

[8]  Gregory D. Schuler,et al.  Database resources of the National Center for Biotechnology Information: update , 2004, Nucleic acids research.

[9]  U. Baumann,et al.  An efficient one-step site-directed and site-saturation mutagenesis protocol. , 2004, Nucleic acids research.

[10]  P. Dürre.  Biobutanol: An attractive biofuel , 2007, Biotechnology journal.

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

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

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

[14]  E. Papoutsakis.  Engineering solventogenic clostridia. , 2008, Current opinion in biotechnology.

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

[16]  E. Papoutsakis,et al.  Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance. , 2008, Metabolic engineering.

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

[18]  P. Dürre.  Fermentative Butanol Production , 2008, Annals of the New York Academy of Sciences.

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

[20]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

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

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

[23]  Y. Jang,et al.  Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of Clostridium pasteurianum , 2011, Applied Microbiology and Biotechnology.

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

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

[26]  Evan E. Bolton,et al.  Database resources of the National Center for Biotechnology Information , 2011, Nucleic acids research.

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

[28]  Y. Jang,et al.  Butanol production from renewable biomass by clostridia. , 2012, Bioresource technology.

[29]  Shangtian Yang,et al.  Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production , 2014, Applied Microbiology and Biotechnology.

[30]  Sang Yup Lee,et al.  Recent advances in microbial production of fuels and chemicals using tools and strategies of systems metabolic engineering. , 2015, Biotechnology advances.

[31]  R. Phillips,et al.  Controlling Substrate Specificity and Stereospecificity of Alcohol Dehydrogenases , 2015 .

[32]  Weihong Jiang,et al.  Current status and prospects of industrial bio-production of n-butanol in China. , 2015, Biotechnology advances.

[33]  Ex situ product recovery and strain engineering of Clostridium acetobutylicum for enhanced production of butanol , 2015 .

[34]  Jae Sung Cho,et al.  Systems Metabolic Engineering of Escherichia coli , 2016, EcoSal Plus.

[35]  Yanping Zhang,et al.  Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum , 2016, Scientific Reports.

[36]  Y. Jang,et al.  One hundred years of clostridial butanol fermentation. , 2016, FEMS microbiology letters.

[37]  P. Soucaille,et al.  Elucidation of the roles of adhE1 and adhE2 in the primary metabolism of Clostridium acetobutylicum by combining in-frame gene deletion and a quantitative system-scale approach , 2016, Biotechnology for Biofuels.

[38]  Yanping Zhang,et al.  A systematically chromosomally engineered Escherichia coli efficiently produces butanol. , 2017, Metabolic engineering.

[39]  S. Lee,et al.  Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs , 2017, Biotechnology and bioengineering.