The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis.

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639g/(lh).

[1]  A Danchin,et al.  Analysis of a Bacillus subtilis genome fragment using a co-operative computer system prototype. , 1995, Gene.

[2]  Z. Rao,et al.  Mutation breeding of acetoin high producing Bacillus subtilis blocked in 2,3-butanediol dehydrogenase , 2013, World journal of microbiology & biotechnology.

[3]  A. Goffeau,et al.  The complete genome sequence of the Gram-positive bacterium Bacillus subtilis , 1997, Nature.

[4]  He Huang,et al.  Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae , 2013, Biotechnology for Biofuels.

[5]  D. Weuster‐Botz,et al.  Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability , 2012, Applied Microbiology and Biotechnology.

[6]  S. Atsumi,et al.  Cyanobacterial conversion of carbon dioxide to 2,3-butanediol , 2013, Proceedings of the National Academy of Sciences.

[7]  B. Rao,et al.  Enhanced acetoin production by Serratia marcescens H32 using statistical optimization and a two-stage agitation speed control strategy , 2012, Biotechnology and Bioprocess Engineering.

[8]  W. D. de Vos,et al.  Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus. , 2003, European journal of biochemistry.

[9]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[10]  J. Coppee,et al.  Genetic analysis and overexpression of lipolytic activity in Bacillus subtilis , 1994, Applied and environmental microbiology.

[11]  G. Suzzi,et al.  Acetoin production in Saccharomyces cerevisiae wine yeasts. , 1993, FEMS microbiology letters.

[12]  E. Cabiscol,et al.  Oxidative stress in bacteria and protein damage by reactive oxygen species. , 2000, International microbiology : the official journal of the Spanish Society for Microbiology.

[13]  Jacky L. Snoep,et al.  Effect of the energy source on the NADH/NAD ratio and on pyruvate catabolism in anaerobic chemostat cultures of Enterococcus faecalis NCTC 775 , 1991 .

[14]  V. Massey,et al.  Purification and properties of milk xanthine dehydrogenase. , 1992, The Journal of biological chemistry.

[15]  Jinwon Lee,et al.  Selective Production of 2,3-Butanediol and Acetoin by a Newly Isolated Bacterium Klebsiella oxytoca M1 , 2013, Applied Biochemistry and Biotechnology.

[16]  M. Kleerebezem,et al.  Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase , 1998, Journal of bacteriology.

[17]  M. Nakano,et al.  Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth , 1997, Journal of bacteriology.

[18]  Liao-Yuan Zhang,et al.  Enhanced acetoin production by Serratia marcescens H32 with expression of a water-forming NADH oxidase. , 2012, Bioresource technology.

[19]  Z. Rao,et al.  Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation , 2011 .

[20]  V. Phalip,et al.  Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides , 1997, Applied Microbiology and Biotechnology.

[21]  Koichi Tanaka,et al.  New reduction method of α-diketones, oxo amides, and quinones with Zn–EtOH in the presence of a salt , 1989 .

[22]  G. Bennett,et al.  Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. , 2002, Metabolic engineering.

[23]  J. Gao,et al.  Production of S‐acetoin from diacetyl by Escherichia coli transformant cells that express the diacetyl reductase gene of Paenibacillus polymyxa ZJ‐9 , 2013, Letters in applied microbiology.

[24]  J. Hugenholtz,et al.  Purification and characterisation of the water forming NADH-oxidase from Lactococcus lactis , 2001 .

[25]  A. Neves,et al.  High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling , 2011, Applied and Environmental Microbiology.

[26]  J. Rydström.  Assay of nicotinamide nucleotide transhydrogenases in mammalian, bacterial, and reconstituted systems. , 1979, Methods in enzymology.

[27]  P. Loubière,et al.  New insights into Lactococcus lactis diacetyl- and acetoin-producing strains isolated from diverse origins. , 2013, International journal of food microbiology.

[28]  Deokjin Jahng,et al.  Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes , 2013, Applied Microbiology and Biotechnology.

[29]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[30]  Manuel Liebeke,et al.  Regulation of acetoin and 2,3-butanediol utilization in Bacillus licheniformis , 2010, Applied Microbiology and Biotechnology.

[31]  P. Xu,et al.  Acetoin Metabolism in Bacteria , 2007, Critical reviews in microbiology.

[32]  P. Xu,et al.  Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate , 2007, Applied Microbiology and Biotechnology.

[33]  Akihiko Kondo,et al.  Enhanced production of 2,3-butanediol by engineered Bacillus subtilis , 2011, Applied Microbiology and Biotechnology.

[34]  W. Nicholson.  The Bacillus subtilis ydjL (bdhA) Gene Encodes Acetoin Reductase/2,3-Butanediol Dehydrogenase , 2008, Applied and Environmental Microbiology.

[35]  D. Webster,et al.  Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene , 2004, Bioprocess and biosystems engineering.

[36]  Z. Rao,et al.  Moderate expression of the transcriptional regulator ALsR enhances acetoin production by Bacillus subtilis , 2013, Journal of Industrial Microbiology & Biotechnology.

[37]  Cuiqing Ma,et al.  Efficient bioconversion of 2,3-butanediol into acetoin using Gluconobacter oxydans DSM 2003 , 2013, Biotechnology for Biofuels.