title: Efficient whole-cell biocatalyst for acetoin production with NAD+ regeneration system through homologous co-expression of 2,3-butanediol dehydrogenase and NADH oxidase in engineered Bacillus subtilis.
abstract: Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD+ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products.
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Teng
authors.initials: T
authors.lastname: Bao
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Zhao
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Rongzhen
authors.initials: R
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Taowei
authors.initials: T
authors.lastname: Yang
authors.affiliation: School of Medicine and Pharmaceuticals, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Chemical Engineering, Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Shangtian
authors.initials: S
authors.lastname: Yang
mesh_terms: D000093:Acetoin / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D001412:Bacillus subtilis / Q000378:metabolism*; D055162:Biocatalysis; D002072:Butylene Glycols / Q000378:metabolism*; D005285:Fermentation / Q000502:physiology; D060847:Metabolic Engineering / Q000379:methods; D009097:Multienzyme Complexes / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D012038:Regeneration / Q000502:physiology*
chemical_list: D002072:Butylene Glycols; D009097:Multienzyme Complexes; D009243:NAD; C026978:2,3-butylene glycol; D000093:Acetoin; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases