NADH plays the vital role for chiral pure D-(-)-2,3-butanediol production in Bacillus subtilis under limited oxygen conditions.
Compared with traditional pathogenic producers, Bacillus subtilis as a Class I microorganism offers many advantages for industrial-scale 2,3-butanediol production. Unlike previous reports in which two stereoisomers (with a ratio of 3:2) were produced, we first found that wild type B. subtilis 168 generates only D-(-)-2,3-butanediol (purity >99%) under low oxygen conditions. The total high yield of 2,3-butanediol and acetoin, and acetoin reductase enzyme assay indicate that it is the high level of NADH availability, instead of high acetoin reductase activity, contributes more to 2,3-butanediol production in B. subtilis. The strategy for increasing the pool of NADH availability, the key factor for 2,3-butanediol production, was designed through low dissolved oxygen control, adding reducing substrates and rationally metabolic engineering. A transhydrogenase encoded by udhA was introduced to provide more NADH from NADPH and allowed enhanced 2,3-butanediol production. Finally, BSF20 produced 49.29 g/L D(-)-2,3-butanediol. These results demonstrated that B. subtilis is a competitive producer for chiral 2,3-butanediol production.
School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China; Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, 300072, People's Republic of China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, 300072, People's Republic of China.
Jing
J
Fu
Zhiwen
Z
Wang
Tao
T
Chen
Weixi
W
Liu
Ting
T
Shi
Guanglu
G
Wang
Ya-jie
YJ
Tang
Xueming
X
Zhao
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D002072:Butylene Glycols / Q000737:chemistry / Q000378:metabolism*; D007218:Industrial Microbiology; D060847:Metabolic Engineering; D009243:NAD / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D013237:Stereoisomerism
D002072:Butylene Glycols; D009243:NAD; C026978:2,3-butylene glycol; D009250:NADP Transhydrogenases; D010100:Oxygen
Bacillus subtilis; D-(−)-2,3-butanediol; UdhA; acetoin reductase; cofactor engineering; metabolic engineering