Wang, Qinghui; Gu, Jinjie; Shu, Lin; Jiang, Weiyan; Mojovic, Ljiljana; Knezevic-Jugovic, Zorica; Shi, Jiping; Baganz, Frank; Lye, Gary J.; Xiang, Wensheng; Hao, Jian

WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY

2022, 38, 0959-3993

Wang, Qinghui; Gu, Jinjie; Shu, Lin; Jiang, Weiyan; Mojovic, Ljiljana; Knezevic-Jugovic, Zorica; Shi, Jiping; Baganz, Frank; Lye, Gary J.; Xiang, Wensheng; Hao, Jian

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of l-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of alpha-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the l-valine production. The budA coded an alpha-acetolactate decarboxylase and catalyzed the acetoin formation from alpha-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the l-valine synthesis. 2-Ketoisovalerate is the precursor of l-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of l-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to alpha-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of l-valine production. The brnQ encodes a branched-chain amino acid transporter, and l-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor l-valine production. Based on these findings, l-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4 g/L of l-valine was produced by the engineered K. pneumoniae Delta budA-Delta ldhA-Delta ipdC-Delta brnQ-ilvE after 55 h of cultivation, with a substrate conversion ratio of 0.27 mol/mol glucose.