Wang, Qinghui; Jiang, Weiyan; Cai, Yaoyu; Tisma, Marina; Baganz, Frank; Shi, Jiping; Lye, Gary J.; Xiang, Wensheng; Hao, Jian

ENZYME AND MICROBIAL TECHNOLOGY

2024, 172, 1879-0909

Wang, Qinghui; Jiang, Weiyan; Cai, Yaoyu; Tisma, Marina; Baganz, Frank; Shi, Jiping; Lye, Gary J.; Xiang, Wensheng; Hao, Jian

2-Hydroxyisovalerate is a valuable chemical that can be used in the production of biodegradable polyesters. In nature, it was only produced at a very low level by Lactococcus lactis. 2-Ketoisovalerate is an intermediate metabolite of the branched-chain amino acid biosynthesis pathway, and Klebsiella pneumoniae Delta budA Delta ldhA (Kp Delta budA Delta ldhA) was a 2-ketoisovalerate producing strain. In this research, 2-hydroxyisovalerate was identified as a metabolite of Kp Delta budA Delta ldhA, and its synthesis pathway was revealed. It was found that 2-ketoisovalerate and 2-hydroxyisovalerate were produced by Kp Delta budA and Kp Delta budA Delta ldhA, but not by Kp Delta budA Delta ldhA Delta ilvD in which the 2-ketoisovalerate synthesis was blocked. budA, ldhA, and ilvD encode alpha-acetolactate decarboxylase, lactate dehydrogenase, and dihydroxy acid dehydratase, respectively. Thus, it was deduced that 2-hydroxyisovalerate was synthesized from 2-ketoisovalerate. Isoenzymes of ketopantoate reductase PanE, PanE2, and IlvC were suspected of being responsible for this reaction. Kinetic parameters of these enzymes were detected, and they all hold the 2-ketoisovalerate reductase activities. PanE and PanE2 use both NADH and NADPH as co-factors. While IlvC only uses NADH as a co-factor. Over-expression of panE, panE2, or ilvC in Kp Delta budA Delta ldhA all enhanced the production of 2-hydroxyisovalerate. Accordingly, 2-hydroxyisovalerate levels were reduced by knocking out panE or panE2. In fed-batch fermentation, 14.41 g/L of 2-hydroxyisovalerate was produced by Kp Delta budA Delta ldhA-panE, with a substrate conversion ratio of 0.13 g/g glucose.