2-Hydroxyisovalerate production by Klebsiella pneumoniae

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

doi:10.1016/j.enzmictec.2023.110330

2-Hydroxyisovalerate production by Klebsiella pneumoniae

Enzyme Microb Technol. 2024 Jan:172:110330. doi: 10.1016/j.enzmictec.2023.110330. Epub 2023 Sep 29.

Authors

Qinghui Wang¹, Weiyan Jiang², Yaoyu Cai², Marina Tišma³, Frank Baganz⁴, Jiping Shi⁵, Gary J Lye⁴, Wensheng Xiang⁶, Jian Hao⁷

Affiliations

¹ Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai 201210, People's Republic of China; Key Laboratory of Agricultural Microbiology of Heilongjiang Province, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin 150030, People's Republic of China.
² Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai 201210, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
³ Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhača 18, Osijek HR-31000, Croatia.
⁴ Department of Biochemical Engineering, University College London, Gordon Street, London WC1H 0AH, UK.
⁵ Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai 201210, People's Republic of China.
⁶ Key Laboratory of Agricultural Microbiology of Heilongjiang Province, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin 150030, People's Republic of China.
⁷ Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai 201210, People's Republic of China; Department of Biochemical Engineering, University College London, Gordon Street, London WC1H 0AH, UK; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China. Electronic address: haoj@sari.ac.cn.

PMID: 37866134
DOI: 10.1016/j.enzmictec.2023.110330

Abstract

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 ΔbudAΔldhA (Kp ΔbudAΔldhA) was a 2-ketoisovalerate producing strain. In this research, 2-hydroxyisovalerate was identified as a metabolite of Kp ΔbudAΔldhA, and its synthesis pathway was revealed. It was found that 2-ketoisovalerate and 2-hydroxyisovalerate were produced by Kp ΔbudA and Kp ΔbudAΔldhA, but not by Kp ΔbudAΔldhAΔilvD in which the 2-ketoisovalerate synthesis was blocked. budA, ldhA, and ilvD encode α-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 ΔbudAΔldhA 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 ΔbudAΔldhA-panE, with a substrate conversion ratio of 0.13 g/g glucose.

Keywords: Klebsiella pneumoniae·2-Hydroxyisovalerate·2-Ketoisovalerate·panE·ilvC.

MeSH terms

Keto Acids / metabolism
Klebsiella pneumoniae* / genetics
Klebsiella pneumoniae* / metabolism
NAD* / metabolism

Substances

alpha-ketoisovalerate
2-hydroxyisovaleric acid
NAD
Keto Acids