Directed evolution of leucine dehydrogenase for improved efficiency of L-tert-leucine synthesis

Applied Microbiology and Biotechnology
Lin ZhuShuang-Yan Tang

Abstract

L-tert-Leucine and its derivatives are used as synthetic building blocks for pharmaceutical active ingredients, chiral auxiliaries, and ligands. Leucine dehydrogenase (LeuDH) is frequently used to prepare L-tert-leucine from the α-keto acid precursor trimethylpyruvate (TMP). In this study, a high-throughput screening method for the L-tert-leucine synthesis reaction based on a spectrophotometric approach was developed. Directed evolution strategy was applied to engineer LeuDH from Lysinibacillus sphaericus for improved efficiency of L-tert-leucine synthesis. After two rounds of random mutagenesis, the specific activity of LeuDH on the substrate TMP was enhanced by more than two-fold, compared with that of the wild-type enzyme, while the activity towards its natural substrate, leucine, decreased. The catalytic efficiencies (k cat/K m) of the best mutant enzyme, H6, on substrates TMP and NADH were all enhanced by more than five-fold as compared with that of the wild-type enzyme. The efficiency of L-tert-leucine synthesis by mutant H6 was significantly improved. A productivity of 1170 g/l/day was achieved for the mutant enzyme H6, compared with 666 g/l/day for the wild-type enzyme.

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Citations

Jul 31, 2018·Expert Opinion on Drug Metabolism & Toxicology·Shenglei YanJingqing Zhang
Feb 28, 2020·Applied Microbiology and Biotechnology·Jun YaoShuang-Yan Tang
Jan 25, 2018·Chemical Society Reviews·Ya-Ping XueYu-Guo Zheng
Apr 10, 2019·World Journal of Microbiology & Biotechnology·Tanja NarancicKevin E O'Connor
Apr 27, 2019·International Journal of Molecular Sciences·Ying-Yu WangLi-Ming Liu
Dec 17, 2020·Chemical Society Reviews·Sjoerd Slagman, Wolf-Dieter Fessner
Feb 6, 2020·Journal of the American Chemical Society·Hui ChenShelley D Minteer
Jan 28, 2021·Annual Review of Physical Chemistry·Yoo Seok LeeShelley D Minteer
Jun 8, 2021·Applied Biochemistry and Biotechnology·Lisheng XuBianling Jiang
Jul 14, 2021·ChemMedChem·Andrzej S SkwareckiMaria J Milewska

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