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

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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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Acknowledgments

This work was supported by the Ministry of Science and Technology of China Grant 2013CB734003 and the National Natural Science Foundation of China (Grant No. 21172095, 31160017, 21472234). We are grateful to Dr. Rongsheng Tao from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, for providing us gene gdh from B. subtilis 168.

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Authors and Affiliations

  1. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

    Lin Zhu, Zhe Wu & Shuang-Yan Tang

  2. Beijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, Beijing, 100048, China

    Jian-Ming Jin

  3. Key Laboratory of Ethnic Medicine Resource Chemistry, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming, 650500, China

    Jian-Ming Jin

  4. University of Chinese Academy of Sciences, Beijing, 100049, China

    Lin Zhu

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  1. Lin Zhu
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  2. Zhe Wu
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  4. Shuang-Yan Tang
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Correspondence to Shuang-Yan Tang.

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The authors declare that they have no competing interests.

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Lin Zhu and Zhe Wu contributed equally to this work.

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Zhu, L., Wu, Z., Jin, JM. et al. Directed evolution of leucine dehydrogenase for improved efficiency of l-tert-leucine synthesis. Appl Microbiol Biotechnol 100, 5805–5813 (2016). https://doi.org/10.1007/s00253-016-7371-5

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  • DOI: https://doi.org/10.1007/s00253-016-7371-5

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