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Rational design of the carbonyl reductase EbSDR8 for efficient biosynthesis of enantiopure (R)-3-chloro-1-phenyl-1-propanol

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Abstract

(R)-3-Chloro-1-phenyl-1-propanol ((R)-CPPO) is an important chiral intermediate for antidepressants. For its efficient biosynthesis, the carbonyl reductase EbSDR8 was engineered to asymmetrically reduce the unnatural substrate 3-chloro-1-phenyl-1-propanone (3-CPP) at high concentrations. Molecular docking and molecular dynamics simulations of the resulting mutants suggested enlarged substrate binding pocket and more reasonable interactions between the enzyme and the substrate or cofactor as the reasons for the enhanced catalytic activity and thus the remarkably improved conversion of high-concentration 3-CPP. Using the best mutant EbSDR8G94A/L153I/Y188A/Y202M as the whole-cell biocatalyst, reduction of 3-CPP (1.0 M) was conducted using 100% isopropanol as both the solvent and co-substrate for NADH regeneration, delivering (R)-CPPO with ˃ 99% eep and 95.5% conversion. This result suggests EbSDR8G94A/L153I/Y188A/Y202M as a potential biocatalyst for green production of (R)-CPPO at the industrial scale.

Key points

• Rational design of EbSDR8 by modulating steric hindrance and molecular interactions;

• Non-aqueous biocatalysis using isopropanol as both the solvent and co-substrate;

• Whole-cell catalyzed production of 161 g/L enantiopure (R)-CPPO from 1.0 M of 3-CPP.

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Funding

This work was financially supported by the Natural Science Foundation of China (Grant No. 21776244), the Zhejiang Provincial Natural Science Foundation of China (Grant Nos. LY18B060001 and LZ20B060002), and the Science and Technology Department of Zhejiang Province (Grant Nos. 2018C02017, 2020C02041, and 2020C02055). We are grateful to the support of the Discovery Studio software from the Fuzhou University platform.

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Authors and Affiliations

  1. College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, China

    Ze-Hui Shao & Sheng-Li Yang

  2. College of Chemical Engineering, Fuzhou University, Fuzhou, 350116, China

    Bing-Mei Su

  3. Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China

    Li-Dan Ye & Hong-Wei Yu

Authors
  1. Ze-Hui Shao
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  2. Bing-Mei Su
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  3. Sheng-Li Yang
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  4. Li-Dan Ye
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  5. Hong-Wei Yu
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Contributions

The manuscript was written through contributions of all authors. ZHS, BMS, LDY, HWY, and SLY designed the experiments and prepared the manuscript. ZHS performed mutagenesis, protein expression, whole-cell reduction, protein purification, and determination of catalytic kinetics. ZHS and BMS conducted the molecular dynamics simulations. All authors analyzed and discussed the results. All authors have given approval to the final version of the manuscript.

Corresponding authors

Correspondence to Sheng-Li Yang, Li-Dan Ye or Hong-Wei Yu.

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The authors declare no conflicts of interest.

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Shao, ZH., Su, BM., Yang, SL. et al. Rational design of the carbonyl reductase EbSDR8 for efficient biosynthesis of enantiopure (R)-3-chloro-1-phenyl-1-propanol. Appl Microbiol Biotechnol 104, 9219–9228 (2020). https://doi.org/10.1007/s00253-020-10904-5

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