Rational design of the carbonyl reductase EbSDR8 for efficient biosynthesis of enantiopure (R)-3-chloro-1-phenyl-1-propanol.,Applied Microbiology and Biotechnology

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Rational design of the carbonyl reductase EbSDR8 for efficient biosynthesis of enantiopure (R)-3-chloro-1-phenyl-1-propanol.
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2020-09-21 , DOI: 10.1007/s00253-020-10904-5
Ze-Hui Shao ₁ , Bing-Mei Su ₂ , Sheng-Li Yang ₁ , Li-Dan Ye ₃ , Hong-Wei Yu ₃

Affiliation

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 EbSDR8^{G94A/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% ee_p and 95.5% conversion. This result suggests EbSDR8^{G94A/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.

中文翻译：

合理设计羰基还原酶EbSDR8，以有效地生物合成对映纯（R）-3-氯-1-苯基-1-丙醇。

摘要

（R）-3-氯-1-苯基-1-丙醇（（R）-CPPO）是抗抑郁药的重要手性中间体。为了有效地进行生物合成，羰基还原酶EbSDR8经过精心设计，可在高浓度下不对称还原非天然底物3-氯-1-苯基-1-丙酮（3-CPP）。所得突变体的分子对接和分子动力学模拟表明，扩大的底物结合口袋和酶与底物或辅因子之间更合理的相互作用是增强催化活性并因此显着改善高浓度3-CPP转化的原因。使用最佳的突变EbSDR8 ^{G94A / L153I / Y188A / Y202M}作为全细胞生物催化剂，使用100％异丙醇作为溶剂和NADH再生的共底物进行3-CPP（1.0 M）的还原，提供（R）-CPPO的˃99％ee _p和95.5％的转化率。该结果表明，EbSDR8 ^{G94A / L153I / Y188A / Y202M}是在工业规模上绿色生产（R）-CPPO的潜在生物催化剂。