Structural‐guided design to improve the catalytic performance of aldo‐keto reductase KdAKR

Author:

Dai Chen123,Cao Hai‐Xing123,Tian Jia‐Xin123,Gao Yan‐Chi123,Liu Hua‐Tao123,Xu Shen‐Yuan123,Wang Ya‐Jun123ORCID,Zheng Yu‐Guo123ORCID

Affiliation:

1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou People's Republic of China

2. Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education Zhejiang University of Technology Hangzhou Zhejiang People's Republic of China

3. The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals Zhejiang University of Technology Hangzhou People's Republic of China

Abstract

AbstractAldo‐keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH‐dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t‐butyl 6‐chloro (5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) were improved by mutating its residues in the loop regions around the substrate‐binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67‐fold higher catalytic efficiency compared to the wild‐type (WT) KdAKR, and improved R‐selectivity toward (5S)‐CHOH (dep value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3‐fold increase in half‐life (t1/2) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)‐CHOH to diastereomeric pure t‐butyl 6‐chloro‐(3R, 5S)‐dihydroxyhexanoate ((3R, 5S)‐CDHH) within 8.0 h, with a space‐time yield of 300.7 g/L/day. Our results deepen the understandings of the structure−function relationship of AKRs, providing a certain guidance for the modification of other AKRs.

Funder

National Natural Science Foundation of China

Science and Technology Department of Zhejiang Province

Publisher

Wiley

Subject

Applied Microbiology and Biotechnology,Bioengineering,Biotechnology

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