Premium
Fluorescence‐based screening for engineered aldo‐keto reductase Km AKR with improved catalytic performance and extended substrate scope
Author(s) -
Qiu Shuai,
Xu ShenYuan,
Li ShuFang,
Meng KangMing,
Cheng Feng,
Wang YaJun,
Zheng YuGuo
Publication year - 2021
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.202100130
Subject(s) - thermostability , chemistry , biocatalysis , substrate (aquarium) , steric effects , stereochemistry , directed evolution , catalysis , reductase , aldo keto reductase , enantiomeric excess , combinatorial chemistry , enzyme , enantioselective synthesis , organic chemistry , biochemistry , biology , reaction mechanism , ecology , gene , mutant
Abstract Background Aldo‐keto reductases‐catalyzed transformations of ketones to chiral alcohols have become an established biocatalytic process step in the pharmaceutical industry. Previously, we have discovered an aldo‐keto reductase (AKR) from Kluyveromyces marxianus that is active to the aliphatic tert ‐butyl 6‐substituted (5 R / S )‐hydroxy‐3‐oxohexanoates, but it is inactive to aromatic ketones. In order to acquire an excellent Km AKRmutant for ensuring the simultaneous improvement of activity‐thermostability toward tert ‐butyl 6‐cyano‐(5 R )‐hydroxy‐3‐oxohexanoate ((5 R )‐ 1 ) and broadening the universal application prospects toward more substrates covering both aliphatic and aromatic ketones, a fluorescence‐based high‐throughput (HT) screening technique was established. Main Methods and Major Results The directed evolution of Km AKR variant M5 ( Km AKR‐W297H/Y296W/K29H/Y28A/T63M) produced the “best” variant M5‐Q213A/T23V. It exhibited enhanced activity‐thermostability toward (5 R )‐ 1 , improved activity toward all 18 test substrates and strict R ‐stereoselectivity toward 10 substrates in comparison to M5. The enhancement of enzymatic activity and the extension of substrate scope covering aromatic ketones are proposed to be largely attributed to pushing the binding pocket of M5‐Q213A/T23V to the enzyme surface, decreasing the steric hindrance at the entrance and enhancing the flexibility of loops surrounding the active center. In addition, combined with 0.94 g dry cell weight (DCW) L −1 glucose dehydrogenase from Exiguobacterium sibiricum ( Es GDH) for NADPH regeneration, 2.81 g DCW L −1 M5‐Q213A/T23V completely converted (5 R )‐ 1 of up to 450 g L −1 at 120 g g –1 substrates/catalysts (S/C), yielding the corresponding optically pure tert ‐butyl 6‐cyano‐(3 R ,5 R )‐dihydroxyhexanoate ((3 R ,5 R )‐ 2 , > 99.5% d.e . p ) with a space‐time yield (STY) of 1.08 kg L −1 day −1 . Conclusions A fluorescence‐based HT screening system was developed to tailor Km AKR's activity, thermostability and substrate scope. The “best” variant M5‐Q213A/T23V holds great potential application for the synthesis of aliphatic/aromatic R ‐configuration alcohols.