Fluorescence‐based screening for engineered aldo‐keto reductase KmAKR with improved catalytic performance and extended substrate scope. Issue 9 (26th June 2021)

• Record Type: Journal Article
• Authors: Qiu, Shuai; Xu, Shen‐Yuan; Li, Shu‐Fang; Meng, Kang‐Ming; Cheng, Feng; Wang, Ya‐Jun; Zheng, Yu‐Guo
• Is Part Of: Biotechnology journal. Volume 16:Issue 9(2021)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2021-06-26
• Subjects: aldo‐keto reductase -- anti‐prelog alcohols -- biocatalysis -- directed evolution -- fluorescence screening; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.202100130
• Languages: English
• ISSNs: 1860-6768

Abstract

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. Abstract : Aldo‐keto reductases (AKRs)‐catalyzed transformations of ketones to chiral alcohols have become an established biocatalytic process step in the pharmaceutical industry. An AKR from Kluyveromyces marxianus ( Km AKR) ZJB14056 has been discovered that is active to the aliphatic tert ‐butyl 6‐substituted (5R/S)‐hydroxy‐3‐oxohexanoates, but is inactive to aromatic ketones. In this study, a fluorescence‐based high‐throughput screening technique was established to co‐evolve Km AKR's activity, thermostability and substrate scope. Finally, the "best" variant M5‐Q213A/T23V was acquired, and it had great potential application for the synthesis of aliphatic/aromatic R ‐configuration alcohols.