Fluorescence‐based screening for engineered aldo‐keto reductase KmAKR with improved catalytic performance and extended substrate scope

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 (5R/S)‐hydroxy‐3‐oxohexanoates, but it is inactive to aromatic ketones. In order to acquire an excellent KmAKRmutant for ensuring the simultaneous improvement of activity‐thermostability toward tert‐butyl 6‐cyano‐(5R)‐hydroxy‐3‐oxohexanoate ((5R)‐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 KmAKR variant M5 (KmAKR‐W297H/Y296W/K29H/Y28A/T63M) produced the “best” variant M5‐Q213A/T23V. It exhibited enhanced activity‐thermostability toward (5R)‐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 (EsGDH) for NADPH regeneration, 2.81 g DCW L−1 M5‐Q213A/T23V completely converted (5R)‐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‐(3R,5R)‐dihydroxyhexanoate ((3R,5R)‐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 KmAKR'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.

High-Throughput Screening Techniques for the Selection of Thermostable Enzymes

“…Qiu et al. developed a FHT screening system to evaluate engineered aldo-keto reductase KmAKR by converting the fluorescent substrate tert -butyl 6-cyano-(5 R )-hydroxy-3-oxohexanoate ((5 R )-1) into a fluorescent product (3 R ,5 R )-2, ultimately identifying two beneficial substitutions with enhanced catalytic efficiency and thermostability . On the other hand, Zhu et al introduced 4-UMP into a phytase mutant library during culturing in 96-well plates .…”

Engineering ketoreductases for the enantioselective synthesis of chiral alcohols

“…43 Zheng and Wang and coworkers used a variant of the Km AKR from Kluyveromyces marxianus , obtained following multiple rounds of directed evolution, for the efficient asymmetric reduction of 1 . 44,67–71 The variant exhibited a longer half-life, an enhanced thermostability and activity, and afforded 2 with a space-time yield of 1.8 kg L −1 d −1 and a d e p >99.5%. 70 Similarly, Lb CR protein from Lactobacillus brevis catalysed the reduction of 1 to the 3R, 5R-diol.…”