Efficient stereoselective synthesis of aryl lactones using engineered ketoreductase ChKRED20 from Chryseobacterium sp. CA49

Abstract

Enzymatic asymmetric reduction of γ,δ-keto esters offers a convenient and efficient approach for producing optically active lactones. However, aryl lactones with bulky structures may not be readily prepared using natural ketoreductases. Herein, we successfully realized the asymmetric reduction of aromatic γ,δ-keto esters through engineering a wild-type anti-Prelog ketoreductase (KRED) ChKRED20 which exhibits no activity toward γ, δ-keto esters. In our semi-rational design strategy, keto esters were able to access the active site of variant M2 (Q97L-S153L) by redesigning hydrophilic pockets into hydrophobic pockets. Further variants with enlarged active cavities were obtained through the engineering of M2 at residues Y188 and we found that mutations of the key residue Y188 greatly increased the yield of lactones by about 5-fold over M2. Moreover, we explained the interaction between the amino acid at position 188 and the substrate by saturation mutagenesis experiments performed on Y188 in combination with computational simulations. In this way, we successfully realized the asymmetric reduction of a broad range of bulky aromatic γ,δ-keto esters and prepared a series of optically active lactones with high enantioselectivity (96–99%). This research provides an efficient and promising biocatalytic method for the preparation of chiral lactones.