Semi-rational engineering of a Kluyveromyces lactis aldo-keto reductase KlAKR for improved catalytic efficiency towards t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important building-block of atorvastatin. In our previous work, a variant KlAKR-Y295W-W296 L (designated as M1) of Kluyveromyces lactis aldo-keto reductase (wild type (WT) KlAKR, M0) was developed, which possessed strict diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). To further improve its catalytic performance, semi-rational engineering of M1 was performed in present work, and the "best" varaint KlAKR-Y295W-W296L-1125V-S30P-Q212R-163W (M8) was developed. M8's K-m(B) towards (5R)-1 was 2.02 mM, and the catalytic efficiency (k(cat)/K-m(B)) value was 36.31 s(-1) mM(-1), which was 1.9-fold higher than that of the parent M1. Compared with M1, the half-life t(1/)(2), T(S50)(50)and T(P50)(50)of M8 were improved. Under the optimized conditions, (5R)-1 at load of up to 80 g L-1 was completely reduced in 1.5 h by M8 along with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for cofactor regeneration, producing (3R,5R)-2 in de(p) > 99.5% and space-time yields (STY) of 660.0 g L(-1)d(-1).