Tailoring an aldo-keto reductase KmAKR for robust thermostability and catalytic efficiency by stepwise evolution and structure-guided consensus engineering

Abstract

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an advanced chiral diol intermediate of the cholesterol-lowering drug atorvastatin. KmAKR_M5 (W297H/Y296W/K29H/Y28A/T63M) constructed in our previous work, displayed good biocatalytic performance on (3R,5R)-2. In the present work, stepwise evolution was applied to further enhance the thermostability and activity of KmAKR_M5. For thermostability enhancement, N109 and S196 located far from the active site were picked out by structure-guided consensus engineering, and mutated by site-directed mutagenesis (SDM). For catalytic efficiency improvement, the residues A30 and T302 adjacent to the substrate-binding pocket were subjected to site-saturation mutagenesis (SSM). As a result, the "best" mutant KmAKR_M9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C) was developed, of which T₅₀¹⁵ and T_m were 5.0 °C and 8.2 °C higher than those of KmAKR_M5. Moreover, compared to KmAKR_M5, KmAKR_M9 displayed a 1.9-fold (846 vs 2436 min) and 6.7-fold (126 vs 972 min) longer half-lives at 40 and 50 °C, respectively. Structural analysis suggested that beneficial mutations introduced additional hydrophobic interactions and hydrogen bonds, contributing rigidification of the flexible loops and the increase of internal forces, hence increasing the thermostability and activity. 5 g DCW (dry cell weight) L^-1KmAKR_M9 completely reduced 350 g L^-1t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate ((5R)-1), within 3.7 h at 40 °C, yielding optically pure (3R,5R)-2 (d.e._p > 99.5%) with a space-time yield (STY) of 1.82 kg L^-1 d^-1. Hence, KmAKR_M9 is a robust biocatalyst for the synthesis of (3R,5R)-2.

Keywords: Aldo-keto reductase; Catalytic efficiency; Stepwise evolution; Thermostability; t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate.