Co-evolution of activity and thermostability of an aldo-keto reductase KmAKR for asymmetric synthesis of statin precursor dichiral diols

Aldo-keto reductase KmAKR-catalyzed asymmetric reduction offers a green approach to produce dichiral diol tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoates, which are important building blocks of statins. In our previous work, we cloned a novel gene of NADPH-specific aldo-keto reductase KmAKR (WT) from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 and a mutant KmAKR-W297H/Y296W/K29H (Variant III) has been constructed and displayed strict diastereoselectivity towards tert-butyl 6-cyano-(5R)-hydroxy 3 ox ohexanoate ((5R)-1) but moderate activity and stability. Herein, to further co-evolve its activity and thermostability, we performed semi-rational engineering of Variant III by using a combinational screening strategy, consisting of tertiary structure analysis, loop engineering, and alanine scanning. As results, the best variant KmAKR-W297H/Y296W/K29H/Y28A/T63M (Variant VI) was acquired, whose K-m, k(cat)/K-m towards (5R)-1 was 0.66 mM and 210.77 s(-1) mM(-1), respectively, with improved thermostability (half-life of 14.13 h at 40 degrees C). Combined with 1.5 g dry cell weight (DCW) L-1 Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for NADPH regeneration, 4.5 g DCW L Variant VI completely reduced (5R)-1 of up to 450 g L-1 within 7.0 h at 40 degrees C, yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)3, > 99.5% d.e.(p)) with a space-time yield (STY) of 1.24 kg L-1 day(-1), and this was the highest level documented in literatures so far on substrate loading and STY of producing (3R,5R)-3. Besides (5R)-1, Variant VI displayed strong activity on tert-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-2). 4.5 g DCW L-1 Variant VI completely reduced 400 g L-1 (5S)-2, within 5.0 h at 40 degrees C, yielding optically pure tert-butyl 6-chloro-(3R,5S)dihydroxyhexanoate ((3R,5S)-4, > 99.5% cl.e.(p)) with a STY of 1.34 kg L-1 day(-1). In summary, Variant VI displayed industrial application potential in statins biomanufacturing.