Cloning And Molecular Modification Of Aldo-keto Reductase K1AKR And Its Application In The Synthesis Of T-butyl 6-Cyano-(3R,5R)-Dihydroxyhexanoate

Optically pure t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate((R)-1b)is the key precursor of atorvastatin calcium,the most widely used cholesterol-lowering drug.Biocatalysis,as an important supplement of chemical process,has many advantages such as mild reaction conditions,high yield,excellent stereoselectivity,few by-products,and so on,making it more efficient and environmentally friendly.This paper is devoted to the screening aldo-keto reductase(AKR)producer,AKR molecular modification and bioprocess development for(R)-1b.Two strains ZJB-09224 and XP1461,capable of asymmetrically reducing t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate(1a)to corresponding optically pure(R)-1b,were successfully isolated from nature.They were identified belonging to Rhodotorula glutinis and Kluyveromyces lactis based on the morphology,physiological tests,and the 18 S r DNA sequence analysis.It was found that heat treatment of cell suspension at 45°C for 25 min significantly improved R.glutinis ZJB-09224 stereoselectivity.The asymmetric bioreduction of 1a was the most efficient at p H 7.5,35°C,50 m M(15.0 g·L-1)substrate concentration,40.0 g·DCW·L-1 cell loading size,0.54 M(60.0 g·L-1)sodium lactate acting as co-substrate.Under these optimal conditions,0.046 M(R)-1b was produced with d.e.(diastereomeric excess)value of99.2% after 40 h conversion.Moreover,R.glutinis ZJB-09224 has a broad substrate spectrum,making it a potential tool for some valuable chiral alcohol pharmaceutical intermediates synthesis.An AKR gene(klakr)from K.lactis XP1461 was cloned and heterologously expressed in Escherichia coli.The K1AKR was purified and found to be NADH dependent with a molecular weight of approximately 36 k Da.It is active and stable at 30°C and p H 7.0.The maximal reaction rate(vmax),apparent Michaelis-Menten constant(Km)for NADH and 1a and catalytic number(kcat)were calculated to be 7.63U·mg-1,0.204 m M,4.42 m M and 697.4 min-1,respectively.Addtionally,the K1AKR has broad substrate spectrum convering a range of aldehydes,ketones and keto-esters,producing chiral alcohol with e.e.or d.e.>99%for the majority of test substrates.A rational engineering was used to improve the K1AKR activity.Based on homology modeling and molecular docking,two amino acid residues(295 and 296)were selected as mutation sites,and two rounds of site-saturation mutagenesis were performed.Among the mutants,K1AKR-Y295W/W296 L exhibited the highest catalytic efficiency(kcat/Km)toward 1a up to 12.37 s-1·m M-1,which was 11.25-fold higher than that of wild-type K1AKR.Moreover,the mutation has no negative impact on stereoselectivity.1a was docked into the active sites of wild-type K1AKR and the mutant Y295W/W296 L,respectively,which aims to illustrate the difference in terms of activity toward 1a.The enlarged binding pocket for1 a and the shortened distance between the C4 atom of the nicotinamide ring of NADH and the carbonyl carbon atom of 1a play crucial roles in determining the AKR catalytic activity toward 1a.Different parameters of protein expression of E.coli/p ET28a-klakr-Y295W/W296 L were evaluated,lactose 9.0 g·L-1,28°C and 12 h was considered as the optimal induction condition.The K1AKR-Y295W/W296 L production and biomass were 1445 U·g-1(DCW)and 2.44 g(DCW),respectively.Using K1AKR-Y295W/W296 L coupled with Exiguobacterium sibiricum glucose dehydrogenase(Es GDH)for cofactor regeneration,(R)-1b was accumulated up to 162.7 m M with d.e.value above 99.5%.The space-time yield was 670.6 g·L-1·d-1,which exceeded the previous report.K1AKR-Y295W/W296 L represents a robust tool for(R)-1b synthesis.