Cloning And Expression Of NADPH Keto Reductase And Its Application In Chiral Synthesis

Ethyl(R)-4-chloro-3-hydroxybutanoate ester((R)-CHBE) is a precursor of enantiopure intermediates used for the production of chiral drugs,including the cholesterol-lowering 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitors(statins). The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate ester(COBE) to(R)-CHBE by biocatalysis has several positive attributes, including low cost, mild reaction conditions, high yield, and a high level of enantioselectivity.The main process of biosynthesis of(R)-CHBE: screening of microorganisms that catalyze the reduction of COBE to(R)- CHBE(I); gene cloning, expression, and characterization of carbonyl reductases for the production of(R)-CHBE in host(II); development of cofactor generation systems for regenerating cofactors(III); and biocatalysis of COBE to(R)-CHBE by recombinant(IV).The new gene of keto reductase(KR) cloned from the genome of Lodderomyces elongisporus has been overexpressed in Yeast. The research on enzymatic properties of KR has been systematically performed. As NADPH dependent keto reductase,KR can be used to catalyze asymmetric reduction of ethyl 4-chloro-3-oxobutanoate(COBE) to(R)-CHBE. The optimal temperature for reaction is 30?and the optimal pH for reaction is 6.0.The biotransformation for production of CHBE has been performed. In order to low cost in the large-scale industrial production, the recombinant strain was designed to coexpress the two enzymes,Keto reductase and glucose dehydrogenase(GDH), to obtain NADPH regeneration. KR and GDH genes were cloned into the plasmid pPIC3.5k,and then the recombinant plasmid was transformed into GS115. Using 23 mM COBE and 99 mM glucose as substrates, and the recombinant GS115 as catalyst,the transformation result showed that the enantiomeric excess and conversion yield of(R)-CHBE was 99% and 90%, respectively. Comparing the two patterns of coenzyme regeneration, the KR-GDH coupled conversion pattern significantly improved CHBE yield with higher substrate concentration and shorter reaction period. Especially it is not necessary for this transformation process to add NADPH additionally.To sum up,a novel KR and GDH were cloned and overexpressed in BL21(DE3). This NADPH regeneration system can be added to the reductase library as a promising catalyst for multi-purpose bio-reduction systems.