Increasing Catalytic Efficiency of SceCPR by Semi-rational Engineering towards the Asymmetric Reduction of D-Pantolactone.

D-pantolactone (D-PL) is one of the important chiral intermediates in the synthesis of D-pantothenic acid. Our previous study has revealed that ketopantolactone (KPL) reductase in Saccharomyces cerevisiae (SceCPR) could asymmetrically reduce KPL to D-PL with a relatively weak activity. In this study, engineering of SceCPR was performed using a semi-rational design to enhance its catalytic activity. Based on the computer-aided design including phylogenetic analysis and molecular dynamics simulation, Ser158, Asn159, Gln180, Tyr208, Tyr298 and Trp299 were identified as the potential sites. Semi-saturation, single and combined-site mutagenesis was performed on all six residues, and several mutants with improved enzymatic activities were obtained. Among them, the mutant SceCPRS158A/Y298H exhibited the highest catalytic efficiency in which the kcat /Km value is 2466.22s-1 ·mM-1 , 18.5 times higher than that of SceCPR. The 3D structural analysis showed that the mutant SceCPRS158A/Y298H had an expanded and increased hydrophilicity catalytic pocket, and an enhanced π-π interaction which could contribute to faster conversion efficiency and higher catalytic rate. The whole cell system containing SceCPRS158A/Y298H and glucose dehydrogenase (GDH), under the optimized condition, could reduce 490.21mM D-PL with e.e.≧99%, conversion rate = 98%, and the space-time yield = 382.80g·L-1 ·d-1 , which is the highest level reported so far.