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Increasing catalytic efficiency of SceCPR by semi-rational engineering towards the asymmetric reduction of
Man Zhao1, Bin Zhou1, Xiaoli Jia1
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
Abstract:
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 2466.22 s-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 490.21 mM D-PL with e.e.≧ 99%, conversion rate = 98%, and the space-time yield = 382.80 g·L-1·d-1, which is the highest level reported so far.
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