Increasing catalytic efficiency of SceCPR by semi-rational engineering towards the asymmetric reduction of D-pantolactone

Man Zhao; Bin Zhou; Xiaoli Jia; Meinan Wang; Zhiqiang Liu; Yuguo Zheng

doi:10.1016/j.jbiotec.2023.06.007

Increasing catalytic efficiency of SceCPR by semi-rational engineering towards the asymmetric reduction of D-pantolactone

J Biotechnol. 2023 Aug 20:373:34-41. doi: 10.1016/j.jbiotec.2023.06.007. Epub 2023 Jun 29.

Authors

Man Zhao¹, Bin Zhou¹, Xiaoli Jia¹, Meinan Wang¹, Zhiqiang Liu², Yuguo Zheng¹

Affiliations

¹ Key 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.
² Key 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. Electronic address: microliu@zjut.edu.cn.

PMID: 37392996
DOI: 10.1016/j.jbiotec.2023.06.007

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 SceCPR^S158A/Y298H exhibited the highest catalytic efficiency in which the k_cat/K_m value is 2466.22 s^-1·mM^-1, 18.5 times higher than that of SceCPR. The 3D structural analysis showed that the mutant SceCPR^S158A/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 SceCPR^S158A/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.

Keywords: Catalytic pocket; D-pantolactone; Ketopantolactone reductase; Site-directed mutagenesis; Whole cell catalysis.

MeSH terms

Catalysis
Kinetics
Mutagenesis, Site-Directed
Oxidoreductases*
Phylogeny
Protein Engineering
Saccharomyces cerevisiae* / genetics

Substances

pantolactone
Oxidoreductases