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Improved Production of (R)-1-phenyl-1,2-ethanediol by a Codon-optimized R-specific Carbonyl Reductase from Candida parapsilosis in Escherichia coli

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Abstract

An R-specific carbonyl reductase from Candida parapsilosis (CprCR) catalyzes the transformation of (R)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone. The gene rcr coding CprCR contains a few codons rarely used by Escherichia coli. In order to improve chiral alcohol production, three codon variants Δ24, aRCR, and mRCR of CprCR were designed through truncation of 4–27 bp disorder sequence at the 5′-terminus or/and adaption of nine rare codons. The effects of codon optimization on enzyme activity, protein production, and biotransformation were studied. Among these three types, the disorder sequence-truncated and rare codon-adapted variant mRCR presents the highest enzyme activity. When compared with CprCR, mRCR showed an increase of 35.6% in the total activity of cell-free extracts. The specific activity of mRCR presented similar increase in the cell-free extract with purified protein, which suggested that the codon optimization caused positive effect on protein productivity of variant enzyme. When microbial cells concentration was 30% (w/v), the molar conversion yield and enantiomeric excess of the mRCR variant reached 86.4% and 93.6%, which were increased 36.5% and 15.8% than those of wild-type at a high substrate concentration of 5 g/L. The work will supply a new method for improving chiral alcohol preparation with codon engineered microorganisms.

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Abbreviations

ADHs:

alcohol dehydrogenases

CprCR:

R-specific carbonyl reductase from Candida parapsilosis

e.e.:

enantiomeric excess

MDRs:

medium-chain dehydrogenases/reductases

NAD(H):

nicotinamide adenine dinucleotide

PED:

1-phenyl-1, 2-ethanediol

rcr :

R-specific carbonyl reductase gene

SCR:

S-specific carbonyl reductase

WT:

wild-type

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Acknowledgements

This project was supported in part from the National Science Foundation of China (No. 20776060), the National Key Basic Research and Development Program of China (973 Program Nos. 2003CB716008 and 2009CB724706) and Hi-Tech Research and Development Program of China (863 Program No. 2007AA02Z226). This work was also carried out as part of the Project for the Ministry of Education, People’s Republic of China under the Program for Changjiang Scholars, Innovative Research Team in University (PCSIRT, IRT0532), and the Program of Introducing Talents of Discipline to Universities 111-2-06.

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Rongzhen Zhang, Yan Xu, Yawei Geng, Shanshan Wang & Ying Sun

  2. Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ, 08854, USA

    Rong Xiao

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  1. Rongzhen Zhang
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Correspondence to Yan Xu.

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Zhang, R., Xu, Y., Geng, Y. et al. Improved Production of (R)-1-phenyl-1,2-ethanediol by a Codon-optimized R-specific Carbonyl Reductase from Candida parapsilosis in Escherichia coli . Appl Biochem Biotechnol 160, 868–878 (2010). https://doi.org/10.1007/s12010-009-8528-9

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