Improved production of (R)-1-phenyl-1,2-ethanediol by a codon-optimized R-specific carbonyl reductase from Candida parapsilosis in Escherichia coli.
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 Delta24, 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.
Zhang
Rongzhen
R
Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Xu
Yan
Y
Geng
Yawei
Y
Wang
Shanshan
S
Sun
Ying
Y
Xiao
Rong
R
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002175:Candida / Q000201:enzymology*; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005026:Ethylene Glycols / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017384:Sequence Deletion; D013237:Stereoisomerism; D013379:Substrate Specificity
D003062:Codon; D005026:Ethylene Glycols; C031474:styrene glycol; D000429:Alcohol Oxidoreductases