[Construction of an enzyme-coupled system consisting of (R)- and (S)-specific carbonyl reductases for one-step preparation of (S)-1-phenyl-1,2-ethanediol].
OBJECTIVE
To prepare (S)-1-phenyl-1,2-ethanediol by a one-step method, we constructed an enzyme coupled system consisting of (R)- and (S)-specific carbonyl reductases in Escherichia coli.


METHODS
The genes coding (R)- and (S)-specific carbonyl reductases from Candida parapsilosis were inserted into a co-expression vector pETDuet-1. The positive plasmid was transformed into codon optimized E. coli Rosetta and an enzyme-coupled recombinant strain E. coli Rosetta / pETDuet-rcr-fdh was constructed. When the OD600 value of the culture reached 0.6-0.8, IPTG with a final concentration of 1 mmol/L was added to induce both target proteins at 30 degrees C for 10 h.


RESULTS
SDS-PAGE analysis showed that two target enzymes were expressed simultaneously with the relative molecular weights of 37 kDa and 30 kDa. When 5 mol/L Zn2+ was added into a phosphate buffer (pH7.0), the biotransformation results showed that the product (S)-1-phenyl-1,2-ethanediol was produced with the optical purity of 91.3% enantiomeric excess and a yield of 75.9% by E. coli Rosetta/pETDuet-rcr-scr.


CONCLUSIONS
The functions of two redox enzymes were integrated by molecular recombinant technique and a one-step method for preparation of (S)-1-phenyl-1,2-ethanediol was developed through an enzyme-coupled system. The method may supply a new procedure in chiral alcohols preparation.
Zhang
Rongzhen
R
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. rzzhang666@yahoo.com.cn
Xu
Yan
Y
Sun
Ying
Y
Geng
Yawei
Y
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002175:Candida / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005026:Ethylene Glycols / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D008970:Molecular Weight
D005026:Ethylene Glycols; D005656:Fungal Proteins; C031474:styrene glycol; D000429:Alcohol Oxidoreductases