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

Abstract:

[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 pETDuetTM-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 oC 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 75.8% by E. coli Rosetta/pETDuet-rcr-scr. [Conclusion] 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 simplify procedure in chiral alcohols preparation.