[Co-expression of formate dehydrogenase from Candida boidinii and (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011 in Escherichia coli].
OBJECTIVE
To overcome coenzyme restriction in the asymmetric reduction at high substrate concentration, we constructed a recombinant Escherichia coli with (R)-sepcificity carbonyl reductase coupled with formate dehydrogenase for cofactor regeneration.


METHODS
The R-carbonyl reductase gene (rcr) and formate dehydrogenase gene (fdh) were amplified from Candida parapsilosis and Candida boidinii genomes by PCR technique respectively. Then the purified PCR products were inserted into a co-expression vector pETDuet-1 to construct plasmid pETDuet-rcr-fdh. The positive plasmid was transformed into codon optimized E. coli Rosetta, and a recombinant strain E. coli Rosetta/pETDuet-rcr-fdh was obtained.


RESULTS
SDS-PAGE analysis showed that two enzymes were expressed simultaneously. Isopropyl-BETA-D-thiogalactopyranoside (1 mmol/L) induced at 30 degrees C the expression of both proteins encoded by rcr and fdh genes with the molecular weights of 37 kDa and 40 kDa. The biotransformation experiments were done using 2-hydroxyacetophenone and sodium formate as substrates. When the concentration of 2-hydroxyacetophenone was 6 g/L, the (R)-1-phenyl-1,2-ethanediol was produced with the high optical purity of 100% enantiomeric excess and a yield of 85.9%. Compared with the recombinant strain E. coli Rosetta/pETDuet-rcr withoutfdh gene for cofactor regeneration, the optical purity and yield of product from the asymmetric reduction of 2-hydroxyacetophenone by E. coli Rosetta/pETDuet-rcr-fdh were increased by 1.3 and 2.7 times, respectively.


CONCLUSIONS
This method supplied a foundation for biosynthesis of (R)-1-phenyl-1,2-ethanediol for the cofactor regeneration by genetic engineering.
Sun
Ying
Y
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. sunyfamily.1984@yahoo.com.cn
Zhang
Rongzhen
R
Xu
Yan
Y
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D001711:Biotransformation; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; D005560:Formate Dehydrogenases