Cloning and overexpression of an NADH-dependent alcohol dehydrogenase gene from Candida maris involved in (R)-selective reduction of 5-acetylfuro[2,3-c]pyridine

Biosci Biotechnol Biochem. 2011;75(11):2155-61. doi: 10.1271/bbb.110453. Epub 2011 Nov 7.

Abstract

5-((R)-1-Hydroxyethyl)-furo[2,3-c]pyridine ((R)-FPH) is a useful chiral building block in the synthesis of pharmaceuticals. An NADH-dependent alcohol dehydrogenase (AFPDH) isolated from Candida maris catalyzed the reduction of 5-acetylfuro[2,3-c]pyridine (AFP) to (R)-FPH with 100% enantiomeric excess. The gene encoding AFPDH was cloned and sequenced. The AFPDH gene comprises 762 bp and encodes a polypeptide of 27,230 Da. The deduced amino acid sequence showed a high degree of similarity to those of other members of the short-chain alcohol dehydrogenase superfamily. The AFPDH gene was overexpressed in Escherichia coli under the control of the lac promoter. One L of the cultured broth of an E. coli transformant coexpressing AFPDH and the glucose dehydrogenase (GDH) gene reduced 250 g of AFP to (R)-FPH in an organic solvent two-phase system. Under coupling with NADH regeneration using 2-propanol, 1 L of the cultured broth of an E. coli transformant expressing the AFPDH gene reduced 150 g of AFP to (R)-FPH. The optical purity of the (R)-FPH formed was 100% enantiomeric excess under both reaction conditions.

MeSH terms

  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism*
  • Amino Acid Sequence
  • Base Sequence
  • Candida / enzymology*
  • Candida / genetics
  • Cloning, Molecular
  • Gene Expression Regulation, Fungal
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Pyridines / chemical synthesis
  • Pyridines / chemistry
  • Pyridines / metabolism*

Substances

  • 5-((R)-1-hydroxyethyl)-furo(2,3-c)pyridine
  • 5-acetylfuro(2,3-c)pyridine
  • Pyridines
  • Alcohol Oxidoreductases
  • alcohol dehydrogenase (NAD(P)+)