Cloning, sequence analysis, and expression in Escherichia coli of gene encoding N-Benzyl-3-pyrrolidinol dehydrogenase from Geotrichum capitatum.

The gene encoding N-benzyl-3-pyrrolidinol dehydrogenase (DDBJ/EMBL/GenBank accession no. AB294179), a useful biocatalyst for producing (S)-N-benzyl-3-pyrrolidinol, was cloned from the genomic DNA of Geotrichum capitatum JCM 3908. The gene contained an open reading frame consisting of 1023 nucleotides corresponding to 340 amino acid residues. The subunit molecular weight was calculated to be 39,000. The predicted amino acid sequence did not have significant similarity to those of N-benzyl-3-pyrrolidinone reductases reported previously. From 30 mM N-benzyl-3-pyrrolidinone, (S)-N-benzyl-3-pyrrolidinol was obtained with a yield >99.9% and an enantiomeric excess >99.9% in 1-h and 2-h reactions without NADH addition by the resting cells of Escherichia coli HB 101 strains harboring the expression plasmids pSG-POBS and pSF-POBS that possess the glucose dehydrogenase gene and formate dehydrogenase gene as an NADH-reproducing system, respectively, besides the N-benzyl-3-pyrrolidinol dehydrogenase gene. N-Benzyl-3-pyrrolidinol dehydrogenase activity (0.56 U/mg) was observed in E. coli (pSG-POBS), which was 17-fold the specific activity observed in G. capitatum JCM 3908.