MOLECULAR CHARACTERIZATION OF GENES OF PSEUDOMONAS SP STRAIN HR199 INVOLVED IN BIOCONVERSION OF VANILLIN TO PROTOCATECHUATE

The gene loci vdh, vanA, and vanB, which are involved in the bioconver sion of vanillin to protocatechuate by Pseudomonas sp. strain HR199 (D SM 7063), were identified as the structural genes of a novel vanillin dehydrogenase (vdh) and the two subunits of a vanillate demethylase (v anA and vanB), respectively. These genes were localized on an EcoRI fr agment (E230), which was cloned from a Pseudomonas sp. strain HR199 ge nomic library in the cosmid pVK100. The vdh gene was identified on a s ubfragment (HE35) of E230, and the vanA and vanB genes were localized on a different subfragment (H110) of E230. The nucleotide sequences of fragment HE35 and part of fragment H110 were determined, revealing op en reading frames of 1062, 951, and 1446 bp, representing vanA, vanB, and vdh, respectively. The vdh gene was organized in one operon togeth er with a fourth open reading frame (ORF2), of 735 bp, which was locat ed upstream of vdh. The deduced amino acid sequences of vanA and vanB exhibited 78.8 and 62.1% amino acid identity, respectively, to the cor responding gene products from Pseudomonas sp. strain ATCC 19151 (F. Br unel and J. Davison, J. Bacteriol. 170:4924-4930, 1988). The deduced a mino acid sequence of the vdh gene exhibited up to 35.3% amino acid id entity to aldehyde dehydrogenases from different sources. The deduced amino acid sequence of ORF2 exhibited up to 28.4% amino acid identity to those of enoyl coenzyme A hydratases. Escherichia coli strains harb oring fragment E230 cloned in pBluescript SK- converted vanillin to pr otocatechuate via vanillate, indicating the functional expression of v dh, vanA, and vanB in E. coli. High expression of vdh in E. coli was a chieved,vith HE35 cloned in pBluescript SK-. The resulting recombinant strains converted vanillin to vanillate at a rate of up to 0.3 mu mol per min per ml of culture. Transfer of vanA, vanB, and vdh to Alcalig enes eutrophus and to different Pseudomonas strains, which were unable to utilize vanillin or vanillate as carbon sources, respectively, con ferred the ability to grow on these substrates to these bacteria.