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 bioconversion of vanillin to protocatechuate by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a novel vanillin dehydrogenase (vdh) and the two subunits of a vanillate demethylase (vanA and vanB), respectively. These genes were localized on an EcoRI fragment (E230), which was cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The vdh gene was identified on a subfragment (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 open reading frames of 1062, 951, and 1446 bp, representing vanA, vanB, and vdh, respectively. The vdh gene was organized in one operon together with a fourth open reading frame (ORF2), of 735 bp, which was located upstream of vdh. The deduced amino acid sequences of vanA and vanB exhibited 78.8 and 62.1% amino acid identity, respectively, to the corresponding gene products from Pseudomonas sp. strain ATCC 19151 (F. Brunel and J. Davison, J. Bacteriol. 170:4924-4930, 1988). The deduced amino acid sequence of the vdh gene exhibited up to 35.3% amino acid identity 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 harboring fragment E230 cloned in pBluescript SK- converted vanillin to protocatechuate via vanillate, indicating the functional expression of vdh, vanA, and vanB in E. coli. High expression of vdh in E. coli was achieved with HE35 cloned in pBluescript SK-. The resulting recombinant strains converted vanillin to vanillate at a rate of up to 0.3 micromol per min per ml of culture. Transfer of vanA, vanB, and vdh to Alcaligenes eutrophus and to different Pseudomonas strains, which were unable to utilize vanillin or vanillate as carbon sources, respectively, conferred the ability to grow on these substrates to these bacteria.