Characterization of the eugenol hydroxylase genes (ehyA/ehyB) from the neweugenol-degrading Pseudomonas sp strain OPS1

During the screening for bacteria capable of converting eugenol to vanillin . strain OPS1 was isolated. which was identified as a new Pseudomonas speci es by 16 s rDNA sequence analysis. When this bacterium was grown on eugenol , the intermediates. coniferyl alcohol, ferulic acid, vanillic acid, and pr otocatechuic acid, were identified in the Culture supernatant. The genes en coding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudom onas sp. strain OPS1 by complementation of the eugenol-negative mutant SK61 65 of Pseudomonas sp. strain HR199. EhyA and EhyB exhibited 57% and 85% ami no acid identity to the eugenol hydroxylase subunits of Pseudomonas sp. str ain HR199 and up to 34% and 54% identity to the corresponding subunits of p -cresol methylhydroxylase from P. putida. Moreover, the amino-terminal sequ ences of the alpha- and beta -subunits reported recently for an eugenol deh ydrogenase of P fluorescens E118 corresponded well with the appropriate reg ions of EhyA and EhyB. Downstream of ehyB, an open reading frame was identi fied, whose deduced amino acid sequence exhibited up to 71% identity to azu rins, representing most probably the gene (azu) of the physiological electr on acceptor of the eugenol hydroxylase. The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.