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

• Published in: Applied microbiology and biotechnology Vol. 56; no. 5/6; pp. 724 - 730
• Main Authors: Brandt, K, Thewes, S, Overhage, J, Priefert, H, Steinbuchel, A
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01-09-2001; Springer Nature B.V
• Subjects: Amino acid sequence; amino acid sequences; Amino acids; azu gene; Bacteria; Bacterial Proteins; Bacteriology; Biodegradation, Environmental; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Biotransformation; Cloning, Molecular; coniferyl alcohol; cresol methylhydroxylase; DNA, Ribosomal - analysis; DNA, Ribosomal - genetics; E coli; ehyA gene; EhyA protein; ehyB gene; EhyB protein; Escherichia coli; eugenol; Eugenol - metabolism; eugenol dehydrogenase; ferulic acid; Fundamental and applied biological sciences. Psychology; genes; Genes, Bacterial; Genetics; genomic libraries; Microbiology; Mission oriented research; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; mutants; open reading frames; Ozone; p-cresol; p-cresol methylhydroxylase; polymerase chain reaction; protocatechuic acid; Pseudomonas; Pseudomonas - classification; Pseudomonas - enzymology; Pseudomonas - genetics; Pseudomonas - isolation & purification; Pseudomonas fluorescens; Pseudomonas putida; ribosomal DNA; RNA, Ribosomal, 16S - genetics; screening; sequence analysis; Sequence Analysis, DNA; Soil Microbiology; vanillic acid; vanillin; Pseudomonadales; Biodegradation; Gene; Nucleotide sequence; Enzyme; Bacteria; Pseudomonadaceae; Homology; Oxidoreductases; Pseudomonas; Eugenol
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530100698

During the screening for bacteria capable of converting eugenol to vanillin, strain OPS1 was isolated, which was identified as a new Pseudomonas species by 16 s rDNA sequence analysis. When this bacterium was grown on eugenol, the intermediates, coniferyl alcohol, ferulic acid, vanillic acid, and protocatechuic acid, were identified in the culture supernatant. The genes encoding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudomonas sp. strain OPS1 by complementation of the eugenol-negative mutant SK6165 of Pseudomonas sp. strain HR199. EhyA and EhyB exhibited 57% and 85% amino acid identity to the eugenol hydroxylase subunits of Pseudomonas sp. strain HR199 and up to 34% and 54% identity to the corresponding subunits of p-cresol methylhydroxylase from P. putida. Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for an eugenol dehydrogenase of P. fluorescens E118 corresponded well with the appropriate regions of EhyA and EhyB. Downstream of ehyB, an open reading frame was identified, whose deduced amino acid sequence exhibited up to 71% identity to azurins, representing most probably the gene (azu) of the physiological electron acceptor of the eugenol hydroxylase. The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.