Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene

• Published in: Applied microbiology and biotechnology Vol. 52; no. 6; pp. 820 - 828
• Main Authors: Overhage, J, Priefert, H, Rabenhorst, J, Steinbuchel, A
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01-11-1999; Springer Nature B.V
• Subjects: Aldehyde Oxidoreductases - antagonists & inhibitors; Aldehyde Oxidoreductases - genetics; Aldehyde Oxidoreductases - metabolism; Aldehydes; Bacteria; Benzaldehydes - metabolism; Biological and medical sciences; Biotechnology; Biotransformation; Chromatography, High Pressure Liquid; Dehydrogenase; Dehydrogenases; Electrophoresis, Polyacrylamide Gel; enyzme inactivation; eugenol; Eugenol - metabolism; Fundamental and applied biological sciences. Psychology; Gene Deletion; gene targeting; Genetic technics; metabolism; Methods. Procedures. Technologies; Microbiology; Mutagenesis, Insertional; Mutant screening; Prokaryotes; Pseudomonas; Pseudomonas - enzymology; Pseudomonas - genetics; Time Factors; vanillin; vanillin dehydrogenase; vdh gene; Pseudomonadales; Enzymatic synthesis; Purification; Enzyme; Pseudomonas; Enzymatic activity; Biotransformation; Bacteria; Pseudomonadaceae; Aromatic compound; Oxidoreductases; Mutation; Kinetic parameter; Vanillin
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530051598

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD(+ -dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (omegaKm) or gentamycin (omegaGm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3'-O-phosphotransferase gene and the gentamycin-3-acetyl-transferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by omegaKm or omegaGm, and the functional vdh gene was replaced by vdhomegaKm or vdhomegaGm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HRvdhomegaKm, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identity of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase.