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

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceed s via coniferyl alcohol coniferyl aldehyde, ferulic acid, vanillin, vanilla te and protocatechuate, which is further degraded by the ortho-cleavage pat hway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which cat alyses the NAD (+) dependent oxidation of vanillin to vanillate, was inacti vated by the insertion of omega elements into the vdh gene, which was chara cterized recently. Omega elements conferring resistance against kanamycin ( Omega Km) or gentamycin (Omega Gm) were constructed by polymerase chain rea ction amplification of the aminoglycoside 3'-O-phosphotransferase gene and the gentamycin-3-acetyltransferase gene, using the plasmids pSUP5011 and pB BR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by Omega Km or nGm, and the functional vdh gene was re placed by vdh Omega Km or vdh Omega Gm in Pseudomonas sp. strain HR199 by h omologous recombination. Cells of the mutant Pseudomonas sp, strain HRvdh O mega Km, pregrown on gluconate, accumulated up to 2.9 mM vanillin during in cubation in mineral medium with 6.5 mM eugenol. As a result of another vani llin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characteri zation of the purified VDH-II revealed the identity of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase.