The catabolism of ferulic acid in Pseudomonas fluorescens AN103 proceeds via vanillin, vanillic acid, and protocatechuic acid. The inactivation of the vanillin dehydrogenase of P. fluorescens AN103, which catalyzes the oxidation of vanillin to vanillic acid, was pursued as an approach for developing a biotechnological process for the production of biovanillin. In order to inactivate the vdh gene, a plasmid construction was made in which an omega element conferring resistance to kanamycin (Omega Km) was inserted into a truncated vdh gene (Delta vdh). Subsequently, this construct was used to replace the functional vdh gene with the disrupted Delta vdh Omega Km in R fluorescens AN 103 by homologous recombination using a triparental mating approach. The successful inactivation of the gene was confirmed both by spectrophotometric and specific activity staining of crude cell extracts after native electrophoresis. However, inactivation of the vdh gene in P fluorescens AN103 did not result in the expected accumulation of vanillin in media despite the absence of other enzymes exhibiting vanillin dehydrogenase activity. Further characterization showed that the P. fluorescens AN103vdh Omega Km mutant was unable to grow on vanillin as well as on ferulic acid as a sole carbon source. The inability to degrade ferulic acid was attributed to the lack of 4CL activity resulting from the inactivation of the vdh gene. (c) 2005 Elsevier Inc. All rights reserved.