Functional analysis of the vanillin pathway in a vdh-negative mutant strain of Pseudomonas fluorescens AN103

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 (ΩKm) was inserted into a truncated vdh gene (Δvdh). Subsequently, this construct was used to replace the functional vdh gene with the disrupted ΔvdhΩKm in P. fluorescens AN103 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 AN103vdhΩKm 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.