Biochemical and genetic analyses of ferulic acid catabolism in Pseudomonassp strain HR199

The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding beta-ketothio lase, which are involved in the catabolism of ferulic acid and eugenol in P seudomonas sp, strain HR199 (DSM7063), were localized on a DNA region cover ed by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp, strain HR199 genomic library in the cosmid pVK100, The nuc leotide sequences of parts of fragments E230 and E94 were determined, revea ling the arrangement of the aforementioned genes. To confirm the function o f the structural genes fcs and ech, they were cloned and expressed in Esche richia coli, Recombinant strains harboring both genes were able to transfor m ferulic acid to vanillin, The feruloyl-CoA synthetase and enoyl-CoA hydra tase/aldolase activities of the fcs and ech gene products, respectively, we re confirmed by photometric assays and by high-pressure liquid chromatograp hy analysis. To prove the essential involvement of the fcs, ech, and aat ge nes in the catabolism of ferulic acid and eugenol in Pseudomonas sp, strain HR199, these genes were inactivated separately by the insertion of omega e lements. The corresponding mutants Pseudomonas sp, strain HRfcs Omega Gm an d Pseudomonas so, strain HRech Omega Km were not able to grow on ferulic ac id or on eugenol, whereas the mutant Pseudomonas sp, strain HRaat Omega Km exhibited a ferulic acid- and eugenol-positive phenotype like the wild type , In conclusion, the degradation pathway of eugenol via ferulic acid and th e necessity of the activation of ferulic acid to the corresponding CoA este r was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a beta-oxidation analogous degradation pathway f or ferulic acid. Moreover, the function of the ech gene product as an enoyl -CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomon as sp. strain HR199 proceeds via a similar pathway to that recently describ ed for Pseudomonas fluorescens AN103.