Functional analyses of genes involved in the metabolism of ferulic acid in Pseudomonas putida KT2440

Pseudomonas putida KT2440 is a physiologically extremely versatile non-pathogenic bacterium that is applied as a "biosafety strain" in biotechnological processes, as authorized by the USA National Institute of Health. Analysis of the P. putida KT2440 whole-genome sequence revealed the genetic organization of the genes fcs, ech, and vdh, which are essential for ferulic acid conversion to vanillic acid via vanillin. To confirm the physiological function of these structural genes as feruloyl-CoA synthetase (Fcs), enoyl-CoA hydratase/aldolase (Ech), and vanillin dehydrogenase (Vdh), respectively, they Were cloned and expressed in Escherichia coli. Recombinant strains harboring fcs and ech were able to transform ferulic acid to vanillin. The enzyme activities of Fcs and Vdh were determined in protein extracts of these cells. The essential involvement of fcs, ech and vdh in the catabolism of ferulic acid in P. putida KT2440 was proven by separately inactivating each gene by insertion of Omega-elements. The corresponding mutant strains KT2440fcsOmegaKm, KT2440echOmegaKm, and KT2440vdhOmegaKm were not able to grow on ferulic acid. The potential application of P. putida KT2440 and the mutant strains in biotechnological vanillin production process is discussed.