Bioconversion of ferulic acid into vanillic acid by means of a vanillate-negative mutant of Pseudomonas fluorescens strain BF13

From a ferulic-acid-degrading Pseudomonas fluorescens strain (BF13), we hav e isolated a transposon mutant, which retained the ability to bioconvert fe rulic acid into vanillic acid but lost the ability to further degrade the L atter acid. The mutant, BF13-97, was very stable, and therefore it was suit able to be used as a biocatalyst for the preparative synthesis of vanillic acid from ferulic acid. By use of resting cells we determined the effect on the bioconversion rate of several parameters, such as the addition of nutr itional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtain ed with cells pregrown on M9 medium containing p-coumaric acid (0.1% [wt/vo l]) as a sole carbon source and yeast extract (0.001% [wt/vol]) as a source of nutritional factors. Under these conditions, 1 mg (wet weight) of bioma ss produced 0.23 mg of vanillic acid per h, The genomic region of BF13-97 f lanking the transposon's site of insertion was cloned and sequenced reveali ng two open reading frames of 1,062 (varA) and 954 (vanB) bp, respectively. The van genes are organized in a cluster and encode the subunits of the va nillate-O-demethylase, which catalyzes the first step of the vanillate cata bolism, Amino acid sequences deduced from vanA and vanB genes were shown to have high identity with known VanAs and VanBs from Pseudomonas and Acineto bacter spp, Highly conserved regions known to exist in class IA oxygenases were also found in the vanillate-O-demethylase components from P.fluorescen s BF13, The terminal oxygenase VanA is characterized by a conserved Rieske- type [2Fe-2S](R) ligand center, The reductase VanB contains a plant-type fe rredoxin [2Fe-2S](Fd), flavin mononucleotide, and NAD-ribose binding domain s which are located in its C-terminal and N-terminal halves, respectively. Transfer of wild-type vanAB genes to BF13-97 complemented this mutant, whic h recovered its ability to grow on either vanillic or ferulic acid.