Open AccessBioconversion of Ferulic Acid into Vanillic Acid by Means of a Vanillate-Negative Mutant of Pseudomonas fluorescens Strain BF13
Author(s) -
Claudio Civolani,
Paolo Barghini,
Anna Rita Roncetti,
Maurizio Ruzzi,
A. Schiesser
Publication year - 2000
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.66.6.2311-2317.2000
Subject(s) - vanillic acid , ferulic acid , pseudomonas fluorescens , biochemistry , bioconversion , biology , mutant , strain (injury) , transposon mutagenesis , transposable element , pseudomonas , chemistry , bacteria , gene , food science , genetics , fermentation , anatomy
From a ferulic-acid-degradingPseudomonas fluorescens strain (BF13), we have isolated a transposon mutant, which retained the ability to bioconvert ferulic acid into vanillic acid but lost the ability to further degrade the latter acid. The mutant, BF13-97, was very stable, and therefore it was suitable 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 nutritional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with cells pregrown on M9 medium containingp -coumaric acid (0.1% [wt/vol]) 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 biomass produced 0.23 mg of vanillic acid per h. The genomic region of BF13-97 flanking the transposon's site of insertion was cloned and sequenced revealing two open reading frames of 1,062 (vanA ) and 954 (vanB ) bp, respectively. Thevan genes are organized in a cluster and encode the subunits of the vanillate-O -demethylase, which catalyzes the first step of the vanillate catabolism. Amino acid sequences deduced fromvanA andvanB genes were shown to have high identity with known VanAs and VanBs fromPseudomonas andAcinetobacter spp. Highly conserved regions known to exist in class IA oxygenases were also found in the vanillate-O -demethylase components fromP. fluorescens BF13. The terminal oxygenase VanA is characterized by a conserved Rieske-type [2Fe-2S]R ligand center. The reductase VanB contains a plant-type ferredoxin [2Fe-2S]Fd , flavin mononucleotide, and NAD-ribose binding domains which are located in its C-terminal and N-terminal halves, respectively. Transfer of wild-typevanAB genes to BF13-97 complemented this mutant, which recovered its ability to grow on either vanillic or ferulic acid.