Molecular characterization of genes of Pseudomonas sp. strain HR199 involved in bioconversion of vanillin to protocatechuate

Abstract

The gene loci vdh, vanA, and vanB, which are involved in the bioconversion of vanillin to protocatechuate by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a novel vanillin dehydrogenase (vdh) and the two subunits of a vanillate demethylase (vanA and vanB), respectively. These genes were localized on an EcoRI fragment (E230), which was cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The vdh gene was identified on a subfragment (HE35) of E230, and the vanA and vanB genes were localized on a different subfragment ( Pseudomonas strains, which were unable to utilize vanillin or vanillate as carbon sources, respectively, conferred the ability to grow on these substrates to these bacteria.Vanillin is one of the most important aromatic flavor compounds in the production of flavors for foods and fragrances for perfumes. Synthetic vanillin is currently produced from petrochemicals and from lignin (11). Vanillin is also wellknown as a metabolic intermediate in the catabolism of phenolic stilbenes such as eugenol, ferulic acid, and lignin (10,47,51,53). However, the degradation of this widely distributed metabolite has not been examined in detail so far.The pathway for the biodegradation of vanillic acid is well understood. The aromatic methyl ether is demethylated via hydroxylation, generating an unstable hemiacetal, which decomposes to protocatechuic acid and formaldehyde (8,9,41,42). The genes vanA and vanB from Pseudomonas sp. strain ATCC 19151, which code for the monooxygenase, which catalyzes this reaction, were cloned and sequenced (5). This vanillate demethylase consisted of two different subunits. The vanB gene product seemed to be related to ferredoxins but was considerably larger. The identity of the vanA gene product remained unknown.In contrast, no molecular data are available for the enzymes catalyzing the conversion of vanillin to vanillate. In the present paper, we describe the cloning, molecular characterization, and heterologous expression of Pseudomonas sp. strain HR199 genes which are responsible for the bioconversion of vanillin to protocatechuate (Fig. 1). MATERIALS AND METHODSBacterial strains and plasmids. The Pseudomonas, Alcaligenes eutrophus, and Escherichia coli strains and the plasmids used in this study are listed in Table 1.Growth of bacteria. Cells of E. coli were grown at 37ЊC in Luria-Bertani (LB) medium or in M9 minimal medium (43). Cells of Pseudomonas and A. eutrophus were grown at 30ЊC either in a nutrient broth (NB) medium (0.8%, wt/vol) (43) or in mineral salts medium (MM) (45) supplemented with carbon sources as indicated in the text. Vanillin, vanillate, and protocatechuate were dissolved in dimethyl sulfoxide and added to the medium at final concentrations of 0.1% (wt/vol). Tetracycline and kanamycin were used at final concentrations of 25 and 300 g/ml, respectively, for the Pseudomonas sp. strains.Nitrosoguanidine mutagenesis. The nitrosoguanidine mutagenesis of the Pseudomonas sp. strains was performed by a mo...

Evaluating the lingering effect of livestock grazing on functional potentials of microbial communities in Tibetan grassland soils

“…In contrast, the abundance of amyA, nplT, pulA, pectinase, ara_fungi, mannanase, and xylA associated with the degradation of labile C (starch, pectin, or hemicellulose) decreased in post-grazing grassland soils of the 3400-m site, whereas those of vdh, vanA, glx, and lip associated with degradation of recalcitrant C (aromatics or lignin) increased (Fig. 3b), suggesting a shift from use of labile C toward recalcitrant C. The vdh and vanA genes associated with the bioconversion of vanillin to protocatechuate for aromatics degradation were abundant in Pseudomonas sp., which was consistent with the results of a previous study (Priefert et al 1997). The most abundant lip gene was similar to the sequence from Trametes versicolor, which was shown to play an important role in lignin oxidation (Bourbonnais et al 1995;Johansson and Nyman 1993).…”

Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358

“…l), which undergoes ring fission by the ortho pathway. T h e genetics of the pathway from vanillic acid to protocatechuic acid have been described by Brunel & Davison (1988) and also recently by Priefert et al (1997). Both studies showed that t w o proteins (VanA and VanB) form a vanillate demethylase complex which demethylates vanillic acid to protocatechuic acid.…”

Molecular characterization of the phenolic acid metabolism in the lactic acid bacteria Lactobacillus plantarum

“…Phenolic acids also serve as a signal and induce virulence gene expression in the plant-associated Agrobacterium tumefaciens [20]. Some Pseudomonas strains as well as Acinetobacter calcoaceticus are able to use these acids as the sole source of carbon for growth [17,24,28,30]. Moreover, ferulic acid is exploited to produce value-added aromatic compounds, such as vanillin.…”