Vanillin is one of the most important aromatic flavor compounds used in foods, beverages, perfumes, and pharmaceuticals and is produced on a scale of more than 10 thousand tons per year by the industry through chemical synthesis. Alternative biotechnology-based approaches for the production are based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids, and on de novo biosynthesis, applying fungi, bacteria, plant cells, or genetically engineered microorganisms. Here, the different biosynthesis routes involved in biotechnological vanillin production are discussed.

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...

The industrial application of biocatalysis for the production of natural flavour compounds is illustrated by a discussion of the production of vanillin, gamma-decalactone, carboxylic acids, C6 aldehydes and alcohols ('green notes'), esters, and 2-phenylethanol. Modern techniques of molecular biology and process engineering, such as heterologous expression of genes, site-directed mutagenesis, whole-cell biocatalysis in biphasic systems, and cofactor regeneration for in vitro oxygenation, may result in more biocatalytic processes for the production of flavour compounds in the future.

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD(+)-dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (omega Km) or gentamycin (omega Gm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3'-O-phosphotransferase gene and the gentamycin-3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by omega Km or omega Gm, and the functional vdh gene was replaced by vdh omega Km or vdh omega Gm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HR vdh omega Km, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identify of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase.

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