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...

Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude ex tracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of a and , subunits (Mrs, 35,5 00 and 36,000, respectively), and a tetrameric a2,12 structure was most likely for the native protein. The mole cular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were det ermined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the a and ,1 subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, aco)B, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been clonei recently (C. Frund, H. Priefert, A. Steinbuchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, an d the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together woith the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading fr ame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial a54-dependent promoter consensus sequence.The amino acid sequences deduced from aco. 4 and acoB for the a subunit (Mr,35,243) and the 0 subunit (Mr, 35,788) exhibited significant homologies to th e primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whei 'eas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltrarw,ferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed.Alcaligenes eutrophus has been studied with respe ct to the degradation of acetoin. Acetoin-grown cells of A. eb rtrophus are devoid of 2,3-butanediol dehydrogenase and acetoin dehydrogenase (diacetyl forming; EC 1.1.1.5), and mutants which lack 2,3-butanediol dehydrogenase (73) are stilil able to utilize acetoin as the sole carbon source for grovvth (72). Therefore, this bacterium lacks the 2,3-butanedi oil cycle which was described by Juni and Heym (36) (23,59). As insertional inactivation of this gene caused a pleiotropic effect and as these mutants were unable to synthesize proteins which are essential for degradation of acetoin, it was concluded that expression of the corresponding genes is rpoN dependent (23).Physiological studies localized the genes for Ao:DCPIP OR and FMP on restriction fragments A and C, which are closely linked in the genome (23), whereas the gene for acetaldehyde dehydrogenase II was localized on restriction fragment D (52a). The present study was aimed at isolation of FMP and Ao:DCPIP OR and identification and characterization of their structural genes. ...

The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding β-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol inPseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, andaat genes in the catabolism of ferulic acid and eugenol inPseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsΩGm andPseudomonas sp. strain HRechΩKm were not able to grow on ferulic acid or on eugenol, whereas the mutantPseudomonas sp. strain HRaatΩKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a β-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103.

The gene loci ech, encoding enoyl-CoA hydratase/aldolase, and fcs, encoding an unusual feruloyl-CoA synthetase, which are involved in the bioconversion of ferulic acid to vanillin by the gram-positive bacterium Amycolatopsis sp. strain HR167, were localized on a 4,000 bp PstI fragment (P40). The nucleotide sequence of P40 was determined, revealing open reading frames of 864 bp and 1,476 bp, representing ech and fcs, respectively. The deduced amino acid sequences of ech exhibited 62% amino acid identity to the enoyl-CoA hydratase/aldolase from Pseudomonas sp. strain HR199 and the enoyl-CoA hydratase/lyase from P. fluorescens strain AN103. The deduced amino acid sequences of fcs exhibited up to 37% amino acid identity to long-chain fatty acid coenzymeA ligases but no significant similarity to the feruloyl-CoA synthetase of Pseudomonas sp. strain HR199. Fragment P40 was cloned in pBluescript SK- and fcs and ech were expressed in Escherichia coli. Recombinant strains were able to transform ferulic acid to vanillin. In crude extracts of these recombinant strains, feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities were detected by photometric assay and high-performance liquid chromatography. The obtained data suggest that ferulic acid degradation in the gram-positive Amycolatopsis sp. strain HR167 proceeds via a pathway similar to that recently described for the gram-negative P. fluorescens strain AN103 and Pseudomonas sp. strain HR199.