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Characterization of a vanillate non-oxidative decaroxylation gene cluster from streptomyces sp.d7 Chow, Kevin Toshio

Abstract

The genetics of non-oxidative decarboxylation of aromatic acids to phenolic compounds are poorly understood in both prokaryotes and eukaryotes. Although such reactions have been observed in numerous microorganisms acting on. a variety of substrates, genetic analyses of these processes have not, to my knowledge, been reported in the literature. Previously, I isolated a streptomycete from soil (Streptomyces sp. D7), which efficiently converts 4-hydroxy-3-methoxybenzoic (vanillic) acid to 2-methoxyphenol (guaiacol). Protein two-dimensional gel electrophoresis revealed that several proteins are synthesized in response to vanillic acid, one of which was characterized by partial amino-terminal sequencing, leading to the cloning of a gene cluster from a genomic lambda phage library of Streptomyces sp. D7. This cluster consists of four open reading frames, vdcA (sequencing in progress), vdcB (602 bp), vdcC (1424 bp) and vdcD (239 bp). Protein sequence comparisons suggest that the product of vdcB (201 aa) is similar to phenylacrylate decarboxylase of yeast; the putative products of vdcC (475 aa) and vdcD (80 aa) are similar to hypothetical proteins of unknown function from various microorganisms, and are found in a similar gene cluster in Bacillus subtilis. VdcA is a putative transcriptional regulatory gene. VdcB, vdcC and vdcD homologues are also clustered, along with putative />-cresol methylhydroxylase and vanillin oxidoreductase genes, on the 184 kb catabolic plasmid pNLl of Sphingomonas aromaticivorans F199. Northern blot analysis revealed the synthesis of a 2.5 kb mRNA transcript, which hybridized strongly to a vdcC gene probe, in vanillic acid-induced cells, suggesting that the cluster is under the control of a single inducible promoter. Expression of the entire vdc gene cluster in Streptomyces lividans 1326, as a heterologous host, resulted in that strain acquiring the ability to decarboxylate vanillic acid to guaiacol non-oxidatively. Both Streptomyces strain D7 and recombinant S. lividans 1326 expressing the vdc gene cluster do not, however, decarboxylate structurally similar aromatic acids, suggesting that the system is specific for vanillic acid. By Southern blot hybridization, we detected the presence of the vdc gene cluster in several streptomycetes, including Streptomyces setonii 75Vi2, which has been previously shown to decarboxylate vanillic acid in a nonoxidative reaction. The vanillate decarboxylase catabolic system may be useful as a component for pathway engineering research focused towards the production of valuable chemicals from forestry and agricultural byproducts.

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