Characterization of a vanillic acid non-oxidative decarboxylation gene cluster from Streptomyces sp D7

The genetics of non-oxidative decarboxylation of aromatic acids are poorly understood in both prokaryotes and eukaryotes. Although such reactions have been observed in numerous micro-organisms acting on a variety of substrates, the genes encoding enzymes responsible for these processes have not, to our knowledge, been reported in the literature. Here, the isolation of a streptomycete from soil (Streptomyces sp. D7) which efficiently converts 4-hydroxy-3-methoxybenzoic acid (vanillic acid) to 2-methoxyphenol (guaiacol) is described. Protein two-dimensional gel analysis revealed that several proteins were synthesized in response to vanillic acid. One of these was characterized by partial amino-terminal sequencing, leading to the cloning of a gene cluster from a genomic DNA lambda phage library, consisting of three ORFs, 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 micro-organisms, and are found in a similar cluster in Bacilius subtilis. Northern blot analysis revealed the synthesis of a 2.5 kb mRNA transcript 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 sp, 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. This catabolic system may be useful as a component for pathway engineering research focused towards the production of valuable chemicals from forestry and agricultural by-products.