Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (؊)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min ؊1 M ؊1 ) for L-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-L-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, ⌬iptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-L-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.Natural products have been an important resource for drug discovery and development. Actinomycetes have been a rich source of natural products, and a wide variety of these chemicals have been used as medicinal drugs (7, 40) and as bioprobes (56) for the elucidation of biological functions. Recently, the screening of bioactive compounds from microorganisms has often resulted in the identification of previously isolated compounds. The decreasing hit rate for new chemicals has reduced the advantage of natural product screening. However, genome sequencing of Streptomyces species highlighted numerous potential areas with metabolic diversity (4, 25, 42). The number of cryptic gene clusters was much larger than that of secondary metabolites identified from each strain. In addition, the cryptic gene clusters contained genes encoding plenty of unique modification enzymes that had the potential to expand the chemical diversity in drug seeds.To uncover cryptic gene clusters that might code for biosynthesis of secondary metabolites, genome sequence-guided metabolite identification has been performed in combination with heterologous expression, gene knockout, and complementation analyses ...
