Guanitrypmycin Biosynthetic Pathways Imply Cytochrome P450-mediated Regio- and Stereospecific Guaninyl Transfer Reactions.

Mining microbial genomes including those of Streptomyces reveals the presence of a large number of biosynthetic gene clusters. Unraveling this genetic potential was proven to be a useful approach for novel compound discovery. Here, we report the heterologous expression of two similar P450-associated cyclodipeptide synthases-containing gene clusters in Streptomyces coelicolor and identification of eight rare and novel natural products, C3-guaninyl indole alkaloids guanitrypmycins. Expression of different gene combinations proved that the cyclodipeptide synthases assemble cyclo-L-Trp-L-Phe and cyclo-L-Trp-L-Tyr, which are consecutively and regiospecifically modified by cyclodipeptide oxidases, cytochrome P450 enzymes, and N-methyltransferases. In vivo and in vitro results proved that the P450 enzymes function as key biocatalysts and catalyze the regio- and stereospecific 3α-guaninylation at the indole ring of the tryptophanyl moiety. Isotope exchange experiments provided evidence for the non-enzymatic epimerization of the biosynthetic pathway products via keto-enol tautomerism. This post-pathway modification during cultivation increases furthermore the structural diversity of guanitrypmycins.