Rebeccamycin and staurosporine are natural products with antitumor properties, which belong to the family of indolocarbazole alkaloids. An intense effort currently exists for the generation of indolocarbazole derivatives for the treatment of several diseases, including cancer and neurodegenerative disorders. Here, we report a biological process based on combinatorial biosynthesis for the production of indolocarbazole compounds (or their precursors) in engineered microorganisms as a complementary approach to chemical synthesis. We have dissected and reconstituted the entire biosynthetic pathway for rebeccamycin in a convenient actinomycete host, Streptomyces albus. This task was achieved by coexpressing different combinations of genes isolated from the rebeccamycin-producing microorganism. Also, a gene (staC) was identified in staurosporine-producing microbes and was shown to have a key role to differentiate the biosynthetic pathways for the two indolocarbazoles. Last, incorporation of the pyrH and thal genes, encoding halogenases from different microorganisms, resulted in production of derivatives with chlorine atoms at novel positions. We produced >30 different compounds by using the recombinant strains generated in this work. (Fig. 1). Various biological activities have been reported for indolocarbazoles, but the greatest interest is focused on compounds that possess antitumor and neuroprotective properties (2-4). These activities may be due to different mechanisms of action, including DNA intercalation, inhibition of DNA topoisomerases, and inhibition of protein kinases. Great efforts are made to generate indolocarbazole derivatives with improved properties for the treatment of cancer, neurodegenerative disorders, and diabetes-associated pathologies, and several analogs have entered clinical trials (2-7).Studies on the biosynthesis of rebeccamycin and staurosporine in the producing microorganisms have shown that the indolocarbazole core is formed by decarboxylative fusion of two tryptophan-derived units, whereas the sugar moiety is derived from glucose (8, 9). Recently, we cloned and characterized the rebeccamycin biosynthetic gene cluster from the actinomycete Lechevalieria aerocolonigenes (formerly Saccharotrix aerocolonigenes) (10). Expression of the entire gene cluster and of different subsets of genes in a heterologous host yielded rebeccamycin and three biosynthetic intermediates (10). The same cluster was later isolated by other researchers (11, 12) and expressed at a low level in Escherichia coli (12), and different insertional inactivation mutants were generated in the producer organism (11). The entire staurosporine gene cluster has been isolated from Streptomyces sp. TP-A0274 (13), although a previous patent application reported the identification of some genes involved in biosynthesis of the staurosporine sugar moiety in Streptomyces longisporoflavus (14).Combinatorial biosynthesis is a recent addition to the metabolic engineering toolbox by which genes responsible for individual metabolic reacti...

SummaryThe indolocarbazole staurosporine is a potent inhibitor of a variety of protein kinases. It contains a sugar moiety attached through C-N linkages to both indole nitrogen atoms of the indolocarbazole core. Staurosporine biosynthesis was reconstituted in vivo in a heterologous host Streptomyces albus by using two different plasmids: the 'aglycone vector' expressing a set of genes involved in indolocarbazole biosynthesis together with staG (encoding a glycosyltransferase) and/or staN (coding for a P450 oxygenase), and the 'sugar vector' expressing a set of genes responsible for the biosynthesis of the sugar moiety. Attachment of the sugar to the two indole nitrogens of the indolocarbazole core was dependent on the combined action of StaG and StaN. When StaN was absent, the sugar was attached only to one of the nitrogen atoms, through an N-glycosidic linkage, as in the indolocarbazole rebeccamycin. The StaG glycosyltransferase showed flexibility with respect to the sugar donor. When the 'sugar vector' was substituted by constructs directing the biosynthesis of L -rhamnose, Ldigitoxose, L -olivose and D -olivose, respectively, StaG and StaN were able to transfer and attach all of these sugars to the indolocarbazole aglycone.

We report the generation of novel glycosylated indolocarbazoles by combinatorial biosynthesis, and the identification of two novel potent and selective compounds inhibitors of JAK2 and Ikkb kinases.