The blm gene cluster is characterized by a hybrid NRPS-PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide-polyketide metabolites.
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...
The indolocarbazole family of natural products, including the biosynthetically related bisindolylmaleimides, is reviewed (with 316 references cited). The isolation of indolocarbazoles from natural sources and the biosynthesis of this class of compounds are thoroughly reviewed, including recent developments in molecular genetics, enzymology and metabolic engineering. The biological activities and underlying modes of action displayed by natural and synthetic indolocarbazoles is also presented, with an emphasis on the development of analogs that have entered clinical trials for its future use against cancer or other diseases.
PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.
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