Combinatorial biosynthesis of antitumor indolocarbazole compounds

Abstract

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...

Enzymatic Generation of the Chromopyrrolic Acid Scaffold of Rebeccamycin by the Tandem Action of RebO and RebD

“…Subsequent N-glycosylation of the aglycone through the anomeric carbon of a glucose moiety (in the case of rebeccamycin) and through both C 1 and C 5 of L-ristosamine, the deoxyhexose sugar of staurosporine, followed by the action of methyltransferases (11), leads to the active natural products (Scheme 1). In the case of staurosporine, the enzymatic generation of the N-C linkage between one indole nitrogen and C5 of the aminodeoxyhexose is of notable interest as a novel transformation (11,12).The biosynthetic gene clusters for rebeccamycin and staurosporine have been sequenced and functionally expressed in the heterologous host Escherichia coli, validating the minimal gene clusters and confirming the identity of key intermediates (7,8). However, little is known about the in vitro behavior, enzymology and mechanistic detail of many of the relevant biosynthetic enzymes.…”

“…The biosynthetic gene clusters for rebeccamycin and staurosporine have been sequenced and functionally expressed in the heterologous host Escherichia coli, validating the minimal gene clusters and confirming the identity of key intermediates (7,8). However, little is known about the in vitro behavior, enzymology and mechanistic detail of many of the relevant biosynthetic enzymes.…”

“…On the basis of previous in vivo studies (8), RebD was proposed to catalyze the formation of CPA (4) from IPA (3) during rebeccamycin biosynthesis. We were interested in determining the activity of RebD in forming CPA (4), and in ascertaining the substrate requirements of this enzyme.…”

“…Genetic studies indicate that RebD is required for formation of chromopyrrolic acid (CPA) 4 (8), and RebP and RebC are then responsible for the oxidative decarboxylation and ring fusion reactions that create the six-ring indolopyrrolocarbazole rebeccamycin aglycone (8). The transformation of compound 4 to the natural product 1 involves conversion of the dicarboxypyrrole ring to a maleimide moiety and represents unusual enzyme chemistry.…”

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

“…Brominated rebeccamycin products have also been detected in culture broths from the producer L. aerocolonigenes and in genetically reconstituted hosts (28,29), indicating the ability of RebH halogenase to accept Br Ϫ in lieu of Cl Ϫ as the halide substrate. When RebF͞ RebH activity was assayed in presence of excess bromide, a Trp-derived product was observed with [M ϩ H] ϩ of 283 and 285 in a 1:1 ratio characteristic of a monobrominated species (Fig.…”