Expansion of bisindole biosynthetic pathways by combinatorial construction

Abstract

Cladoniamides are indolotryptoline natural products that derive from indolocarbazole precursors. Here, we present a microbial platform to artificially redirect the cladoniamide pathway to generate unnatural bisindoles for drug discovery. Specifically, we target glycosyltransferase, halogenase, and oxidoreductase genes from the phylogenetically related indolocarbazole rebeccamycin and staurosporine pathways. We generate a series of novel compounds, reveal details about the substrate specificities of a number of enzymes, and set the stage for future efforts to develop new catalysts and compounds by engineering of bisindole genes. The strategy for structural diversification we use here could furthermore be applied to other natural product families with known biosynthetic genes.

Keywords: bisindole biosynthesis; combinatorial engineering; flavin-dependent monooxygenase; glycosyltransferase; halogenase; synthetic pathways.

Figure 1

Analytical HPLC profiles of various genetic mutants constructed for the combinatorial study (detection wavelength = 348 nm).

Figure 2

Production of non-chlorinated cladoniamides C (5), H (13) and I (14) from engineered strain S. coelicolor YD17. S. coelicolor YD17 produces 5 as a major clone-specific metabolite along with cladoniamide H (13) and I (14) (detection wavelength = 211 nm). Selective ion monitoring at m/z = 404 reveals 5 as clone-specific metabolite. Selective ion monitoring at m/z = 398 reveals 13 and 14 as clone-specific metabolites.

Figure 3

In vivo biotransformation of 17 and 18/19 with claX1. (A) HPLC traces for in vivo biotransformation assay. Insert, only the peak at the left was consumed in the in vivo biotransformation assay of E. coli/pET28a-claX1 + 18/19, which is assumed to be 19, based on the corresponding production of 21. Blue, E. coli/pET28a + 18/19. Red, E. coli/pET28a-claX1 + 18/19. Detection wavelength: 348 nm. (B) Scheme for conversion by ClaX1.

Scheme 1

Biosynthesis of bisindoles. Two molecules of L-tryptophan are converted to an indolocarbazole by the action of four enzymes (RebODPC/StaODPC/ClaODPC). Pathway-specific enzymes then generate the final rebeccamycin (1), staurosporine (2), and cladoniamide (3-5) structures. All gene clusters are shown with the O-D-P-C genes in blue. Genes investigated in the combinatorial engineering study are chlorinase gene rebH (in green), glucosyltransferase gene rebG (in pink), and oxidoreductase gene staC (light blue). The unique cladoniamide genes are shown in red (methyltransferase genes), orange (flavoenzyme genes claX1 and claX2), green (halogenase and associated reductase gene), gray (unknown, claY), and black (regulator and transporter genes). For the rebeccamycin pathway: R₁=Cl, R₂=R₃=H, R₄=O; for the staurosporine pathway: R₁=R₂=R₃=H, R₄=2H; for the cladoniamide pathway: R₁=R₅=H, R₂=R₃=H/Cl, R₄=O.

Scheme 2

A microbial platform for combinatorial construction of new bisindole compounds. (A) Natural product and its native producer. (B) A library of biosynthetic clusters arise from isolation and modification of the natural biosynthetic gene cluster. Gray boxes indicate inactivated genes. (C) Reconstitution of the artificial pathways in optimized heterologous hosts. (D) Metabolic analysis for strain-specific metabolite(s). (E) Large-scale fermentation for target compound(s) purification. (F) Structure elucidation.

Scheme 3

Combinatorial biosynthesis of bis-indole metabolites.