Biosynthetic Gene Cluster for the Cladoniamides, Bis-Indoles with a Rearranged Scaffold

Abstract

The cladoniamides are bis-indole alkaloids isolated from Streptomyces uncialis, a lichen-associated actinomycete strain. The cladoniamides have an unusual, indenotryptoline structure rarely observed among bis-indole alkaloids. I report here the isolation, sequencing, and annotation of the cladoniamide biosynthetic gene cluster and compare it to the recently published gene cluster for BE-54017, a closely related indenotryptoline natural product. The cladoniamide gene cluster differs from the BE-54017 gene cluster in gene organization and in the absence of one N-methyltransferase gene but otherwise contains close homologs to all genes in the BE-54017 cluster. Both gene clusters encode enzymes needed for the construction of an indolocarbazole core, as well as flavin-dependent enzymes putatively involved in generating the indenotryptoline scaffold from an indolocarbazole. These two bis-indolic gene clusters exemplify the diversity of biosynthetic routes that begin from the oxidative dimerization of two molecules of l-tryptophan, highlight enzymes for further study, and provide new opportunities for combinatorial engineering.

Site-Selective C–H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling

“…Only two of the nine enzymes in this activity cluster were present in the Indole Subnetwork, whereas four were in the Phenol Subnetwork. Halogenase 1-F11, from an unannotated subnetwork within the Indole Subnetwork (38% identical to tryptophan-5 halogenase ClaH 72 ), and 2-C01, a halogenase in the same subnetwork as the lanthipeptide indole halogenase MibH (36% identical 54 ), were capable of chlorinating multiple phenols, 2,4-dihydroxyacetophenone (2,4-DHAP) and 7-hydroxycoumarin. The FDH 2-C01 was particularly versatile in halide scope.…”

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

“…In RebH and PrnA, L-tryptophan binds in the active site such that the C7–H bond is positioned proximal to the "Cl + " species and undergoes electrophilic aromatic substitution (41, 42). In addition to 7-Trp-FDHs (45, 46), both 5- (47–49) and 6-Trp-FDHs (50–54) have been characterized. As previously noted, site-selectivity for a single benzo-position, especially in the presence of the more electronically activated pyrrolo position, is remarkable.…”

Submerged fermentation of Streptomyces uncialis providing a biotechnology platform for uncialamycin biosynthesis, engineering, and production

“…It spans loci AB852_16715 to AB852_16630 (GenBank accession no. LFBV01000003), including the whole cladoniamide (CLA) BGC (Ryan, 2011 ). In E. coli BW25113/pIJ790, neomycin resistance cassette of cosmid pBS18008 was replaced with an apramycin resistance cassette as S. uncialis is naturally resistant to kanamycin, to give pBS18009.…”