Discovery of indolotryptoline antiproliferative agents by homology-guided metagenomic screening

Abstract

Natural product discovery by random screening of broth extracts derived from cultured bacteria often suffers from high rates of redundant isolation, making it ever more challenging to identify novel biologically interesting natural products. Here we show that homology-based screening of soil metagenomes can be used to specifically target the discovery of new members of traditionally rare, biomedically relevant natural product families. Phylogenetic analysis of oxy-tryptophan dimerization gene homologs found within a large soil DNA library enabled the identification and recovery of a unique tryptophan dimerization biosynthetic gene cluster, which we have termed the bor cluster. When heterologously expressed in Streptomyces albus, this cluster produced an indolotryptoline antiproliferative agent with CaMKIIδ kinase inhibitory activity (borregomycin A), along with several dihydroxyindolocarbazole anticancer/antibiotics (borregomycins B-D). Similar homology-based screening of large environmental DNA libraries is likely to permit the directed discovery of new members within other previously rare families of bioactive natural products.antitumor | uncultured microbes | bisindole alkaloid | indenotryptoline

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.…”

Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

“…In addition, it couples each observed phenotype to a single fragment of cloned metagenomic DNA, making it possible to simultaneously identify specific effector molecules and the specific effector genes that encode these molecules. Functional metagenomics has been used to isolate small molecules and proteins from soil metagenomes (15)(16)(17)(18)(19)(20)(21)(22)(23)(24); however, its application to the human microbiome has to date been very limited (16,25,26). In this study, a set of arrayed, large-insert cosmid libraries hosted in Escherichia coli was created from DNA isolated from the stool of three patients who, based on their phenotype, were predicted to have different commensal bacteria cohorts.…”

Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters

“…In an effort to expand the observed natural diversity of indolotryptolines and potentially improve their therapeutic prospects, we PCR-screened eDNA cosmid libraries using degenerate primers targeting genes that encode for tryptophan dimerization enzymes (32,38). This led to the discovery of the Lzr gene cluster, which closely resembles the cladoniamide and BE-54017 gene clusters; however, it is predicted to encode tailoring enzymes (e.g., an extra halogenase and a cytochrome P450 oxidase) that are not used in the biosynthesis of any known indolotryptoline, suggesting that it should encode for a novel indolotryptoline congener.…”