The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, which are proposed to be introduced by YcaO-TfuA proteins. Using RiPPER, we show that previously undescribed RiPP gene clusters encoding YcaO and TfuA proteins are widespread in Actinobacteria and encode a highly diverse landscape of precursor peptides that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new structural class of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis .

Thiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very...

Heterologous expression of biosynthetic gene clusters (BGCs) represents an attractive route to the production of new natural products, but is often hampered by poor yields. It is therefore important to develop tools that enable rapid refactoring, gene insertion/deletion, and targeted mutations in BGCs. Ideally, these tools should be highly efficient, affordable, accessible, marker free, and flexible for use with a wide range of BGCs. Here, we present a one-step yeast-based method that enables efficient, cheap, and flexible modifications to BGCs. Using the BGC for the antibiotic bottromycin, we showcase multiple modifications including refactoring, gene deletions and targeted mutations. This facilitated the construction of an inducible, riboswitch-controlled pathway that achieved a 120-fold increase in pathway productivity in a heterologous streptomycete host. Additionally, an unexpected biosynthetic bottleneck resulted in the production of a suite of new bottromycin-related metabolites.

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, and it has been proposed that a YcaO-TfuA pair of proteins also catalyzes their formation. Potential biosynthetic gene clusters encoding YcaO and TfuA protein pairs are common in Actinobacteria but the chemical diversity generated by these pathways is almost completely unexplored. A RiPPER analysis reveals a highly diverse landscape of precursor peptides encoded in previously undescribed gene clusters that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new family of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

Bottromycin is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by several streptomycetes, including the plant pathogen Streptomyces scabies. There is significant interest in this molecule as it possesses strong antibacterial activity against clinically relevant multidrug resistant pathogens and is structurally distinct from all other antibiotics. However, studies into its efficacy are hampered by poor yields. An understanding of how bottromycin biosynthesis is regulated could aid the development of strategies to increase titres. Here, we use 5-tag-RNA-seq to identify the transcriptional organization of the gene cluster, which includes an internal transcriptional start site that precedes btmD, the gene that encodes the bottromycin precursor peptide. We show that the gene cluster does not encode a master regulator that controls pathway expression and instead encodes a regulatory gene, btmL, which functions as a modulator that specifically affects the expression of btmD but not genes up-or downstream of btmD. In order to identify non-cluster associated proteins involved in regulation, proteins were identified that bind to the main promoter of the pathway, which precedes btmC. This study provides insights into how this deceptively complex pathway is regulated in the absence of a pathway specific master regulator, and how it might coordinate with the central metabolism of the cell.

Thiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very few thioamitide biosynthetic steps have been elucidated, even though the gene clusters of multiple thioamitides have been identified. We hypothesised that engineering the thiostreptamide S4 gene cluster in a heterologous host could provide insights into its biosynthesis when coupled with untargeted metabolomics and targeted mutations of the precursor peptide. Modified gene clusters were constructed, and in-depth metabolomics enabled a detailed understanding of the biosynthetic pathway, including the identification of an effector-like protein critical for amino acid dehydration. We use this biosynthetic understanding to bioinformatically identify new widespread families of RiPP biosynthetic gene clusters, paving the way for future RiPP discovery and engineering.

Microbial natural products are specialized metabolites that have long been a rich source of human therapeutics. While the chemical diversity encoded in the genomes of microbes is believed to be large, the productivity of this modality has waned as traditional fermentation-based discovery methods have been plagued by high-rates of rediscovery, inefficient scaling, and incompatibility with target-based drug discovery. Here, we demonstrate a scalable discovery platform that couples dramatically improved assembly of deep-sequenced metagenomic samples with highly efficient, target-focused, in silica search strategies and synthetic biology to discover multiple novel inhibitors of human methionine aminopeptidase-1 (HsMetAP1), a validated oncology target. For one of these novel inhibitors, metapeptin B, we demonstrate sub-micromolar potency, strong selectivity for HsMetAP1 over HsMetAP2 and leverage natural congeners to rapidly elucidate key SAR elements. Our “next-gen” discovery platform overcomes many of the challenges constraining traditional methods, implies the existence of vast, untapped chemical diversity in nature, and demonstrates computationally-enabled precision discovery of modulators of human proteins of interest.