Bacteriocins are antimicrobial peptides produced by bacteria, and their production is regarded as a desirable probiotic trait. We found that Lactobacillus gasseri LM19, a strain isolated from human milk, produces several bacteriocins, including a novel bacteriocin, gassericin M. These bacteriocins were purified from culture and synthesised to investigate their activity and potential synergy. L. gasseri LM19 was tested in a complex environment mimicking human colon conditions; it not only survived, but expressed the seven bacteriocin genes and produced short-chain fatty acids. Metagenomic analysis of these in vitro colon cultures showed that co-inoculation of L. gasseri LM19 with Clostridium perfringens gave 16S ribosomal RNA metagenomic profiles with more similarity to controls than to vessels inoculated with C. perfringens alone. These results indicate that L. gasseri LM19 could be an interesting candidate for maintaining homeostasis in the gut environment.

Heterologous expression, pathway mutations and detailed metabolomic analysis were used to deduce a model for the biosynthesis of thiostreptamide S4, which belongs the thioamitide family of antitumour RiPPs.

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a wide range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor annotation of the short precursor peptides that are ultimately modified into the final molecule. Here, we utilise a previously described genome mining tool, RiPPER, to identify novel RiPP precursor peptides near YcaO-domain proteins, enzymes that catalyse various RiPP post-translational modifications including heterocyclisation and thioamidation. Using this dataset, we identified a novel and diverse family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albidoflavus J1074 (formerly known as Streptomyces albus) was characterised, leading to the discovery of streptamidine, a novel amidine-containing RiPP. This new BGC family highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.

Bacterial secondary metabolites represent an invaluable source of bioactive molecules for the pharmaceutical and agrochemical industries. Although screening campaigns for the discovery of new compounds have traditionally been strongly biased towards the study of soil-dwelling Actinobacteria, the current antibiotic resistance and discovery crisis has brought a considerable amount of attention to the study of previously neglected bacterial sources of secondary metabolites. The development and application of new screening, sequencing, genetic manipulation, cultivation and bioinformatic techniques have revealed several other groups of bacteria as producers of striking chemical novelty. Biosynthetic machineries evolved from independent taxonomic origins and under completely different ecological requirements and selective pressures are responsible for these structural innovations. In this review, we summarize the most important discoveries related to secondary metabolites from alternative bacterial sources, trying to provide the reader with a broad perspective on how technical novelties have facilitated the access to the bacterial metabolic dark matter.

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 .