Biology subjects

Lacret, R.

Publications and source records attributed to Lacret, R..

4 recordsLinked to original sources

bioRxiv2020

Understanding Thioamitide Biosynthesis Using Pathway Engineering and Untargeted Metabolomics

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.

microbiology↗

bioRxiv2020

Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor detection 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, diverse and highly conserved family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albus J1074 was characterised, leading to the discovery of streptamidine, a novel-amidine containing RiPP. This highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.

microbiology↗

bioRxiv2018

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool

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.

microbiology↗

bioRxiv2017

Discovery and biosynthesis of the antibiotic bicyclomycin in distant bacterial classes

Bicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesised by Streptomyces cinnamoneus DSM 41675. BCM is structurally characterized by a core cyclo(L-Ile-L-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesised by a cyclodipeptide synthase and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded in the genomes of hundreds of Pseudomonas aeruginosa isolates distributed globally, and heterologous expression of the pathway from P. aeruginosa SCV20265 demonstrated that the product is chemically identical to BCM produced by S. cinnamoneus. Overall, putative BCM gene clusters have been found in at least seven genera spanning Actinobacteria and Proteobacteria (Alpha-, Beta- and Gamma-). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.\n\nIMPORTANCEBicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known producing organism Streptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. This wide distribution of a complex biosynthetic pathway is very unusual, and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

microbiology↗