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Sulfation is a widely used strategy in nature to modify the solubility, polarity, and biological activities of molecules. The enzymes catalyzing sulfation, sulfotransferases (STs), are typically highly specific to a single sulfation site in a molecule. Herein, the identification and characterization of sulfated adipostatins is reported and reveals a novel sulfotransferase, AdpST, which is responsible for di‐sulfation at two sites of adipostatins. The initial bioinformatic analysis in search of adipostatin analogs fromStreptomyces davaonensisDSM101723 identifiesadpSTand a 3’‐phosphoadenosine‐5’‐phosphosulfate (PAPS) biosynthetic cassette, which are co‐clustered with the adipostatin‐encoding type III polyketide synthase. Mono‐ and di‐sulfated adipostatin analogs are discovered in the extracts ofS. davaonensisDSM101723, whereas di‐sulfated bacterial natural products has not been reported. Using a series of in vivo and in vitro experiments, it is confirmed that AdpST is solely responsible for both mono‐ and di‐sulfation of adipostatins, a catalytic activity which has not been identified in bacterial PAPS‐dependent STs to date. It is further demonstrated that the dedicated PAPS biosynthetic cassette improves di‐sulfation capacity. Lastly, it is determined that AdpST shares similarity with a small group of uncharacterized STs, suggesting the presence of additional unique bacterial STs in nature, and that AdpST is phylogenetically distant from many characterized STs. 

Abstract Lasso peptides are an increasingly relevant class of peptide natural products with diverse biological activities, intriguing physical properties, and unique chemical structures. Most characterized lasso peptides have been from Actinobacteria and Proteobacteria, despite bioinformatic analyses suggesting that other bacterial taxa, particularly those from Firmicutes, are rich in biosynthetic gene clusters (BGCs) encoding lasso peptides. Herein, we report the bioinformatic identification of a lasso peptide BGC fromPaenibacillus taiwanensisDSM18679 which we termedpats. We used a bioinformatics‐guided isolation approach and high‐resolution tandem mass spectrometry (HRMS/MS) to isolate and subsequently characterize a new lasso peptide produced from thepatsBGC, which we named trilenodin, after the tri‐isoleucine motif present in its primary sequence. This tri‐isoleucine motif is unique among currently characterized lasso peptides. We confirmed the connection between thepatsBGC and trilenodin production by establishing the firstBacillus subtilis168‐based heterologous expression system for expressing Firmicutes lasso peptides. We finally determined that trilenodin exhibits potent antimicrobial activity againstB. subtilisandKlebsiella pneumoniae, making trilenodin the first characterized biologically active lasso peptide from Firmicutes. Collectively, we demonstrate that bacteria from Firmicutes can serve as high‐potential sources of chemically and biologically diverse lasso peptides.