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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. 

Axially chiral enamides bearing a N–C axis have been recently studied and were proposed to be valuable chiral building blocks, but a stereoselective synthesis has not been achieved. Here, we report the first enantioselective synthesis of axially chiral enamides via a highly efficient, catalytic approach. In this approach, C(sp 2 )–N bond formation is achieved through an iridium-catalyzed asymmetric allylation, and then in situ isomerization of the initial products through an organic base promoted 1,3-H transfer, leading to the enamide products with excellent central-to-axial transfer of chirality. Computational and experimental studies revealed that the 1,3-H transfer occurs via a stepwise deprotonation/re-protonation pathway with a chiral ion-pair intermediate. Hydrogen bonding interactions with the enamide carbonyl play a significant role in promoting both the reactivity and stereospecificity of the stepwise 1,3-H transfer. The mild and operationally simple formal N -vinylation reaction delivered a series of configurationally stable axially chiral enamides with good to excellent yields and enantioselectivities. 

Abstract Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on hidden enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by unclustered protease genes required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and hidden proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5209 correlations between precursors and hidden proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of previously unreported class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of hidden lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis.