Genome Mining-Driven Discovery of the Sactipeptide Smithiosin in Probiotic, Thermophilic Strain Bacillus smithii

Abstract Sactipeptides are a class of ribosomally synthesised and post-translationally modified peptides (RiPPs), which have a cyclic topology formed by unique thioether crosslinks called sactionines. Sactionines are introduced by radical S-adenosylmethionine (rSAM) enzymes between cysteine residues and the α-carbon of other amino acids. Genome sequencing has revealed many putative sactipeptide biosynthetic gene clusters; however, few sactipeptides have been isolated and characterised. Here, we use genome mining to identify a novel sactipeptide from Bacillus smithii. The novel sactipeptide, named smithiosin, was extracted from the industrially important thermophile B. smithii and characterised as a novel homologue of the previously characterised sactipeptide subtilosin A from B. subtilis. Furthermore, the smithiosin precursor peptide gene was heterologously expressed in the subtilosin A producer Bacillus subtilis 168 without knockout of the native precursor peptide, successfully leading to the production of both subtilosin A and smithiosin. This demonstrates for the first time that a non-native precursor peptide is able to outcompete the subtilosin A precursor for all of the native biosynthetic enzymes in B. subtilis, including the radical SAM enzyme AlbA. It also opens a potential new opportunity for screening non-native precursor peptides with the subtilosin A biosynthetic enzymes via heterologous expression in B. subtilis.