Ribosomally synthesized and posttranslationally modified peptides(RiPPs)are a major class of natural products revealed by the genome sequencing efforts of the past decades.These compounds are produced in all three domains of life and possess vast structural diversity.Sactipeptides are a small but growing class of RiPPs that contain highly unusual thioether bonds bridging the sulfur of a cysteine residue with an alph-carbon of an acceptor amino acid.These peptides are stable at high temperature and to proteolysis,and many of them exhibit potent activities against various multi-drug resistant bacteria,representing promising candidates for antibiotic development.However,structural characterization of sactipeptides is challenging due to their complicated ring systems.We here report genome mining of a series of novel sactipeptides from a bacillus strain.The ring structures of these sactipeptides were revealed by an algorithm named HSEE,which is based on the automated analysis of multiple high resolution mass spectra and evaluation of a collection of predicted hypothetical structures.The resulting structures were further validated by chemical derivatization studies.