Genome Mining And Biosynthetic Studies Of Novel Bacterial RiPPs From Various Habitats

Microbial natural products are an important source of pharmaceuticals.However,conventional bioactivity-oriented natural product discovery strategy led to an increased probability of the discovery of known compounds,and the number of approved antiinfective drugs in recent years has dramatically declined.In recent decades,the rapid development of genome sequencing and bioinformatic technologies have revealed numerous uncharacterized microbial secondary metabolite biosynthetic gene clusters,which have attracted widespread attention from researchers worldwide due to their great potential for the synthesis of novel bioactive natural products.In recent years,the genomics-oriented novel natural product mining strategy,genome-mining strategy,has been greatly improving the efficiency of new compounds discovery and leading to a new and efficient phase of natural product discovery.Ribosomally synthesized and post-translationally modified peptides(RiPPs)are a group of peptide natural products with diverse structures and biological activities.RiPP biosynthetic gene clusters(BGCs)are widely distributed in microorganisms,but most RiPPs identified to date are derived from Gram-positive bacteria,and numerous compounds encoded by potential RiPP BGCs have not been identified and characterized.To explore more RiPPs with novel chemical structures and biological activities,this thesis focused on 3 Gram-negative bacteria originating from various habitats(Myxococcus fulvus,Pseudoalteromonas flavipulchra,and Chitinophagia japonensis),all of which contain a large number of uncharacterized RiPP BGCs in their genomes.Through genome bioinformatic analysis,3 intriguing RiPP BGCs were identified.The heterologous biosynthetic strategies were employed to investigate the biosynthetic pathways,and the chemical structures and bioactivities of corresponding products were characterized.Furthermore,the novel post-translational modification enzymes in 3 BGCs were biochemically and/or structurally characterized,which enriched the knowledge of RiPPs biosynthetic pathway and laid the foundation for their development as catalytic elements for the potential application in synthetic biology.The main content and conclusions of this thesis are described as follows:(1)Biosynthetic study of myxococins,novel lanthipeptides with unique biosynthetic features and anti-inflammatory activity from terrestrial myxobacteria.The genomes of myxobacteria harbor a variety of biosynthetic gene clusters encoding numerous secondary metabolites with diverse structures and bioactivities.There are limited RiPPs identified from myxobacteria thus far,however,bioinformatic analysis revealed the presence of a large number of uncharacterized RiPPs BGCs in their genomes.Herein,we reported a novel Ianthipeptide biosynthetic gene cluster mfu,which was identified from Myxococcus fulvus Mcy8288.The novel lanthipeptides,myxococin A and myxococin B.were obtained through heterologous biosynthesis combined with in vitro leader-removal strategy,and the biosynthetic pathway was described.Myxococin A represents the first example of lanthipeptides,of which the characteristic multiple thioether rings are installed by employing a Class Ⅱlanthipeptide synthetase MfuM and a Class Ⅰ lanthipeptide cyclase MfuC in a cascaded way.Myxococin B was generated by hydrolysis of myxococin A,which was catalyzed by an uncharacterized M61 family peptidase MfuP.MfuP is leader peptideindependent but strictly dependent on the five-ring structure of myxococin A and responsible for hydrolyzing two amide bonds inside two thioether rings.The hydrolyzed peptide fragments are still connected through thioether bonds,therefore,MfuP acts more like an "unwrapping function" instead of the common cleavage function of peptidases.MfuP is the first M61 family peptidase that is involved in the biosynthesis of RiPPs.Subsequently,the crystal structure of MfuP was further resolved and its active site was identified combined with mutant engineering.MfuP shares a similar overall structure with the known M61 family peptidase IloAPN but exhibits a much larger cavity volume than IloAPN,which corresponds to the binding of the complex five-ring structure of myxococin A by MfuP.In addition,we generated a binding model of MfuPsubstrate to investigate the substrate interactions of MfuP.The analysis of MfuP homologous proteins indicated that most proteins are located near putative RiPP BGCs.Thus,future studies on the biochemical functionality of these MfuP homologous involved in RiPPs biosynthetic pathways become highly attractive.Finally,myxococins are evaluated to exhibit anti-inflammatory activity in lipopolysaccharide-induced macrophages without detectable cytotoxicity,which is rare in RiPPs.This indicates their potential as a starting point for anti-inflammatory drug development.The anti-inflammatory mechanism of myxococins deserves further investigation.(2)Biosynthetic study of pseudorosins,novel class Ⅰ lanthipeptides from marine Pseudoalteromonas,reveals a lanthipeptide dehydratase PsfB with dethiolation activity.The biosynthetic potential of marine bacteria remains largely unexplored.In this study,a cryptic class I lanthipeptide biosynthetic gene cluster psf was identified from the marine prawn-isolated Pseudoalteromonasflavipulchra S16,and the biosynthetic pathway was characterized through heterologous expression in E.coli.Three precursor peptides encoded in the psf gene cluster could be modified by a single set of biosynthetic enzymes(PsfB,PsfC,and PsfT),to generate structurally distinct products pseudorosins A-C.This phenomenon of multiple precursor peptides being modified by the same set of post-translational modification enzymes is unusual in class I lanthipeptide.Pseudorosin C contains a large loop spanning 18 amino acid residues(including the methyllanthionine),which is atypical of characterized lanthipeptides.The dehydratase PsfB exhibited a degree of promiscuity,as it could perform extra dehydration at Thr7 in three leader peptides.Interestingly,it could also catalyze the dethiolation of specific Cys residues in all three core peptides through the same mechanism as dehydration,thereby generating dehydroalanines in the absence of LanC cyclase.The dethiolation of specific Cys residues in peptides has rarely been reported to date,and PsfB is the first member of the LanB dehydratase family to perform dethiolation activity.Due to the dethiolation activity and promiscuity of PsfB,it is expected to be developed as an enzymatic tool for synthetic biology.(3)Elucidation of the biosynthetic pathway of chitinoviridins,potent protease-inhibitors,reveals the biosynthetic potentials of microviridins in BacteroidetesMicroviridins are a subclass of RiPPs mainly discovered in cyanobacteria,few microviridins have been discovered from non-cyanobacteria origin so far.The bacterial genomic data revealed a promising biosynthetic potential of secondary metabolites(including microviridins)from Bacteroidetes.Hence,we bioinformatically analyzed microviridin biosynthetic gene clusters from the Bacteroidetes phylum and revealed the unique characteristics of these precursor peptides.In addition,we found a microviridin-like biosynthetic gene cluster chi in the genome of Chitinophagia japonensis DSM13484.There are two precursor peptides,ChiAl and ChiA2 encoded in the chi gene cluster,and the core sequence of ChiAl is consistent with the backbone of the known elastase inhibitor FR901451,which the biosynthetic pathway is still unknown.Here,we characterized the biosynthetic pathway of the chi gene cluster,and obtain the artificial product chitinoviridins through heterologous expression in E.coli,and it was demonstrated that chitinoviridin A1 is the known compound FR901451.Analysis of the ring structure of the products revealed that the core peptide of ChiA2 has an extra macrolactone in addition to the tricyclic structure,forming an unusual tetracyclic structure.The chitinoviridins showed potent inhibitory effects against elastase and chymotrypsin independently.In summary,this thesis focused on 3 Gram-negative bacteria from various habitats,from which 3 novel RiPP BGCs,mfu,psf,and chi were identified via bioinformatic analyses.The biosynthetic pathways of 3 BGCs were elucidated and the corresponding products,myxococins,pseudorosins,and chitinoviridins,were obtained utilizing heterologous biosynthesis combined with in vitro leader-removal strategy.The chemical structures and bioactivities of these products were also characterized and evaluated.In addition,novel catalytic activities of several post-translational modification enzymes were identified as well:MfuM and MfuC encoded in mfu BGC catalyze the production of 5 complex thioether rings in a cascade way,and MfuP,the first M61 family peptidase involved in RiPPs biosynthesis,catalyzes "unwrapping" of myxococin A to produce myxococin B;the dehydratase PsfB encoded in psf BGC is the first member of the LanB dehydratase family to catalyze the dethiolation of specific Cys residues in peptides;ChiC and ChiB encoded in chi BGC can install an unusual tetracyclic structure on the precursor peptide ChiA2.These results deepened the understanding of the biosynthetic pathways of RiPPs,enriched the diversity of chemical structures and biological activities of RiPPs,and laid the foundation for their further development as precursors of lead compounds.