Genome Mining And Biosynthetic Pathway Analyses Of Novel Lanthipeptides

Microbial natural products(NPs)have made remarkable contributions to drug development in history.Representatives could be named penicillin,streptomycin,and avermectin,which have been awarded the Nobel Prize in Physiology or Medicine,respectively.Ribosomally synthesized and post-translationally modified peptides(RiPPs,referred to as ribosomal peptides)are a class of natural products with rich chemical structures and diverse biological activities.With the rapid development of genome mining and other bioinformatics technologies,a large number of natural products of ribosomal peptides have been discovered and characterized,which is conducive to promoting the development of active natural product drugs.Lanthipeptides are a large class of compounds reported in RiPPs,named for the unnatural amino acid lanthionines contained in their structures.Nisin A is the most well-studied lanthipeptide and has been successfully used in food processing or medical fields,because of its special mechanism of action,few resistant pathogens are produced.The newly discovered mathermycin has antitumor activity and labyrinthopeptins have antiviral activity.As a treasure trove of natural products,lanthipeptides have great application prospects and are still a hot frontier research field.In this study,microbial genomes were analyzed and screened by genome mining and bioinformatics technology,two soil-derived microorganisms,Myxococcus sp.MCy 10608 and Actinomadura atramentaria DSM 43919 were studied in the genome of two novel lanthipeptide BGCs,and heterologous expression strategies were used,aiming to explore the biosynthetic pathway of novel lanthipeptides.The mcy BGC derived from Myxococcus sp.MCy 10608 contains a class Ⅰ lanthipeptide dehydratase coding gene mcyB.mcy BGC lacks the lanthipeptide cyclase coding gene,contains a precursor peptide-coding gene mcyA,and the associated McyA amino acid sequence does not contain Cys residues involved in the formation of lanthionine.In this study,the catalytic activity of the dehydratase McyB was optimized by the heterologous expression of Escherichia coli,and the cleavage activity of the precursor peptidase was verified.The structure of Mcya was successfully characterized by HR-MS/MS and classified as a new subclass of lanthipeptides.The fxl BGC derived from Actinomadura atramentaria DSM 43919 contains typical type Ⅰ lanthipeptide synthetase fxlB and fxlC coding genes.Unlike typical classⅠ lanthipeptide synthetases,fxl BGC contains a unique type I lanthipeptide glutamate elimination coding gene fxlBC,which is rarely reported in lanthipeptide BGCs.In addition,fxl BGC also contains a gene pcmt encoding a novel isoaspartate O-methyltransferase,which has been reported to catalyze the transformation of L-Asp in peptides into L-isoAsp to introduce isomeric production in peptides or to form lactam structures in peptides.It is speculated that fxl BGC encodes a class of lanthipeptide with novel chemical structure modifications.Similarly,in this study,the precursor peptide FxlA was successfully expressed,the biochemical activities of dehydratases were verified,and FxlBC was found to assist FxlB in the dehydration of FxlA,and it was guessed that FxlB and FxlBC had a synergistic effect.In summary,this study enriches the chemical structure diversities of lanthipeptide natural products,enriches the diversities of lanthipeptide biosynthesis pathways,and discovers a new linear peptide subclass mediated by class Ⅰ lanthipeptide synthetase.The biosynthetic enzymes characterized in this study provide ideas for the development of catalytic elements of synthetic biology.