Biosynthetic Pathway And Bioactivity Analysis Of Novel Natural Products Of Lanthipeptides

In last decades,the drug resistance of pathogenic microorganisms is ermerging,thus the search for novel anti-infective drugs has become a research hotspot.As the indispensable source of drugs applied in clinics,microbial natural products are produced by microbial secondary metabolism and have made great contributions to the development of novel drugs in the treatment of infectious diseases,Alzheimer’s disease,cancer and other clinical treatment.Recently,with the rapid development of genomesequencing technology and bioinformatics,it has been revealed that the microbial genomes contain a large number of potential natural product biosynthetic gene clusters(BGCs),indicating that microbes as a treasure house of natural product sources still have huge potential for tapping bioactive compouds.Among them,ribosomallysynthesized and post-translationally modified peptides(RiPPs)are a large class of natural products synthesized in ribosomal way and extensively post-translationally modified(PTM).Biosynthetic genes of RiPPs are composed of a precursor peptide encoding gene and various PTM enzymes encoding genes.The precursor peptide commonly includes a N-terminal leader peptide and a C-terminal core peptide.The PTM enzyme specifically recognizes the leader and performs various modifications on the core.In addition,a site-specific protease cleaves the leader and releases the mature core peptide extracellularly to perform its corresponding physiological functions.Abundant PTMs endow RiPPs structure stability,as well as highly diverse chemical scaffolds and biological activities.As a well-investigated representative class of RiPPs,lanthipeptides are a large class of cyclic peptide compounds with thioether bonds.Typical PTM includes dehydrations on serine/threonine residues which results in dehydroalanine(Dha)/dehydrobutyrine(Dhb)and cyclization of cysteine with Dha/Dhb via Michael-addition which results in a unique thioether ring.Lanthipeptides are widely present in nature with a variety of biological activities.The purpose of this study is to discover and characterize two lanthipeptide biosynthetic gene clusters(BGC)sch and mys,which are derived from cyanobacteria Scytonema hofmanni PCC 7110,ATCC29171 and Myxococcus sp.strain MCy9171(Mxbonn171).BGC sch belongs to the class II lanthipeptide BGC containing seven precursor peptide encoding genes.The PTM enzyme encoding genes include a class Ⅱ lanthionine synthetase encoding gene,a specific protease encoding gene,two novel epimerase encoding genes,and one reductase encoding gene.BGC mys belongs to the class Ⅰ lanthipeptide BGC with 4 precursor peptide encoding genes.The PTM enzyme encoding genes include a class Ⅰlanthionine synthethase but lacking the conserved cyclase which are responsible for the formation of the thioether ring,a protease encoding gene and an acetyltransferase encoding gene.We note that only one of the four precorsur peptide in the core region contains one cysteine and the BGC lacks the cyclase encoding gene.This scenario interests us to futher explore whether the core peptides will undergo cyclization reaction.Due to the novel PTM enzymes within this BGC,it is of great interest to investigate the post-modification pathways of the two lanthipeptide BGCs as well as the product structure and physiological functions.Three heterologous expression strategies are employed:sole precursor peptide expression,precursor peptide and PTM enzyme encoding genes co-expression,PTM enzyme encoding genes expression.All the precursors are expressed in E.coli BL21(DE3).The in vitro proteolytic reaction releases the cores to verify that the molecular weight.The activity and function verification of the PTM enzymes will lay the foundation for the analysis of the modification pathway of the PTM enzymes.In this study,recombinant plasmids were constructed using recombineering technology to perform heterologous expression of target genes and PTM enzymes.Finally,mature target products were obtained.The target products were isolated using protein purification techniques combined with in vitro proteolytic reaction.