Study On The Mining Of New Cyclic Peptides By Metagenomics

The development of new food preservatives has always been an important part of food industry research.Food preservatives can be divided into two major categories,including chemical synthesis and natural preservatives.Chemical preservatives cause consumer concerns because of their potential toxic side effects in food residues.Therefore,looking for safe,effective and non-toxic side effects of natural preservatives is the research trend in the field of food preservation.Bio-preservation material which does not have toxic and side effect and is degradable,has attracted attention of both research fields and common public.This technology can extend the shelf life of foods and improve food safety by utilizing naturally or manually controlled microbial flora and/or antibacterial substances they produce.Microbial metabolites have always been a valuable resource for mining new active natural products,especially new antibacterial substances.Non-ribosomal polypeptides are a general term for a class of compounds synthesized by non-ribosomal polypeptide synthetase(NRPS),and are one of the larger families of microbial metabolites to date.Priveous studies have shown that most of these compounds have significant antibacterial activity and can be used as one of the main sources of new food preservatives.Traditional methods based on culture-dependent were influenced by various factors such as laborary conditions,and the cultivable microorganisms account for only 1%of the total microorganisms,which greatly limits the discovery of novel active secondary metabolites.In recent years,with the development of molecular biology and sequencing technology,there has been a metagenomics technology for the study of cultured-independent microorganisms,which bypasses the pure culture process of traditional microorganisms,directly extracts environmental DNA,and expresses it in heterologous hosts.Finally,the target product is obtained,and the research on cultured-independent microorganisms is realized,which provides new resources,new methods and new ways for the mining of new bioactive natural products.In this study,a highly conserved adenylation domain(A)sequence in the NRPS-encoding gene was used as a target,and a sequence-targeted screening method was used to obtain a gene sequence encoding NRPS to achieve the targeted mining of soil microbial metabolites.(1)Construction of Soil Metagenomic Library and Screening of NRPS Gene.The soil was used as a research object.After screening and impurity removal,the DNA in the soil was directly extracted by cetyltrimethylammonium bromide cleavage method,purified and detected by agarose gel electrophoresis.Two soil environment DNA librarys were constructed by phage encapsulation infection.The NRPS gene sequence was used as a target with a pair of degenerate primers for screening.The amino acid phylogenetic trees were established.Specific primers were designed based on these sequences.9 monoclonals containing NRPS sequences were recovered by 96-well plate gradient dilution,marked as DL8-1,DL5-12,DL15-1 DL23-3,DL5-6,DL10-2,DL9-3,BD5-1 and BD3-2,respectively.To find overlapping clones,specific primers based on double-end sequencing analysis of the monoclonal were designed to perform secondary PCR-screening,and none were obtained.(2)Heterologous Expression and Product Prediction of NRPS Clones.Escherichia coli BL21(DE3)was used as a host to heterologously express the clones recovered from soil eDNA library.The fosmids of the above 9 monoclonal clones were electroporated into E.coli BL21(DE3),7 of which were successfully transformed,and the crude fermentation extract was obtained by micro-fermentation and centrifugal extraction,and was analyzed by high performance liquid chromatography profile.The crude extract of only the two clones,named BD5-1 and BD3-2,has new peaks by HPLC profile,suggesting the NRPS gene in the clone were heterologous expressed.The remaining clones products have no different peaks compared with control,indicating the interesting gene of them were not expressed in heterologous host.For this reason,the BD5-1 clone was selected for high-throughput sequencing,and the bioinformatics online software antiSMASH was used to analyze and predict that it contained a complete non-ribosomal polypeptide biosynthetic gene cluster.BD5-1 has potential to synthesize new NRPS cyclic peptides.(3)Isolation and Identification of NRPS Fermentation Products.After large-scale fermentation of E.coli BL21(DE3)/BD5-1,10 monomer compounds were isolated from the crude extract by organic reagent extraction and various column chromatography methods.They are cyclopeptide(1)and cyclopeptide(2),bis(indol-3-yl)sulfide(3),cyclo-(pro-val)(4),cyclo-(pro-tyr)(5),cyclo-(pro-ser)(6),compound(7),3,3-di-(3-indolyl)propane-1,2-diol(8),1-Lino leoylglycerol(9),compound(10),wherein cyclopeptide 1 and 2 are novel cyclic peptides of NRPS metabolite.(4)Evaluation of antibacterial activity of monomeric compounds.The results of antimicrobial activity experiments showed that compound 1 had a strong inhibitory effect on M.tetragenus,and the MIC value was less than 2 ?mol L-1,as well as compound 1 has a good antibacterial effect for S.aureus and Bacillus subtilis.In particular,it has a good inhibitory effect on Methicillin-resistant Staphylococcus aureus(MRSA),such as S.aureus AR1 and S.aureus ZAyT2,while the positive control ampicillin is not resistant to these two MRSAs,suggesting that compound 1 has a strong broad-spectrum antibacterial activity.Other compounds have no obvious bacteriostatic effect.The results from the current research highlight the advantage of phylogeny-guided pipeline for the screening of new non-ribosomal polypeptides from microbial metabolites and lay the foundation for finding new antibacterial compounds.Research strategies have expanded the methods and scope of research on microbial secondary metabolites.