Mining And Heterologous Expression Of Bacteriocins Produced By Lactobacillus Paracasei WX322 And Its Controlling Pepper Bacterial Soft Rot

The yield of pepper in China ranks first in the world.Bacterial soft rot is one of the main diseases that affects crops all over the world,causing huge economic losses to postharvest pepper in China.At present,the main methods used to control bacterial soft rot are agricultural control,pesticides and chemical bactericides.The main methods of agricultural control are environmental management and cultivation control,which act before harvest.Pesticides and chemical bactericides can control the occurrence of bacterial soft rot to a certain extent,but they can induce resistance and drug residues which will cause serious environmental pollution.Therefore,it is very important to seek a green and effective biological control method.Lactic acid bacteria have been widely used in agriculture and food as a kind of GRAS microorganism,and the bacteriocin produced by lactic acid bacteria has good antibacterial effect on some harmful bacteria.However,nisin is the only commercially used bacteriocin,which only has good antibacterial effect on Gram-positive bacteria.Therefore,it is urgent to develop bacteriocins that can kill Gram-negative bacteria at the same time.In order to expand the application of bacteriocins in biological control,the broad-spectrum bacteriocins with activity against both Gram-positive and Gram-negative bacteria are needed.In this study,Lactobacillus paracasei WX322（L.paracasei WX322）isolated in our laboratory was used as the research material to study the bacteriostatic analysis of L.paracasei WX322 against Pectobacterium carotovorum（P.carotovorum）,controlling potentials to bacterial soft rot of pepper by L.paracasei WX322 and its bacteriocin,the growth curve of L.paracasei WX322,the production kinetics of bacteriocin and the stability of bacteriocin.The complete genome sequencing results of L.paracasei WX322 were annotated and analyzed,and the gene sequences encoding bacteriocin were identified.The bacteriocins were heterologously expressed by Escherichia coli expression system and Pichia pastoris expression system.Further,the bacteriocin paracin wx3 expressed by Pichia pastoris X33 was studied for its antibacterial mechanism against P.carotovorum.The main results are as follows:（1）The results of antibacterial spectrum of L.paracasei WX322 showed that it had good inhibitory effect on P.carotovorum,Staphylococcus aureus,Listeria monocytogenes,Enterococcus faecalis,Enterobacter sakazakii,Salmonella,Pseudomonas aeruginosa and Escherichia coli.L.paracasei WX322 could significantly control the occurrence of bacterial soft rot of pepper.The growth curve and bacteriocin production kinetics of L.paracasei WX322 showed that the strain was in delay phase from 0 to 12 h,entered logarithmic phase after 12 h,and reached stable phase at about 66 h.There was no inhibitory activity before 30 h,but bacteriocin had obvious antibacterial activity after 36 h,and its antibacterial activity gradually increased with the extension of fermentation time.The stability of bacteriocin produced by L.paracasei WX322 showed that the bacteriocin produced by L.paracasei WX322 had good thermal stability and p H adaptability,and protease sensitivity analysis showed that the bacteriocin was proteinaceous.（2）Results of Illumina second-generation sequencing and Min ION nanopore third-generation sequencing showed that the L.paracasei WX322 genome consisted of one chromosome and eight plasmids.Based on genome annotation,2794 genes were annotated in COG database and 2539 genes were annotated in KEGG database.L.paracasei WX322 had abundant metabolism and intracellular and extracellular signal transduction genes,which indicated that the strain has strong metabolism and strong environmental adaptability.The phylogenetic tree of L.paracasei WX322 showed that L.paracasei WX322 had high homology with L.paracasei ATCC 393 and L.paracasei ATCC25302.Collinearity results showed that there was no large-scale gene insertion,deletion or inversion in the genome of L.paracasei WX322,only a small range of insertion and inversion was found,the degree of gene recombination and transfer was very small during the evolution of L.paracasei WX322.Finally,through database comparison,there were three bacteriocin gene clusters in L.paracasei WX322 genome,and seven new bacteriocin genes were found in the gene clusters.（3）Seven bacteriocins were heterologously expressed by Escherichia coli expression system and Pichia pastoris expression system.For Escherichia coli expression system,the recombinant plasmids were constructed by ligating the bacteriocins into the expression vector pET-30a（+）and transformed into E.coli BL21（DE3）plys S for heterologous expression.The results showed that all the seven bacteriocins could inhibit the growth of P.carotovorum.For Pichia pastoris expression system,the bacteriocins were ligated to the expression vector p PICZαA,linearized and transformed into Pichia pastoris X33.Only paracin wx3 had significant antibacterial activity against P.carotovorum.The diameter of inhibition zone of paracin wx3 was 30.8 mm and the yield was 0.537 g·L^-1.Tricine SDS-PAGE showed that the molecular weight of paracin wx3 was 4.1-6.5 k Da（theoretical size 5.6 k Da）.（4）The MIC value of the bacteriocin paracin wx3 against P.carotovorum was 16μg·m L^-1.In order to clarify the antibacterial mechanism of the bacteriocin paracin wx3against P.carotovorum,the growth curve and the time-kill curve of paracin wx3against P.carotovorum were determined.The results showed that the action mode of the bacteriocin paracin wx3 against P.carotovorum was bactericidal mode accompanied by partial lysis of the target cells when the concentration was above 4MIC.SEM results showed that paracin wx3 treatment resulted in the formation of holes in the cell membrane,as well as cell collapse and deformation were observed.SYTOX Green fluorescent probe experiments showed that paracin wx3 destroyed the integrity of the cell membrane of P.carotovorum.The results of the NPN probe indicated that paracin wx3 treatment resulted in an increase in the permeability of the outer membrane of P.carotovorum.The results of ONPG probes showed that paracin wx3 treatment resulted in an increase in the permeability of the inner membrane of P.carotovorum cells.That is,the bacteriocin paracin wx3 killed P.carotovorum by causing hydrolysis of cell membrane,destroying the integrity of the cell membrane,and increasing the permeability of both the inner and the outer membrane of cells.