Gene Clone And Expression Of Anti-listerial Bacteriocins And Listeria Phage Endolysins Cell-wall Binding Domains (CBD)

As a foodborne pathogen, Listeria monocytogenes is a great threat to food safety.One effective way of inhibiting Listeria contamination is to add bacteriocin in food.Pediocin produced by Pediococcus acidilactici PA003is a strong anti-listerialbacteriocin. In order to solve low production of pediocin in this wild type strain,pediocin structure gene pedA was cloned into Escherichia coli and food-gradeLactococcus lactis expression systems, respectively, for heterologous expression.Listeria phage endolysin is one of the factors for Listeria lysis. In this study,bacteriocin structure gene and gene encoding Listeria phage endolysin cell-wallbinding domain （CBD） were constructed in the same Lc. lactis expression vector toexamine recombinant strain’s abilities of binding Listeria and secreting bacteriocin.The total DNA of P. acidilactici PA003was used as the template to amplify thestructural gene pedA which was inserted into pET32a（+） vector before transformationof E. coli BL21（DE3） competent cells. The recombinant plasmid containing the pedAgene was100%homologous to the pedA gene published in the NCBI （GenBankAY083244）. This recombinant strain was induced and efficiently expressed22kDafusion protein of thioredoxin and pediocin in the form of inclusion body. A singleprotein band was observed in500mmol/L imidazol elute of Ni-IDA agarose resincolumn after treatment of the renaturation buffer in GSSG-GSH system. One proteinband corresponded with the predicted pediocin was obtained after enterokinasetreatment and the antimicrobial activity against L. monocytogenes CVCC1595is512AU/ml of culture. Same strategy was adopted using pET20b（+） as the expressionvector. The pelB signal peptide in this vector resulted in the soluble expression offusion protein both intracellularly and in the periplasmic space. Pediocin productionwas384AU/ml of culture.The signal sequence of usp45from Lc. lactis and pediocin structure gene pedAwere ligated, and then inserted into multiple clone sites after the promoter P32of E.coli-Lc. lactis shuttle vector pMG36e and promoter P₄₅of food-grade Lc. lactis vectorpLEB688respectively. Recombinant plasmids were electroporated into Lc. lactisNZ9000. Pediocin secretion was detected in the recombinant cultures which showedinhibitory activities against L. monocytogenes.The P₄₅promoter gene, signal sequence of usp45and pediocin structure gene papA (P₄₅-SSusp45-papA), and the P₄₅promoter sequence, signal sequence of usp45and leucocin C structure gene lecC (P₄₅-SSusp45-lecC) were cloned into vectorpBluescript/fliC H7,respectively, and electroporated into E. coli JT1strain.Recombinant E. coli strains secreted anti-listerial expression products of pediocin andleucocin C respectively.CBD genes were cloned into pediocin and leucocin C secretion vectors of Lc.lactis, respectively, and transformed into Lc. lactis GRS5. Bacteriocin structure geneand CBD gene were co-expressed. Recombinant strains were capable of bindingListeria and secretion of bacteriocin. On the basis of it, nisin production and immunegenes were introduced by conjugation and the recombinant strains produced pediocinand nisin, leucocin C and nisin respectively, which realized co-production of twobacteriocins in the same vectors.In this study, thioredoxin-PedA was expressed both as inclusion body and solublesecreted product in E. coli cells at high levels. Pediocin was secreted in food-gradeexpression system of Lc. lactis. CBD gene and bacteriocin structure gene wereco-expressed in Lc. lactis resulting in Listeria-binding and Listeria-killingrecombinant Lc. lactis strains. Nisin and pediocin, or nisin and leucocin C, weresecreted together in the constructed Lc. lactis strain. This research provided novelmethods for bacteriocin production potentially useful for control of Listeria in foodand also for improvement of food safety level.