Genome Sequencing And Analysis Of Enterococcus Faecium BZ2 And Expression Of Bacteriocin Structure Genes

Class II bacteriocins produced by Lactic acid bacteria(LAB),are defined as thermol stability small antipeptides,which can be degraded by human digestive tract and are harmless to health.They are of great significance and potential impact on the food industry.In this study,an antimicrobial strain designated as Enterococcus faecium BZ2,which was previously isolated from koumiss,was used as research subject to locate the bacteriocin gene by genomics and bioinformatics,while the heterologous expression of bacteriocins.This research provided a theoretical basis for the development of new natural food preservatives,and provided new ideas for further exploration of LAB.By using high-throughput second generation sequencing technology and mapped by de Novo.The chromosome of E.faecium BZ2 was 2687278 bp,GC content was 38.1%,2671 coding sequences were obtained with 2292024 bp length,the average length was 858 bp,covering 85.29% of the genome.Also,9 gene islands,2 temperate bacteriophages,and 2 possible CRISPR sequences were predicted.38 candidate genes in 5 area of interest were screened out by bacteriocin gene annotation,and entA and ent B genes encoding enterococin A and enterococin B were selected as the target genes for heterologous expression.p ET-28 a was used as expressing vector,and ent A and ent B gene expression vectors were constructed respectively.Firstly,The inclusion protein was successfully expressed through optimized expression.Then,high concentration target proteins were obtained through denaturation,purification,renaturation,desalting and concentration.Finally,Listeria monocytogenes CMCC54002 was used as indicator to detect the antibacterial activity of His-enterocin A and His-enterocin B.The results showed that His-enterocin A and His-enterocin B had antibacterial activity against the indicator indicative bacteria,and His-enterocin A and His-enterocin B have synergistic effect when combined with volume ratio of 1:1.This study has established a pathway from gene mining to the expression of recombinant products,which will provide bacteriocin heterologous expression and gene function references for furture LAB research.