Study On The Molecular Regulation Mechanism Of Priming Glycosyltransferase In Exopolysaccharides Biosynthesis Of Lactobacillus Plantarum YM-4-3 Strain

Microbial exopolysaccharides?EPS?is high-molecular weight compounds produced by microorganisms.It is widely used because of its hygienical functions.Lactobacillus plantarum YM-4-3 strain is a high EPS-producing strain which isolated from Yunnan traditional douchi was being used in this study.EPS produced by YM-4-3 strain was isolated from common MRS medium and modified MRS medium after 24 h incubation at37?,respectively and purified by anionic chromatography DEAE-cellulose and gel-permeation chromatography Sepharose CL-6B.The antioxidant activity of EPS was evaluated with the in vitro scavenging abilities and oxidative stress of H₂O₂-induced in U2OS,Hela and HCT116cells.After that,the growth inhibitory effect of EPS on U2OS,Hela and HCT116 cancer cells was tested.In addition,analysis YM-4-3 genome,two priming glycosyltransferase genes cps 4E and cps 2E were been found.Homologous recombination was used to construct single-knockout and double-knockout strains of priming glycosyltransferase by using temperature-sensitive plasmids.RNA-seq method was used to investigate the differential expression of transcriptome and genes which associated with EPS synthesis in wild-type strains in different culture conditions and knockout strains in MRS medium to investigate the molecular regulation mechanism of priming glycosyltransferase in the synthesis of EPS.The main research results are as follows:1.The EPS yields produced by L.plantarum YM-4-3 strain in modified MRS medium was significantly higher than that in common MRS medium,from 122.64±1.78mg/L to 322.47±3.08 mg/mL.2.The scavenging activity on DPPH radical,superoxide anion radical and hydroxyl radical was been observed in L.plantarum YM-4-3 strain produced EPS.In addition,it was found in cell experiments that the EPS could alleviate the stress of U2OS and Hela cells under H₂O₂oxidative stress.3.The EPS produced by L.plantarum YM-4-3 strain could inhibit the growth of three cancer cells of U2OS,Hela and HCT116.It shows that the EPS has certain anti tumor capacity in vitro.4.The single-knockout and double-knockout cps 4E and cps 2E mutant of YM-4-3strain were built by homologous recombination,and named YM-4-3?cps 4E,YM-4-3?cps 2E and YM-4-3?cps 2E-4E strain respectively.The EPS yield of double knockout strains was significantly lower than that of wild type and single knockdown strains.5.Using RNA-seq to determine the gene transcriptional changes of L.plantarum YM-4-3strain in MRS medium?WT?,L.plantarum YM-4-3 strain in modified MRS medium?CK?and YM-4-3?cps 4E strain in MRS medium?qc4E?,respectively.There were 1260 differential expression genes were obtained between WT and CK samples,including 589 up-regulated genes and 671 down-regulated genes.13 differential expression genes were obtained between WT and qc4E samples,all of them were down-regulated genes.And the function of differential genes and the metabolic pathway involved were analyzed through bioinformatics.