| Exopolysaccharide(EPS)from lactic acid bacteria was a kind of macromolecule polymer that produced in the process of growth and metabolism of lactic acid bacteria.Because of the diversity gene of strains and diversity structure of EPSs,the physiological activity of EPS were also diverse and have great potential value in various fields.This study focused on two glycosyltransferases(orf1595 and cps4I)involved in the synthesis of oligosaccharide repeat units in the extracellular polysaccharide synthesis of Lactobacillus plantarum YM 4-3 strain.The functions of these two glycosyltransferases were preliminarily determined by gene knockout and gene recovery,and combined with the transcriptome data of the knockout strain.In addition,the growth characteristics and antimicrobial activity of the strains knocked out of the glycosyltransferase gene were tested to evaluate the effects of the two glycosyltransferases on the physiological characteristics of the strains.Extracellular polysaccharides from gene knockout strains and wild-type strains were extracted and their contents,chemical components,antimicrobial and antioxidant activities in vitro were determined to evaluate the effects of these two glycosyltransferases on the physiological activities of extracellular polysaccharides.The function of the two glycosyltransferase genes was confirmed by the results of monosaccharide analysis,which laid a theoretical foundation for the regulation of extracellular polysaccharide biosynthesis.Specific research contents and results are as follows:(1)The gene knockout vectors pFED760-orf1595 and pFED760-cps4 I were successfully constructed based on the knockout fragments of orf1595 and cps4 I genes.Lactobacillus plantarum YM 4-3-orf1595 and YM 4-3-cps4 I were obtained by homologous recombination.Based on the same principle,Lactobacillus plantarum YM 4-3-orf1595 and YM 4-3-cps4 I were successfully obtained.(2)The exopolysaccharide content of YM 4-3-orf1595 strain was 53.7±2.76 mg/L,and that of YM 4-3-cps4 I strain was 23.8±0.364 mg/L,while that of wild YM 4-3 strain was 74.75±0.254 mg/L.After gene knockout,the exopolysaccharide content of YM 4-3 strain decreased.The yield of exopolysaccharide of YM 4-3-orf1595 was 80.85±1.344 mg/mL and that of YM 4-3-cps4 I was 70.4 ±0.973 mg/mL(p>0.05),and the yield of exopolysaccharide was restored.(3)The differential expression of gene knockout strains was analyzed by transcriptome sequencing.Knockout of the glycosyltransferase gene will result in the differential expression of some genes in the exopolysaccharide synthesis gene cluster,as well as the differential expression of genes in other glycometabolic pathways of the strain.(4)The DPPH scavenging capacity,hydroxyl radical scavenging capacity and superoxide anion radical scavenging capacity of exopolysaccharides produced by knockout strains were tested in vitro,and the effect of gene knockout on the antioxidant activity of exopolysaccharides was evaluated.The results showed that the scavenging activity of PPH and superoxide anion radicals of EPS decreased significantly after the deletion of glycosyltransferase gene,but the scavenging ability of hydroxyl radicals increased significantly.When the concentration of EPS reached 400 ug/mL,the scavenging activity was more than 60%.(5)The protein content,glucuronic acid content and sulfate content of exopolysaccharides produced by knockout strains and wild-type strains were detected.The results showed that the chemical composition of exopolysaccharides changed after gene knockout. |
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