Effect Of Overexpression Of Priming Glycosyltransferase On Exopolysaccharide Synthesis Of Lactobacillus Plantarum YM-4-3

Lactobacillus plantarum YM-4-3 strain is a high exopolysaccharides(EPS)producing strain,which isolated from Yunnan traditional douchi was been used in this study.Two priming glycosyltransferase genes(cps 4E and cps 2E)were been investigated the effects on L.plantarum YM-4-3 strain EPS production,molecular weight,fractions and antioxidant activity by constructing single overexpression and double overexpression glycosyltransferase gene strains,respectly:YM-4-3-2E OE,YM-4-3-4E-OE and YM-4-3-2E-4E-OE.In addition,the effect of priming glycosyltransferase overexpression on the activity of strain was been tested by in vitro strain inhibition assays.Meanwhile,RNA-seq method was been used to investigate the differential expression of transcriptome and genes,which associated with EPS synthesis in wild-type strains and overexpression strains to clarify the molecular regulation mechanism of priming glycosyltransferase in the synthesis of EPS.The main research results are as follows:1)Expression vector pSIP409 was used to construct cps 4E and cps 2E single overexpression and double overexpression vectors p SIP409-2E,p SIP409-4E and p SIP409-2E-4E,and the overexpression strains YM-4-3-2E-OE,YM-4-3-4E-OE and YM-4-3-4E-OE were been constructed.The exopolysaccharide production of the overexpression strains was significantly increased compared with the wild-type strains(P<0.05).The boosts were 30.15%,26.84%and 36.29%,respectively.2)The molecular weight and monosaccharide fractions of each EPS were been tested.The molecular of YM-4-3 EPS,YM-4-3-2E-OE EPS,YM-4-3-4E-OE EPS and YM-4-3-2E-4E-OE EPS were 1.43×10~5 Da,3.48×10~5 Da,2.96×10~5 Da and 1.98×10~5Da,respectively.YM-4-3-4E-OE EPS contained D-mannose,D-ribose,L-rhamnose,D-aminoglucose,D-aminogalactose,D-glucose,D-galactose,D-xylose,L-arabinose,L-fucose,L-gulonuronic acid,D-mannuronic acid,D-glucuronic acid and D-galacturonic acid,14 monosaccharides.Compared with YM-4-3-4E-OE EPS,YM-4-3 EPS without L-rhamnose,D-aminogalactose and D-xylose,YM-4-3-2E-OE EPS without L-gulonuronic acid and D-galacturonic acid,YM-4-3-2E-4E-OE EPS without D-galacturonic acid.FT-IR analysis showed that all the EPS produced by the four strains contained C-H,C-O-C and C-O-H bonds,?-glyoxylate,sulfate groups and sulfate group substituent groups.However,except for YM-4-3-2E-4E-OE EPS all other three strains'EPS contain?-glycosidic bonds.3)All strains had significant inhibitory effects on the growth of Fusarium trichosporium,banana anthracnose and Penicillium expansum.In addition the scavenging activity on DPPH radical,superoxide anion radical and hydroxyl radical was been observed in all EPS and there was no difference among the strains.4)Using RNA-seq to determine the gene transcriptional changes of wild-type strain and overexpression strains.There were 1386,1395 and 1458 differential expression genes obtained between YM-4-3 strain with YM-4-3-2E-OE strain,YM-4-3-4E-OE strain and YM-4-3-2E-4E-OE strain,respectively.In the glycolytic pathway,the up-regulation of fructokinase 6-phosphate kinase 1 and glucokinase which may lead to the accumulation of glucose-6-phosphate.On the other hand,in the pentose phosphate pathway,the down-regulation of glucose-1-dehydrogenase 6-phosphate and gluconate dehydrogenase 6-phosphate expression may lead to decrease the consumption of glucose-6-phosphate.Therefore,more glucose-6-phosphate may involve in the gluconeogenic pathway,which promotes the production of EPS.