Extracellular Polysaccharides Synthesis In Bacillus Licheniformis-functional Studies Of Eps Gene(Cluster)

Extracellular polymeric substances(EPS)are extracellular macromolecules secreted by bacteria,which function in cell growth and show potential for mechanism studies and biosynthesis applications.Based on the previous studies,the key genes(eps cluster)in the biosynthetic pathway of extracellular polysaccharides were studied and resolved more systematically in this thesis,using Bacillus licheniformis CGMCC 2876 as the target strain.First,we investigated the glycosyltransferase genes epsG-O in the eps gene cluster,which are important for polysaccharide synthesis,and analyzed the effect of its overexpression on EPS synthesis in Bacillus licheniformis CGMCC 2876 The results showed that the overexpression of epsK and epsL genes had a more pronounced effect on the synthesis of extracellular polymers.Overexpression of epsK decreased EPS yield by 26.54%to 6.56 g/L,while the proportion of protein,total sugar and y-PGA in EPS decreased by 44.94%,35.63%and 86.60%,respectively.In addition,the maximum biomass of the epsK overexpressing strain OEK1 during fermentation decreased significantly compared to the wild strain and its cell became more elongated.Combined with transcriptomic data and protein interaction analysis,it was found that EpsK may be jointly responsible for the transmembrane transport of teichuronic acid repeating units with TuaB and is closely associated with the phosphate starvation response.As for epsL,although overexpression of the epsL gene-caused a decrease in the yield and flocculation activity of the EPS produced by B.licheniformis,it promoted the synthesis of extracellular polysaccharides and increased the content and yield of total sugars in the EPS.Analysis of the monosaccharide composition in the extracellular polymer combined with protein-protein interactions and qRT-PCR analysis led us to hypothesize that EpsL is an arabinoglycosyltransferase in B.licheniformis CGMCC 2876,and in addition we suggest that EpsE and EpsH may be involved in the transport of three monosaccharides,GalN,GalA and Fuc.Further experiments showed that overexpression of the teichuronic acid synthesis genes tuaB and tuaE,as well as phosphate starvation conditions,led to a reduction in EPS,while high concentrations of phosphate promoted EPS synthesis.Analysis of EPS components revealed that overexpression of tuaB and tuaE genes promoted protein and polysaccharide synthesis and inhibited y-PGA synthesis in EPS;phosphate starvation conditions stimulated the cells to synthesize more protein and y-PGA,while increased phosphate concentrations moderately reduced the proportion of protein and y-PGA in EPS.Analysis of gene transcript levels showed that both tuaB and tuaE overexpression and phosphate starvation conditions induced a phosphate starvation response in the cells.Besides,expression of epsK was down-regulated while tuaB was overexpressed and upregulated along with tuaB while tuaE was overexpressed.Therefore,we suggest that epsK functions as a transmembrane transport unit for teichuronic acid repeating units and that its overexpression enhances teichuronic acid synthesis and triggers endogenous phosphate starvation response in cells,which led to a series of changes in cell growth and metabolism,including the decreased EPS synthesis and bacterial biomass,the increased bacterial chemotaxis,and the changes in bacterial morphology.In addition,we found that high phosphate concentration conditions induced the osmotic stress response,which may be responsible for the enhanced EPS synthesis.Based on the above results,we proposed a method to achieve purposeful regulation of cellular metabolism and product synthesis by regulating the expression of stressresponsive genes,and experimented with salt stress fermentation and overexpression of osmotic stress responsive genes.We found that when 3%and 5%KCl were added to the medium,the EPS production increased from 9.43 g/L to 13.44 and 12.91 g/L,the proportion of protein and polysaccharide in its composition decreased from 31.33%to 43.14%,respectively,while the proportion of y-PGA remained stable.Surprisingly,overexpression of osmotic stress-responsive genes inhibited the synthesis of EPS,especially the synthesis of polysaccharide and y-PGA,to different degrees.