Modification Of Polysaccharide Synthesis Pathway Of Saccharomyces Cerevisiae And Its Characteristic Analysis

Natural active polysaccharides have the biological activity of anti-tumor,anti-virus,anti-oxidation and immunoreg ?lation,which can be widely used in medicine,health care products and cosmetics.The polysaccharide with immune activity mainly derived from plants,animals and microorganisms.The biggest advantage of the microbial polysaccharide is not restricted by seasonal,regional,plant diseases and insect pests compared with other sources of polysaccharides.Saccharomyces cerevisiae's cell wall is consist of glucan(55%~65%),and mannan(20%).The FDA has identified the Saccharomyces cerevisiae as biological safety that does not produce toxic substances.Besides,it has a clear genetic background and mature industrial application technology,so it becomes the ideal source of active polysaccharides.In the actual production,through pressure(osmotic pressure,nutrition,extreme pH,etc.)to reg ?late the synthesis of saccharomyces cerevisiae cell wall polysaccharides,not only need higher production costs and the process conditions,but diffic ?lt to achieve the control of the whole process.Therefore,it is partic ?larly necessary to study the polysaccharide synthesis pathway of saccharomyces cerevisiae and construct a non-stress induced yeast strain with high yield.In this study,we first investigated the effects of the beta-1,3-glucan synthase Fks1 and its catalytic subunit Rho1 on the synthesis of cell wall polysaccharides in cell wall glucan synthesis pathway.We amplified the Fks1 and Rho1 from the genome of Saccharomyces cerevisiae BY4743 and constructed the recombinant plasmid p UG6-rDNA-Fks1 and pUG6-rDNA-Rho1.We transferred the recombinant plasmids into saccharomyces cerevisiae by electro transformation and successf ?lly constructed the recombinant S.cerevisiae in which the transcription levels of Fks1 and Rho1 increased by 3.38 and 2.63 times respectively.We amplified the upstream and downstream arm of the mutation area from the genome of BY4743,constructed the Upstream arm–KanMX–Downstream arm cassette by fusion PCR and transferred it into BY4743,and screen out the recombinant yeasts which mutation areas have almost no transcription.The cell wall of recombinant S.cerevisiae was extracted and the content of polysaccharide in cell wall was deter mined by ion exchange chromatography.The res ?lts showed that the overexpression of Fks1 and Rho1 gene had no effect on the content of cell wall polysaccharide,and this method co ?ld not control the excessive synthesis of polysaccharides.In the mutation of Fks1 activity center,glucose levels fell by 25.99%,mannose content increased by 18.09%,glucosa mine content increased by 169.72%,a significant change in cell wall composition.It co ?ld be used in the exploration of cell wall polysaccharides synthesis pathway.Secondly,the growth curve and tolerance of the pressure environment of BY4743/?Fks1 were measured,and the strain showed growth retardation and sensitivity to pressure after the mutant of Fks1.The transcriptomic profiles of BY4743/?Fks1 was deter mined using rna-seq transcriptome technology.These transcriptomic profiles showed that there are 1151 differentially expressed genes(DEGs),in which 662 genes were significantly up-reg ?lated,and 489 genes were significantly down-reg ?lated.Through GO analysis and KEGG pathway analysis of the DEGs,we found that the main effects of the FKS1 mutant are related to carbon metabolism,and the MAPK pathway.In the synthesis pathway of mannan,the synthesis of the precursor of mannan,the elongation of the chain and the process of transferring the mannan to the glycoprotein were enhanced.In the synthesis pathway of glucan,the process of UDP-glucose synthesizing the beta-1,3-glucan,and the process of UDP-glucose conversion to glycogen,trehalose and mannose decreased.The changes in the MAPK pathway indicated that the process for maintaining cell wall integrity was strengthened,whereas processes related to mating,filamentation,and osmolyte synthesis were weakened.Finally,by referring to the reg ?lation mode activated by the CWI signaling pathway in BY4743/F?ks1,we activate CWI signaling pathway of BY4743 to reg ?late the content of the cell wall glucan.We amplified the Msb1,Sph1 and Knr4 from the genome of BY4743 and constructed the recombinant plasmid pUG6-rDNA-Msb1,pUG6-rDNA-Sph1 and pUG6-rDNA-Knr4.We transferred the recombinant plasmids into saccharomyces cerevisiae by electro transformation and successfully constructed the recombinant.The content of beta-1,3-glucan in cell wall was deter mined by aniline blue.Compared with the control BY4743,the content of beta-1,3-glucan was no significant change in BY4743/Sph1 and BY4743/Knr4,and the content of beta-1,3-glucan increased by 38.99% in BY4743/Msb1.A non-stress induced yeast strain with high yield beta-1,3-glucan was constructed.