Study On The Biosynthesis And Affecting Factors Of Hyperbranched Polysaccharide From Pleurotus Tuber-regium Mycelium

Pleurotus tuber-regium(PTR)is a preciously medicinal and edible macro-fungus,which is rich in a variety of bioactive substances.Polysaccharide,as the main active products of PTR,possess hyperbranched structure and exhibit exceptional potential in many fields.The present study applied submerged fermentation and bioinformatics analysis to explore the synthesis of hyperbranched polysaccharide.1.Isolation,purification and characterization of polysaccharides from the mycelium cell wall of PTR.Four kinds of cell wall fractions were obtained from PTR mycelium by different isolation methods,including hot water extraction(MHW),cold alkali extraction(MCA-1,MCA-2),hot alkali extraction(MHA)and alkali insoluble fraction(MAI).MCA-1I,MCA-2I and MHA-I were further purified.The results showed that they were heteropolysaccharides mainly containing glucose.MHA-I nine glycoside bond connections with highly branched degree,which possess spherical structure with molecular weight and R.M.S.radius of 4.502×10~4 g/mol,33.2 nm,respectively.TEM analysis further proved the hyperbranched structure of MHA-I.2.Influence of different carbon source on monosaccharide composition in PTR exopolysaccharide(PTR-EPS).The monosaccharide composition and proportion of PTR-EPS are different based on the carbon source change.The results showed that mannose and glucose were present in the EPS no matter what carbon source used,moreover the proportion of mannose was higher than glucose.3.Genome sequencing of PTR for analysis of hyperbranched polysaccharide synthesis pathway.The genome size of PTR mycelium was 39.4 M,while the number of coding genes was 12255,and a total of 577 genes were annotated by CAZyme.Combined with CAZyme functional annotation,the pathway of polysaccharide synthesis of PTR was initially analyzed.Moreover,protein interaction analysis was carried out to identify 18 key genes in polysaccharide synthesis.4.Effect of vegetable oil on liquid fermentation of PTR.Series of vegetable oil were added in the liquid fermentation of PTR to promote the growth of PTR.When the addition of corn oil was 4%,the production of biomass and PTR-EPS was 2.56 and 1.9 times to control,respectively.The monosaccharide composition of PTR-EPS was changed and the mycelium was stronger with no obvious twining status after the addition of corn oil.Transcriptome analysis showed that the differentially expressed genes were mainly related to carbohydrate metabolism,catalytic activity and membrane components,indicating that corn oil not only promote the growth of PTR,but also alter the activity of enzymes in EPS synthesis pathway.Finally,53 genes with significant differences were screened.