Optimization Of Liquid Fermentation Conditions For Polysaccharide Production By Poria Cocos And Identification Of Key Genes Involved In Polysaccharide Synthesis

Poria cocos(Schw.Wolf)is a traditional Chinese herbal medicine.Its main medicinal ingredients are triterpenoids and polysaccharides among which polysaccharides content can reach more than 80%of dry sclerotium weight.P.cocos polysaccharides exhibit such excellent pharmacological activities such as immunity-enhancing,anti-tumor,anti-inflammation,and thus possess great value for exploitation and utilization.In past years,many studies focused on the P.cocos polysaccharides extraction,structure determination,pharmacological activities.However,less information regarding P.cocos polysaccharides production by submerged fermentation and identification of the key genes involved in P.cocos polysaccharides synthesis is reported.In this study,17 P.cocos strains from different areas were collected and compared by determing their growth rate and the polysaccharide content of the mycelium.The results showed that the SD strain exhibited higher growth rate and polysaccharide content in mycelium.Furthermore,fermentation conditions and medium components for polysaccharide product by SD strain were optimized by using Single Factor Design,Plackett-Burman(PB)Design,and Response Surface Design.In addition,the key enzymes α-phosphoglucomutase(pgm)and UDP-glucose pyrophosphorylase(gpp)involved in the synthesis of polysaccharides were identified,and heeterologously expressed in Pichia GS115.The activities of the recombinant enzymes were determined and confirmed.The main conclusions obtained from this study are as follows:1.Comparative analysis of growth rate and mycelial polysaccharide content of 17 p.cocos strainsThe growth rate of 17 strains were determined and compared on the plate,and the result showed that the fastest growth rate was found by Y1 strain with thinner mycelium layer,while the slowest growth rate was observed by A5 strain with thicker mycelium layer.As for mycelium polysaccharide content,the polysaccharide content of SD among the 17 strains was the highest,up to 48%of the dry weight mycelium.Furthermore,the SD strain was used as the starting strain for submerged fermentation.The changes in the content of polysaccharide in mycelium were measured on different days.It was found that the content of polysaccharide in mycelium rapidly increased during the initial 7 days,and decreased after the 7th day.Finally,the 7th day was chosen as the fermentation period for polysaccharide production by SD strain.2.Optimization of liquid fermentation medium components of polysaccharide product by p.cocos SDSingle Factor Designs were firstly used to analyze the effects of nitrogen sources,carbon sources,and metal ions on polysaccharide production of SD strain.The result showed that sucrose,Angel yeast extract,sodium nitrate and copper ions significantly improved the mycelium polysaccharides content,.To further proceed to confirm the effects of each component of the fermentation medium on the yield of polysaccharides in mycelium,PB Designs were performed for each component and the results showed that sucrose,Angel yeast extract,and copper ions appeared remarkable effects on the yield of SD polysaccharides.Subsequently,the Response Surface Designs were performed to optimize the content of sucrose,Angel yeast extract and copper ions,and the optimal fermentation medium for polysaccharides synthesis was determined,which was composed of sucrose 41.3 g/L,Angel yeast extract 3.3 g/L,copper sulfate 9.7 mM,KH2PO4 1.5 g/L,MgSO4 0.75 g/L,VB1 5 mg/L.Under the condition of the optimized fermentation medium,the yield of polysaccharides produced by SD strain was up to 2.07 g/L with a 1.6-fold increase and the content of polysaccharide in mycelium was increased by 29%when compared to that produced by the SD strain before optimization.3.Cloning,heterologous expression and catalytic analysis of enzymes involved in the synthesis of p.cocos polysaccharidesWith reference to the basic pathway of ganoderma lucidum polysaccharide synthesis,it was found that pgm mainly influenced the yield of ganoderma lucidum polysaccharide,and gpp mainly was involved in ganoderma lucidum polysaccharide type and monosaccharide composition in the ganoderma lucidum polysaccharide synthesis pathway.Hence,in this study pgm and gpp were selected as the entry point,and the candidate genes pgm and gpp were identified by genomic sequence alignment.Total RNA was extracted and then reversed to obtain cDNA.Gene-specific primers were employed to amplify the pgm and gpp to get the target fragments,respectively.Subsequently,the obtained PCR products were cloned into the pPIC9K vector to generate pPIC9K-pgm and pPIC9K-gpp vector,respectively.Two expression vectors were transformed into GS115 and their expression was induced by methanol and the result showed that the gene pgm and gpp succeeded to express by SDS-PAGE ananlysis.In vitro enzymatic assays showed that PGM could catalyze glucose-1-phosphate into glucose-6-phosphate in the presence of Mg2+ and glucose 1,6bisphosphate and the crude enzyme activity was determined to be 1540 U/mL,while GPP could catalyze the reaction between glucose-1-phosphate and UTP to generate UDP-glucose in the presence of Mg2+and the crude enzyme activity was up to 660 U/mL.