The Analysis, Verification And Control Of Anabolism Pathways Of Active Ingredients From Hirsutella Sinensis

Due to its significant medicinal value,Ophiocordyceps sinensis has become a rare and precious traditional Chinese medicine.As a result of the limited growth condition of wild Cordyceps and over-harvesting,the sources of O.sinensis are under threat.It is a feasible and sustainable method for producing the medicinal fungus since its active compounds have shown similar pharmacological efficacy comparable to that of natural O.sinensis,Hirsutella sinensis could serve as a substitute for O.sinensis.In order to improve the contents of cordyceps polysaccharide,cordycepic acid and fatty acid from fermentation mycelia of H.sinensis,the corresponding biosynthetic pathways were analyzed and verified.The differentially expressed genes were analyzed to inform the key enzymes involved in these pathways.Finally,adding substance of these key enzymes was proved effective to promote the production of these active ingredients.In this study,all of the work is based on the datas of genome and transcriptome sequencing of the H.sinensis L0106.The biosynthetic pathways of cordyceps polysaccharide,cordycepic acid and fatty acid inH.sinensis were analyzed and verified.In total,43 functional genes from these pathways were amplified and part of them were expressed in E.coli BL21(DE3).Real-time PCR and RPKM were used to detect the differentially expressed genes involved in these biosynthetic pathways and housekeeping genes during different growth stages of H.sinensis L0106.18 S rRNA was selected as the optimal housekeeping genes,and there are21 up-regulated enzymes and 9 down-regulated enzymes from these pathways.Among these up-regulated genes,8 significantly up-regulated enzymes were confirmed as key enzymes.Our results also indicated that secondary metabolites began to synthesize rapidly after entering stable growth phase.In order to control the liquid fermentation process of H.sinensis L0106,6 kinds of precursors or substance of key enzymes including glucose,ribose,adenosine,glutamine,glycine and mannose were added into the medium with different concentrations.Ribose and glycine were shown more effective for the yield of cordyceps polysaccharide.The results showed that adding 1.0 mg/mL ribose could improve the content of cordyceps polysaccharide up to 5.176% which was 5-fold compared with the control group.Adding 1.0 mg/mL glycine could enhance the content of cordyceps polysaccharide up to 4.752% which was 4.7-fold compared with the control group.Different concentrations of glutamineand mannose could promote the accumulation of cordyceps polysaccharide.Adding glucose over 10 mg/mL and low concentration of mannose and glutamine could inhibit the growth of mycelium.Addition of glucose could improve the content of cordycepic acid,especially with concentration of 3 mg/mL,the content cordycepic acid was increased to40.02%,increasing 11.2-fold compared with the control group with3.56%.