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

As one of the most valued traditional Chinese medicines,Cordyceps sinensis,is honoured as one of the three greatest invigorants together with Panax and Pilose antlers.The relatively limited resources of wild ones and overharvesting make the natural sources of C.sinensis shortage.Mycelial fermentation of Hirsutella sinensis is a feasible and sustainable method for producing the medicinal fungus and its active compounds which have shown similar pharmacological efficacy comparable to that of natural C.sinensis.In order to improve the content of nucleosides from fermentation mycelia of Hirsutella sinensis,we predicted the pathways of nucleosides and verified them.The differentially expressed of functional genes was analyzed to inform the key enzymes of these pathways.Finally,the content of nucleosides was improved by adding exogenous substances to control these pathways.All of the work we do in this paper is based on the datas of genome and transcriptome of the H.sinensis L0106.The metabolism pathways of cordycepin,purine nucleosides and pyrimidine nucleosides in H.sinensis were predicted and drawn.In total,59 functional genes from these pathways were amplified and part of them were expressed in E.coli BL21(DE3).Realtime fluorescence quantitative PCR was used to detect the differentially expressed genes of these three metabolism pathways during different growth stages of H.sinensis L0106.It was shown that the number of down-regulated genes for cordycepin pathway and pyrimidine nucleosides pathway was 5 and 2,respectively.All of genes from purine nucleosides pathway were up-regulated ones.Among these up-regulated genes,14 genes which encoding 13 enzymes appeared significantly up-regulated which indicated the 13 enzymes were key enzymes.The results indicated that secondary metabolites began to synthesize rapidly after entering stable growth phase.In order to control the liquid fermentation process of H.sinensis L0106,10 kinds of precursors and nutrients including ribose,adenosine,glutamine,glycine,adenine,guanine,hypoxanthine,cytosine,thymine and uracil were added into the medium with different concentrations.Ribose,adenosine and glycine were shown to be more effective additives for the yield of nucleosides.The results showed that 2.0 mg/mL ribose improved cordycepin content up to 1.42 mg/g which was 0.27 mg/g in the control group.Different concentration free adenosine promoted the accumulation of intracellular adenosine.The additives showed less effective on the yield of guanosine and inosine.By adding 4.0 mg/mL ribose and 2 mg/mL adenosine,the yield of guanosine raised up to 0.48 mg/g and 0.65 mg/g,respectively.4 mg/mL ribose could improve the yield of cytidine to 7.73 mg/g,and increased 13 times with adding cytosine,adenosine and glycine also had a positive influence.In addition,adenosine,glycine,uracil,thymine could improved the yield of uridine and thymidine,the highest yield of uridine reached 17.06 mg/g which was about increased 4 times,and 1.0 mg/mL glycine enhenced thymidine to a yield of 1.80 mg/g which was an increase of 3.5 times compared to the control group.