| Cordyceps militaris(C.militaris)is rich in cordycepin,cordyceps polysaccharide and other natural active ingredients.It has physiological functions such as anti-inflammatory,antioxidation and lowering blood pressure.It is one of the most widely studied and applied Cordyceps fungi.Cordycepin has become the most important bioactive substance of C.militaris with superior anti-tumor,immune regulation and other medicinal values.However,the low production efficiency and high cost of cordycepin limit its industrial application.In this study,the effect of silkworm pupae grading components on cordycepin synthesis was explored,and the multiple liquid fermentation control strategy of C.militaris was established.Combined with differential transcriptomics,the regulation mechanism of silkworm pupae grading components on cordycepin synthesis was clarified,and the ability of C.militaris to synthesize cordycepin was further improved,providing technical support for its industrial production.The main research contents are as follows:(1)A new liquid fermentation technology for promoting cordycepin production by C.militaris strains was established.The dominant strain HY01 producing cordycepin was identified as C.militaris by comparing the sequence of the transcriptional spacer in the conserved sequence.A series of graded components of silkworm pupae were prepared based on different polarity of solvents,and their effects on cordycepin production by liquid fermentation of C.militaris were investigated.The results showed that the liquid fermentation system of C.militaris was used as the research object,and different polar fractions of petroleum ether extract,ethyl acetate extract,chloroform extract,methanol extract,water extract and residue of silkworm pupae were added in equal proportion to the extraction rate.It was found that the concentration of cordycepin in the fermentation broth reached the maximum value of 0.32 g/L when 13 g/L water extract of silkworm pupa was added,which was 1.6 times higher than that without addition(p<0.05).The main component of the water extract of silkworm pupae was identified as silkworm pupae protein.The protein of silkworm pupa was graded by Osborne method,and the albumin,globulin,prolamin and glutelin of silkworm pupae were prepared respectively.Compared with the control group,the addition of 3.31 g/L silkworm pupae globulin component significantly increased the yield of cordycepin in the fermentation broth(p<0.05),and its cumulative concentration was 0.37 g/L.Through RT-q PCR analysis,it was found that the expression of HY1-HY4 genes in the key gene cluster of cordycepin synthesis was up-regulated by 3.06,3.36,1.82 and 4.57 times,respectively.The total concentration of cordycepin in the fermentation system reached 2.29g/L.Therefore,the silkworm pupae globulin fraction obtained by solvent polarity and protein solubility classification could effectively promote the ability of C.militaris strain to synthesize cordycepin in liquid fermentation.(2)The regulatory mechanism of silkworm pupa globulin components on C.militaris metabolism and cordycepin synthesis was elucidated.By optimizing the culture conditions such as carbon and nitrogen sources,fermentation temperature,rotation speed,and initial p H,the liquid fermentation conditions for cordycepin production by C.militaris strain HY01 were determined as follows: 20 g/L sucrose,20 g/L peptone,1 g/L potassium dihydrogen phosphate,0.5 g/L magnesium sulfate heptahydrate,and 3.31 g/L silkworm pupa globulin.At 25 °C,100 rpm,and an initial p H of 9,the cumulative yield of cordycepin reached 0.41 g/L after 16 days of fermentation,which was 2.1 times higher than the basic system(p<0.05).By comparative transcriptome analysis,there were 764 differentially expressed genes in the silkworm pupae globulin addition group and the control group,mainly related to carbon source and energy metabolism pathways.The RT-q PCR analysis showed that the expression of genes encoding hexokinase,phosphatase,phosphocyclase,succinate lyase,acetyl-Co A synthetase,5’-nucleotidase,formate dehydrogenase and global regulator Ve A increased by 5.09,9.99,2.68,5.41,10.86,2.34,1.93 and 2.49 folds,respectively.Therefore,the addition of silkworm pupae globulin components could promote the expression of genes related to cell energy metabolism and cordycepin synthesis,strengthen ATP synthesis,and enhance energy metabolism,thereby increasing the yield of cordycepin synthesized by C.militaris.(3)A new strategy of fermentation regulation was established based on the promoting effect of silkworm pupa globulin components on cordycepin synthesis.By adding carbon and nitrogen sources,silkworm pupa globulin and controlling p H during the liquid fermentation of C.militaris HY01 strain,a 5 L bioreactor was used for 2 L fed-batch fermentation,and a multiple fermentation strategy was proposed.That is,20 m L of sucrose solution with a concentration of 100 g/L was supplemented every day for 3-16 days,and 20 mL of silkworm pupa globulin with a concentration of 16.55 g/L was supplemented every 2 days for 8-16 days.At the same time,the p H of the system was controlled at 9,and the cordycepin yield obtained after 16 days of fermentation reached a maximum of 0.56 g/L.The total cordycepin concentration in the fermentation system reached 2.61 g/L,which was 2.1 times higher than that of the control group(p<0.05).Compared with the control group,RT-q PCR detection showed that the expression of key genes HY1-HY4 and global regulatory gene ve A were significantly up-regulated during fermentation,indicating that multiple fermentation strategies could enhance the ability of HY01 strain to synthesize cordycepin and the regulation of Ve A on the cell to increase cordycepin production.Compared with the current reported fermentation strategies such as adding precursor substances,controlling fermentation conditions,optimizing fermentation medium and feeding during fermentation,the multiple fermentation strategy for liquid fermentation of C.militaris to synthesize cordycepin has a shorter cycle and higher efficiency,and the yield of 16 d fermentation cycle was increased by 2.1 times.Therefore,the ability of C.militaris to synthesize cordycepin was significantly improved by the multiple fermentation strategy of batch feeding-p H control,which provided a new idea for the industrial production of cordycepin.(4)The effect of regulatory factor VeA on cordycepin synthesis was systematically analyzed.Through comparative transcriptome analysis,it was found that the expression level of ve A,encoding the core protein of the Velvet protein family,was significantly increased.The ve A gene deletion strain(ΔVe A)of C.militaris was constructed,and the cordycepin production,expression of related genes and mycelial growth of ΔVe A strain were analyzed to explore the regulation of Ve A on the growth and cordycepin synthesis of C.militaris.The results showed that the cordycepin production of ΔVe A strain was significantly reduced to0.04 g/L,which was 9 times lower than that of wild-type strain(p<0.05).In addition,the expression of genes related to carbon source metabolism and energy metabolism was significantly down-regulated(p<0.05).The expression levels of genes HY1,HY2 and HY4 related to cordycepin synthesis in ΔVe A strain were 0.42,0.61 and 0.44 times higher than those in WT strain(p<0.05),and the mycelium showed a dispersed flocculent state without forming mycelial pellets.The results showed that Ve A had a positive effect on the synthesis of cordycepin and the growth of the strain.By regulating the carbon source metabolism and energy metabolism of the bacteria,the growth and mass transfer of the bacteria were promoted,and the synthesis of cordycepin was promoted.It shows that Ve A is a positive regulator of cordycepin synthesis in C.militaris,which provides a new idea for genetic engineering to transform C.militaris to efficiently synthesize cordycepin.In summary,based on the effects of silkworm pupae grading components on cordycepin production and metabolic pathway of C.militaris,combined with comparative transcriptional analysis and gene verification results,this thesis constructed a multiple fermentation regulation strategy of C.militaris,clarified the regulation mechanism of silkworm pupa grading components on cordycepin synthesis by liquid fermentation of C.militaris,and provided technical support for cordycepin synthesis regulation pathway and efficient production. |
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