Study On Breeding High-yield Strains Of Cordyceps Militaris And Its Accumulation Of Cordycepin

In previous study, lots of studies to date have focused on the accumulation of cordycepin for Cordyceps militaris, which is a well-known cordyceps species due to it produces an important bioactive compound, cordycepin (3’-deoxyadenosine). Cordycepin (3’-deoxyadenosine), an active ingredient of C.militaris, is a potential anticancer drugs, which has increased greatly its medicinal value. How to enhance the cordycepin production is a bottleneck for commercial and medicinal application of Cordyceps militaris fermentation technology. This paper takes strains Cordyceps militaris FFCC5111 as starting strain, undertaking mutagenesis, sereening double auxotroph mutant which can remove feedback inhibition and lead to an accumulation of cordycepin and optimizing the conditions of mutant strain fermentation, to obtain high yield cordycepin strain and screen a more effective additive to the liquid medium by C.militaris in shake flask. The main results are as follows:(1)Successful mutation of Cordyceps militaris FFCC5111 by combined-mutation technique with 1% DES and 200 W,22 kHz ultrasound was performed, and 7 classes of xanthine and guanine double auxotroph mutants were obtained on mutant screening, of which one class was selected as promising preliminary mutant having an antibacterial ability as an index of cordycepin production. In a shake flask fermentation technique, among the double auxotroph mutants, it was shown that mutant C. militaris FFCC5111-c was much better than the others in the cordycepin productivity. The cordycepin production obtained by the mutant C. militaris FFCC5111-c was 4.142 g/L on 16-day culture, more than the original strain 40.18%. The results indicated that screening Xanthine and Guanine auxotrophic mutant is effective way to obtain a superior production performance of cordycepin, and furthermore, the synthetic pathways of cordycepin and guanine might be linked to each other.(2)Effects of various carbon sources and nitrogen sources were investigated to optimize cordycepin production by C.militaris in shake flasks. Glucose and peptone were found to be the best for cordycepin production. To further enhance the cordycepin production, the effect of glucose, peptone, KH2PO4 and MgSO4 concentrations were studied. Maximum cordycepin production of 4.94 mg/g was achieved in the optimized medium with 30 g/L glucose,12 g/L peptone,2.5 g/LKH2PO4 and 0.7g/L MgSO4. The results obtained are considered useful for the high production of cordycepin on a large scale using artificial media.(3)Effects of purine biosynthesisrelated compounds on liquid cultivation of Cordyceps militaris FFCC5111-c, which is a kind of purine auxotrophic mutants, were investigated in shake flasks on account of their closely linked relationship. Purine biosynthesisrelated compounds i.e. adenosine, adenine and hypoxanthine were separately added to the basal medium in order to improve the cordycepin accumulation and explore the relationship between nucleic acid-related compounds, such as adenosine, adenine, hypoxanthine and cordycepin. Results show that hypoxanthine as an additive leaded to the accumulation of adenosine, rather than cordycepin; besides, adenosine or adenine is one of the end products of purine-metabolic pathway, and plays an important role in cordycepin production, that is, the maximum cordycepin production reached 3614.1 mg/L and 2128.7 mg/L, which corresponds to 2.03 and 1.20 times that in the basal respectively. In conclusion, the nucleotide metabolism system of Cordyceps militaris is closely linked, furthermore, adenine-related compounds can be directly or indirectly convered into cordycepin.