Screening Of High-yield Hypocrellin Aproducing Mutants Of Shiraia Sp. S8by Ultraviolet Irradiation And EMS

The fruit bodies of Shiraia bambusicola P. Hennigs, a rare parasitic medicinal fungus, havebeen used as a valuable traditional Chinese medicine. Hypocrellins, perylenequinoid pigments inS. bambusicola are of excellent anti-inflammatory, antibacterial, analgesic, local anesthetic, andantitumor activities. Due to its light-induced activities of antitumor, antibiosis, hypocrellin hasbeen applied as a clinical drug in photodynamic therapy. At present, S. bambusicola fruit bodiesare collected from the natural world. The production and quality of hyprocrellins, S. bambusicolapolysaccharide and other active ingredients are greatly influenced by climate, distribution andcollecting time. In order to fully use of S. bambusicola resourses, we cultured the anamorph of S.bambusicola and then bred Shiraia sp. mutant strains with higher yield of hypocrellin A throughphysical (ultraviolet irradiation) and chemical (ethyl methane sulphonate) mutagenesis. This canbe helpful to the production and application of hypocrellins and the protection of wild S.bambusicola resourses.In this research, we cultured Shiraia sp. S8as the start strain, then extracted and purified thehypocrellin A from the mycelia. The liquid fermentation medium of S8strain was screened andthe carbon source and nitrogen source were optimized. The optional medium for the hypocrellinproduction was made up of20%potato,2%glucose,0.3%KH2PO4,0.15%MgSO4,0.001%VB1,0.5%yeast extract, and the initial pH was6.0. The mycelia were harvested in ten days afterinoculation.Then the optimum conditions for protoplast preparation were determined as follows. After50h-cultivation the hypha were collected and digested with the mixing enzymes includingcellulase (5mg/mL) and snailase (10mg/mL) for2h under30°C. The protoplast productionreached3.24×106/mL and the regeneration rate was3.5%.The C6mutant with higher hypocrellin A yield was obtained via ultraviolet irradiation (at adistance of30cm from a15W UV lamp) on the isolated protoplasts. The production of themutant was verified to be genetically stable. The hyprocrellin A yield of mutant C6wasincreased by53.7%than that of the start strain S8, which reached28.1mg/L.Ethyl methane sulphonate (EMS) mutation was also applied to the S8strain for higherproduction of hypocrellin A. EMS at0.5-2.5%was used to treat the suspension spores for10-30min. Through the combined methods with the preliminary screening on the diameter of the redpigment ring and HPLC, the10-5mutant with higher HA yield, which reached26.8mg/L, was obtained and the production was verified to be genetically stable. HA yield by mutant10-5wasincreased by46.4%than that of the original strain.