Strain Improvement And Optimization Of The Fermentation Condition Of Taxol-producing Fungus Fusarium Mairei

Taxol, a complex diterpenoid produced by some species of Taxus, was found in 1971. Taxol is marketed as an anticancer drug approved by FDA in 1992. The amount of taxol in yews is relatively small and this has been a major factor contributing to its high price. Recently, many researchers have reported that they had found taxol-producing fungus isolated from phloem of yews.With the goal of increasing the yield of fungal taxol, this study shows that a mutant K178 selected from an endophytic fungus, Fusarium mairei Y1117, obtained from the inner bark of China mairei with protoplast mutation, which produced taxol in a high yield after optimizing the fermentation condition.1. A fast and reliable method of obtaining protoplasts from Fusarium mairei which can produce taxol is studied. After 2 days of still culture and 2 days of shaking-culture at 25℃, the mycelials was treated with a combination of lywallzyme(3%), snailase(2.5%), lysozyme(1%) and cellulase(0.5%) in pH5,and 0.6mol/L KCl as the osmotic stabilizer. The high protoplast yield of 3.2×107/mL is obtained in 4 h at 32℃. The conditions of protoplast regeneration were also investigated. The protoplasts were purified and regenerated on the solid YCM at 25℃, using 0.6mol/L sucrose as the osmotic stabilizer. The regeneration rate reached at 8.9%.2. The condition of mutagenesis was studied and multiple mutations to protoplast were chose as the tool to acquire the strain of high production. Mycostatin (Nystatin) as a screen to get taxol-producing fungus of high yield was added to the screening media. The effect of mycostatin on screening was studied and the result suggested that the degree of P.T. was 13%. A mutant Fusarium mairei K178 was selected by protoplast mutation of UV radiation and DES.3. The optimization of medium components for the production of taxol by the mutant K178 was performed using both Plackett–Burman design and response surface methodology. The results showed that the optimal concentrations were: glucose 80g/l,NH₄NO₃ 7.84g/l,NaOAc 2.02 g/l,MgSO₄ 0.68g/l,KH₂PO₄ 0.5 g/l,ZnSO₄ 1×10^-3 g/l,Cu(NO₃) 2 1×10^-3 g/l,FeCl₃ 2×10^-3 g/l,VB1 50×10^-3 g/l,L-Tyrosine 5×10^-3 g/l. After the optimization of the media the yield of taxol was increased to 225.2μg/l. Several precursors such as phenylalanine, Na benzoate and catechol were studied and proved to have a positive influence. A kinetic model of mycelial growth was constructed based on Logistic formula.