Optimization Of The Fermentation Condition Of Taxol-producing Fungus From Taxus Chinensis

Taxol, a highly derivative diterpenoid, was firstly found in some species of Taxus. In 1992, Taxol was marketed as an anticancer drug approved by FDA. Taxol produced by plant endophytic fungus fermentation has come up to hotspot in recent years. Finding out high yield strains, screening optimal cultivation conditions and controlling metabolism will be effective ways to increase the yield of taxol.With the goal of increasing taxol yield by microorganism fermentation, the author isolated an endophytic fungus, Metarhizium anisopliae LB-10, from Qin-Ba mountainous areas in China, and further conducted the research on culture media, fermentation conditions and growth regulators. Chief experimental methods and results are as follows:1. One hundred and nine endophytic fungi were isolated from wild Taxus chinensis originated in Qin-Ba mountainous areas, and 82 strains were purified, which, in the fermentations, were examined by the method of UV, HPLC and HPLC-MS. The strains were identified by morphological characteristics and ITS sequence analysis. The results showed that 8 strains of endophytic fungi could produce taxol in their metabolites, including a high taxol-producing fungus, LB-10, whose yield of taxol was 846.1μg/L, and the strain was identified fungus Metarhizium anisopliae.2. The yield of taxol was set as an index, the analytical results by HPLC showed that the best conben and nitrogen sources for producing higher yield were glucose and ammonia nitrate respectively. The optimum concentration of glucose, ammonium nitrate and anhydrous magnesium sulfate were determined by single factor esperiments. The experiment in the orthogonal design L₉(3⁴) indicated that the three best combination in the medium was A₁B₂C₁, or glucose 50.0 g/L, nitrate bromide 6.0 g/L, anhydrous magnesium sulfate 0.3 g/L. Consequently, the optimal fermentation medium was obtained that glucose 50.0 g/L, NH₄NO₃ 6.0 g/L, anhydrous MgSO₄ 0.3 g/L, KH₂PO₄ 0.5 g/L, VB1 5.0×10^-2 g/L. Their yield of taxol was 938.6μg/L.3. Using HPLC detection method, the fermentation conditions of Metarhizium anisopliae LB-10 was studied. The optimum conditions of temperature, pH and inoculum were determined by single factor esperiments. The experiment in the orthogonal design L₉(3⁴) indicated that the three best combination in the conditions was A₂B₂C₃, or temperature was 27 oC, initial fermentation pH was 7.0, inoculum was 5%. Their yield of taxol was 949.6μg/L. At the same time, the curve of taxol accumulation and mycelium growth were drawn. The optimal fermentation period was 14 d, with the growth regulators added on the 10th day, the end of logarithmic growth period.4. According to the structural characteristics and synthesis mechanism of taxol, parts of the precursor, elicitor and inhibitors were selected to study its effect on yield of taxol and mycelium growth. The results indicated that when each of the following growth regulators was added to the fermention process on the 10th day, and allowed its initial concentration to be corresponding value, the yield of taxol could be enhanced: sodium benzoate, 30.0 mg/L; tyrosine, 20.0 mg/L; phenylalanine, 3.0 mg/L; sodium acetate, 30.0 mg/L; salicylic acid, 20.0 mg/L; methyl jasmonate, 100.0μmol/L; GA3, 2.0 mg/L; CCC, 2.0 mg/L. The experiment in the orthogonal design L₁₆(4⁵) indicated that the four best combination of precursors in the medium was A₃B₃C₃D₁, or phenylalanine 3.0 mg/L, tyrosine 40.0 mg/L, sodium acetate 30.0 mg/L, sodium acetate 1.0 g/L. Their yield of taxol was 987.3μg/L. Simultaneously, the addition of sugar to fermentation was researched. The results indicated that taxol accumulation and mycelium growth was stimulated when appending sugar no higher than 15.0 g/L at 10th day in the fermentation process. But above this concentration, mycelium growth was inhibited and yield of taxol was decreased.