Producing Taxanes Screening Of Endophytic Fungi And Preliminary Optimization Of Fermentation Conditions

Taxol is a member of diterpene taxane family of nature products and currently used in the treatment of ovarian and breast tumor.taxol and other related taxanes are extracted from the bark and leaves of Taxus family. The concentration of taxol in bark of Taxus family is so low that the supply of taxol can't afford the need of market. In this study, endophytic fungus which can producing taxane were isolated from T. yunnanensis. The purpose is to use the abundant microbes to explore the new taxane resources. The activity of GGPP sythase was assayed, which is a key enzyme of the way to biosynthesis of taxane, and the effect of some trace elements on the yield of taxane was also investigated. All those preliminarily resolve some basic problems to produce taxane by the microbial fermentation.200 strains of filaceous endophytic fungus were isolated from the inner bark, phloem-xylem of the T. yunnanensis. Most of these funguses can be separated into five genuses which belong to Penicillium sp., Aspergillus sp., Nigrospora sp., Alternaria sp. and Mucor sp. according to the morphology of the fungal colony, the mechanism of the spore production and the characteristics of the spores.46 strains were selected and cultured under shaking and then still conditions. The mycelium was extracted with methylene chloride. The crude extract was analyzed by thin-layer chromatography (TLC), UV and high performance liquid chromatography (HPLC), with taxane (taxol and baccatin III as standard. An endophyte 7.4.3 was screened out having the ablity of producing baccatin III.The optimum carbon and nitrogen source in liquid medium were saccharose and peptone separately. Sodium benzoate and sodium acetate could promote the accumulation of baccatin III as they act as the precursor of the baccatin III with the highest concentration at 0.1 mM and 0.01 mM separately. Phenylalanine or leucine has little effect on the biosynthesis of baccatin III. The content of baccatin III increased to 34.6μg/L after the inoculation in the optimum medium containing 20 g/L saccharose, 1.5 g/L peptone, 0.1 mM sodium acetate and 0.01 mM sodium benzoate.The GGPP synthase in the endophytic fungus was qualitative analyzed and compared with the enzyme existing in the leaves of T. yunnanensis.