Isolation And Identification Of Taxanes-producing Endophytic Fungi

Taxol, a chemical substance of tetracyclic diterpene lactam, was first isolated from the bark, roots and branches of the Taxus brevifolia, in the late 1960's by Wall and Wani and their collaborators. It is a highly efficient, low toxicity, broad-spectrum natural anti-cancer drugs, which has been widely used against galactophore cancer, uterine cancer and other cancers. The main natural extract of taxol is from the skin of yew plant (Taxus), and its content is only 0.01%-0.05%. Taxus species is a threatened plant, and grows very slowly. The traditional methods of extracting taxol from the bark of Taxus species consume lots of raw materials. In other words, extracting taxol will cut down a large number of yew trees. According to calculating, 1kg paclitaxel, which could be used by only 5 patients, needs 10 tons bark from 300 trees. For this reason, seeking new ways of obtaining taxol is the key to protect yew resources and reduce the cost of drug therapy. To this end, scientists all over the world have done a lot of research in the areas related to taxol production, such as chemical synthesis, plant tissue cell culture and microbial fermentation and so on. Especially in the microbial fermentation, it is demonstrated that isolation and identification of taxol-producing endophytic fungi is a new approach to produce taxol.In this study, 123 endophytic fungi from different tissues of Taxus chinensis (Pilger) Rehd.were isolated, among which 4 taxanes-producing endophytic fungi named Cbr37-2,Cbr44-3,Cbr34-1 and Cbr52-1 were identified by HPLC analyses respectively. After PCR amplification based on ITS sequence, Cbr1-1, Cbr31-1, Cbr31-3, Cbr34-1, Cbr37-2, Cbr38-2, Cbr44-3, Cbr46-2 and Cbr51-2's species were preliminarily classified.The contents of taxanes in branches of Taxus chinensis var. mairei, Taxus chinensis Rehd, and Taxus cuspidata Sieb.et Zucc were determined and analysed by HPLC after optimization conditions. This method compared the contents of 3 kinds of taxanes (10-deacetyl-baccatin III, baccatin III, taxol) detected through the same chromatographic condition, which lay a foundation for developing and making use of Taxus resources, and lay a platform for extracting and detecting fermentation products of taxanes -producing endophytic .Fermentation condition of a gene engineering strain EFY-21 was researched, experimental comparison study by selecting different original medium, antibiotic, temperature and pH to determine optimum culture condition, the growth curve of EFY-21 was drawn and the culture conditions were optimized. The optimum carbon and nitrogen sources medium was determined, and optimum proportion of carbon and nitrogen sources was determined based on orthogonal experimen. This study paves the way for the massive production of taxol using transgenic endophytic fungi in the future.