Molecular Breeding Of Fungus Strains And Taxus Cell Lines With High Yield Of Taxol

Taxol is one kind of highly effective, low cytotoxic and broad-spectrum natural anticancer drugs. However, due to the relative scarcity of yew trees, the supply of taxol is very limited. Producing taxol through cell cuture of Taxus and fermentation of taxol-producing fungi may be two promising ways to solve the scare resource of taxol. However, high production of taxol in the large-scale Taxus cell cultures is unstable. Low yield of taxol generated by current taxol-producing fungal strains is one problem in the application of taxol-producing fungi. Taxol-producing fungi and Taxus cell lines will be transformed with the key gene encoding the taxol biosynthase and the transcription factors in order to resolve these problems. In this study, some taxol-producing fungi were isolated from the bark of Taxus, the preliminary optimization of the fermentation conditions was done for the isolated taxol-producing fungi, the Taxus media cell line and the isolated taxol-producing fungi were transformed with the dbat gene and orca3 gene in order to obtain tansgenic Taxus media cell line and transgenic taxol-producing fungi with high yield of taxol. In addition, the putative promoter of dbat gene was cloned, and some transcription factors regulating the biosynthesis of taxol were transformed into taxol-producing fungi and Taxus cells.The results of this study were as follows:(1) A new and rapid screening method for taxol-producing fungi based on PCR amplification was developed by using the genes encoding for 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) and C-13 phenylpropanoid side chain-CoA acyltransferase (BAPT) as molecular markers. This method greatly simplifies the screening process of taxol-producing fungi.(2) Three taxol-producing endophytic fungi, strain MD2, MD3 and YN6, were obtained from T. media and T. yunnanesis by this method, and they were identified as Cladosporium cladosporioides, Aspergillus candidus, and Pestalotiopsis sp. according to microscopic morphological characteristics, physiological and biochemical characteristics and their 18S rDNA gene sequence analysis, etc. C. cladosporioides MD2 and A. candidus MD3 are first described as taxol-producing endophytic fungi in this study. The mean yield of fungal taxol from C. cladosporioides MD2, A. candidus MD3 and P. sp YN6 was about 160μg/L, 112μg/L and 140μg/L, respectively, when they were cultured in 300 ml potato dextrose liquid media at 25℃with 180 rpm for 10 days. The three strains are relatively high yield of taxol in the current wild strains.(3) The dbat genes were cloned for the first time from C. cladosporioides MD2, A. candidus MD3 and P. sp YN6, and the blast results showed that they possesses high homology to the same gene found in Taxus spp. It is the first time to report the dbat gene sequence from taxol-producing fungi.(4) The fermentation conditions of C. cladosporioides MD2 and A. candidus MD3 were investigated including culture temperature, the initial pH of media, the rotational speed of the incubator and the culture time, and 4 precursors were studied by added in the media. The optimized media allowed the yield of taxol to be increased to 569.5μg/L for C. cladosporioides MD2, and 497.3μg/L for A. candidus MD3.(5) The genetic transformation system of the condinia of C. cladosporioides MD2 by Agrobacetrium tumefaciens LBA4404-mediated transformation was established after optimizing some factors, including co-culture temperature, MD2 conidia concentration, the growth period and the initial quantity of LBA4404, the rotational speed of the shaker, co-cultivation time, the period induced by AS and AS concentration.(6) The dbat gene and orca3 gene were cloned. The pCAMBIA1303 vectors as the the basic skeleton were used to construct the expression vectors containing dbat gene, orca3 gene, and dbat gene and orca3 gene, respectively, and successfully transformed into C. cladosporioides MD2, A. candidus MD3 and T. media cell lines. The mean yield of taxol of C. cladosporioides MD2, A. candidus MD3 and T. media cells transformed with dbat gene was significantly improved, and was 860μg/L, 750μg/L, 923.5μg/L, respectively, and the results also showed that the dbat gene is a key taxol biosynthase gene for C. cladosporioides MD2 and A. candidus MD3.(7) The mean yield of taxol of C. cladosporioides MD2, A. candidus MD3 and Taxus media cells transformed with orca3 gene was decreased, and the results showed that the transcription factor, ORCA3, was negative regulation for the taxol biosynthesis in C. cladosporioides MD2, A. candidus MD3 and T. media cells for the first time.(8) The arbitrary primers PCR method and one-side specific primers PCR method was combined to clone the 5'-flanking sequence of dbat gene for the first time. The putative promoter sequence of dbat gene was transformed into the tobacco leaves for transient expression, and the results indicated that the 5'-flanking sequence of dbat gene could drive expression of GUS gene. The transcription factors regulating the biosynthesis of taxol were cloned and transformed into taxol-producing fungi and Taxus cells to obtained taxol-producing fungi and Taxus cells with stable and high-yield taxol production.