Construction Of High Efficient Genetic Transformation System And Evaluation Of Promoter Activity For Cladosporium Cladosporioides MD2

Taxol is one kind of high efficient, low cytotoxic and broad-spectrum naturalanticancer drugs, however the supply of taxol is currently limitted due to lack of Taxus.Production of taxol with fermentation of taxol-producing fungi is considered as one of themost effective ways to solve the problem of the shortage of taxol. However, the taxol yieldof most of taxol-producing endophytic fungi is so low that it still cannot meet therequirements of large-scale industrial production. Genetic engineering technique is aneffective method to enhance the production of microbial secondary metabolites. Thegenetic transformation methods and screening of strong promoters are two of the keyfactors for genetic engineering of microorganism. In this study, the genetic transformationmethods and screened high efficient promoters for Cladosporium cladosporioides MD2, ataxol-producing fungi.were study to laid a fine groundwork for the construction of taxolhigh-yield transgenic strains of C. cladosporioides MD2. The results are as follows:(1) An efficient and stable micro-projection transformation protocol using particledelivery system was successfully developed for C. cladosporioides MD2by optimizedseveral parameters in this study. The the highest transformation efficiency was100±5transformants per microgram of plasmid DNA under the following conditions: vaccumpressure at20mg Hg, spore concentration at108/mL, target distance at7cm and0.3μgplasmid DNA per bombardment.(2) Highly efficient promoters were screened through analyzing the expression levelsof the green fluorescent protein (gfp) gene drived by trpC promoter, gpdA promoter andCaMV35S promoter in C. cladosporioides MD2, and the results showed that the drivingability of the gpdA promoter derived from fungus was highest and was2.95times of thatof the CaMV35S promoter, which was minimum.(3) The expression vector containing pgpdA promoter and a gene encoding10-deacetyibaccatin III-10β-O-acetyltransferase, which was a key enzyme for taxolbiosynthesis, was successfully constructed and transformed into C. cladosporioides MD2by gene gun. The biomass and the taxol production of the transgenic strains were analyzed,and the results showed that the taxol yield of the transgenic strains was1.67times of that of the wild strains; however the biomass was decreased by10%compared with the wildstrains.