Transformation Of Filamentous Fungi Mediated By Agrobacterium Tumefaciens

Recombinant DNA technology has been developed and applied in filamentousfungi to improve production of target products. However, lack of efficient transforma-tion system always hampered studies of genetic engineering in filamentous fungi.In order to obtain new resistant gene and construct new selection marker fortransformation of filamentous fungi mediated by A. tumefaciens, strains being able todegrade PPT from soil were isolated. The strain could grow on medium containingPPT as sole carbon source and resist PPT up to 2.7 g/L. In addition to generalphysiologial and biochemical characterization, the strain was identified by 16S rDNAsequence and systematic analysis. The results showed that the sequence of 16S rDNAof the strain had similarity of 99.3% with that of Enterobacter cancerogenus, andsuggested the strain belong to Enterobacter. The strain was nominated asEnterobacter sp. PPT.The binary vector pCAM3300 was introduced into A.tumefaciens byelectroporation. Effects of different electroporation conditions on transformationfrequency were studied. Researches suggested transformation frequency was thehighest, up to 9.8×103 transformants/μg DNA, when the electrical field strength was11kV·cm-1. When OD600 was about 1.0, transformation frequency was the highest, upto 9.54×103 transformants /μg DNA. Ttransformation frequency is related with celldensity, and increase with rise of cell concentration. But the size of plasmid has nospecific relation with electroporation. The A. tumefaciens containing the binaryvectors pCAM3300 was co-cultured with filamentous fungi. Then, transformants weregained on plate containing PPT. PCR results demonstrated that Bar gene wasintegrated into filamentous fungi. Research indicated Bar gene can be utilized asselection marker in filamentous fungi and PPT can be used as selection pressure.Transformation system of filamentous fungi mediated by A. tumefaciens in which Bargene as selection marker was constructed, and this system can provide bases forresearch of filamentous fungi in the future.Based on above researches, The transformation system was utilized to transfergenes encoding the key enzymes for glycerol metabolism in candida glycerol genesisinto filamentous fungi in the first time. In construction of recombinant binary plasmidpCAM3300-h-D and pCAM3300-h-P, GPD and GPP gene were inserted into locus inwhich CaMV35S promoter and Tnos terminator was located in upstream anddownstream of target gene, respectively. Genes resisting to PPT and hygromycinBwere used for selection located in upstream and downstream of promoter. In presenceof acetosyringone (AS), A. tumefaciens containing pCAM3300–h-D andpCAM3300–h-P was co-cultivated with hypha and spore of filamentous fungi,respectively. Transformants being able to grow in the plate containing PPT orhygromycin B were selected, then, PCR indicated target gene had been transferredinto filamentous fungi. Fermentation was studied with transfermants of Aspergillusniger and Neurspora crassa, respectively. The results indicated that capability ofproducing glycerol of transformants didn't both improve greatly.