Department of Microbiology, VNUHN-University of Science, Vietnam
Title of the talk: A new approach for construction of the auxotrophic mutants in the filamentous fungus Aspergillus oryzae by using Agrobacterium-mediated transformation
Aspergillus oryzae is a safe filamentous fungus widely used for production of traditional foods and beverages. This fungus is currently considered as microbial cell factory for recombinant protein production. However, A. oryzae is naturally resistant to most of the common antibiotics used for fungal transformation. Therefore, construction of auxotrophic mutants and relevant expression vectors plays an important role in genetic manipulation of this fungus. In this study, the first time uridine/uracil auxotrophic strains of A. oryzae were successfully generated by deletion of the pyrG gene using Agrobacterium tumefaciens-mediated transformation (ATMT). With this method, the pyrG gene was easily removed from the fungal genome by homologous recombination of the pyrG deletion cassette at the pyrG locus in all the obtained transformants. The pyrG mutants were first screened on the selective medium containing 5-fluoroorotic acid (5-FOA), then on the minimal medium lacking uridine/uracil prior to be confirmed by PCR analyses. The auxotrophic phenotype of the pyrG deletion mutant could be recovered to prototrophy by exogenous uridine/uracil supplementation or by complementation of an intact copy of the pyrG gene. In addition, we have developed a new versatile binary vector carrying the native pyrG cassette as nutritional marker and a gene expression cassette under regulation of the constitutive Aspergillus nidulansgpdA promoter. This binary vector is well functional for heterogenous expression of DsRed fluorescent reporter gene and phytase-encoding phyA gene from Aspergillus fumigatus in the auxotrophic A. oryzae strains. Our study provides a new and efficient approach for construction of uridine/uracil auxotrophic mutants in A. oryzae using Agrobacterium tumefaciens as the gene transfer tool and fungal spores (instead of protoplasts) as the material for transformation. This strategy is also applicable to other filamentous fungi.
Tran Van Tuan has completed his PhD and postdoctoral studies on molecular microbiology/fungal genetics from Georg-August University Göttingen (Germany). He is the head of Genomics Unit, National Key Laboratory of Enzyme & Protein Technology and the deputy head of Department of Microbiology, Faculty of Biology, VNU University of Science in Hanoi. He has published 7 papers in reputed journals such as PLoS Pathogens, New Phytologist, Applied Microbiology and Biotechnology, Molecular Plant-Microbe Interactions, etc.