The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae

• Published in: World journal of microbiology & biotechnology 2016-12, Vol.32 (12), p.204-204, Article 204
• Main Authors: Nguyen, Khuyen Thi, Ho, Quynh Ngoc, Pham, Thu Ha, Phan, Tuan-Nghia, Tran, Van-Tuan
• Format: Article
• Language: English
• Subjects: Agrobacterium tumefaciens - physiology; Antibiotic resistance; Antibiotics; Applied Microbiology; Aspergillus oryzae - genetics; Biochemistry; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Drug resistance; Environmental Engineering/Biotechnology; Enzymes; Fungi; Gene expression; Genes, Fungal; Genes, Reporter; Genetic engineering; Genetic Vectors - genetics; Genotype & phenotype; Glucose; Laboratories; Life Sciences; Microbiology; Original Paper; Plasmids; Polyethylene glycol; Promoter Regions, Genetic; Protein Engineering; Proteins; Recombinant Proteins - metabolism; Transformation, Genetic; Virulence
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-016-2168-3

Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens -mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans . All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.