Journal Article

A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation

Nguyen, Khuyen Thi, Tran, Huyen Thi Thanh, Mai, Linh Thi Dam, Do, Loc Thi Binh Xuan, Ho, Quynh Ngoc, Le, Diep Hong, Nguyen, Huy Quang, Pham, Duc-Ngoc, Tran, Van-Tuan

2017

Overview

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens -mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens , we could get about 1060 transformants per 10 6 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae . This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

• Publisher: Springer Netherlands,Springer Nature B.V
• Subject: Acid resistance / Agar / Agrobacterium radiobacter / Agrobacterium tumefaciens - genetics / Agrobacterium tumefaciens - physiology / antifungal agents / Applied Microbiology / Aspergillus oryzae / Aspergillus oryzae - genetics / auxotrophs / Bacteria / Biochemistry / Biomedical and Life Sciences / Biotechnology / Clonal deletion / Construction industry / Deletion mutant / Environmental Engineering/Biotechnology / Fluorescence / Food industry / Food processing industry / fungal spores / Fungi / Fungicides / Gene Deletion / Gene expression / Genes / Genes, Fungal / Genetic Engineering / Genetic transformation / Life Sciences / Markers / Microbiology / Microorganisms / Mutagenesis, Insertional / Original Paper / Plates / PyrG gene / recombinant proteins / Reporter gene / reporter genes / Spores / Strategy / Transformation, Genetic / Uracil / Uracil - metabolism / Uridine / Uridine - metabolism