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

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Abstract

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 106 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.

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Acknowledgements

We thank Dr. Jun-ichi Maruyama (The University of Tokyo, Japan) for kindly providing the A. oryzae AUT1-PlD strain and Prof. Dr. Ursula Kües (Georg-August-University Göttingen, Germany) for helpful comments to improve the manuscript. We are grateful to Prof. Dr. Tuan-Nghia Phan (VNU University of Science, Hanoi, Vietnam) for his valuable support and inspiration. We thank the members of the Genomics Unit, National Key Laboratory of Enzyme and Protein Technology (VNU University of Science, Hanoi, Vietnam) for their helpful discussions and technical assistance. This work was funded by the National Foundation for Science and Technology Development of Vietnam (NAFOSTED) under grant 106-NN.04-2014.75.

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  1. National Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

    Khuyen Thi Nguyen, Quynh Ngoc Ho, Loc Thi Binh Xuan Do, Huy Quang Nguyen & Van-Tuan Tran

  2. Faculty of Biology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

    Linh Thi Dam Mai, Duc-Ngoc Pham, Huyen Thi Thanh Tran, Diep Hong Le, Huy Quang Nguyen & Van-Tuan Tran

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  1. Khuyen Thi Nguyen
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Nguyen, K.T., Ho, Q.N., Do, L.T.B.X. et al. A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation. World J Microbiol Biotechnol 33, 107 (2017). https://doi.org/10.1007/s11274-017-2275-9

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