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Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae

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Abstract

Genetic engineering of the filamentous fungus Aspergillus oryzae still requires more suitable selection markers for fungal transformation. Our previous work has shown that Agrobacterium tumefaciens-mediated transformation (ATMT) based on the uridine/uracil auxotrophic mechanism with pyrG as the selection marker is very efficient for gene transfer in A. oryzae. In the present study, we delete the hisB gene, which is essential for histidine biosynthesis, in A. oryzae via homologous recombination and demonstrate that hisB is a reliable selection marker for genetic transformation of this fungus. Under optimal conditions, the ATMT efficiency of the histidine auxotrophic A. oryzae reached 515 transformants per 106 spores. Especially, we have succeeded in constructing a new ATMT system based on dual auxotrophic A. oryzae mutants with two different selection markers including hisB and pyrG. This dual auxotrophic ATMT system displayed a transformation efficiency of 232 transformants per 106 spores for the hisB marker and 318 transformants per 106 spores for the pyrG marker. By using these selectable markers, the co-expression of the DsRed and GFP fluorescent reporter genes was implemented in a single fungal strain. Furthermore, we could perform both the deletion and complementation of the laeA regulatory gene in the same strain of A. oryzae to examine its function. Conclusively, the ATMT system constructed in our work represents a promising genetic tool for studies on recombinant expression and gene function in the industrially important fungus A. oryzae.

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Acknowledgements

The authors thank Dr. Tao Xuan Vu for the technical assistance and Dr. Thu V. Vuong (The University of Toronto, Canada) for critical reading of the manuscript. This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant Number 106.04-2018.36.

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Authors and Affiliations

  1. Department of Microbiology, Faculty of Biology, University of Science, Vietnam National University, Hanoi (VNU), 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

    Hanh-Dung Thai & Van-Tuan Tran

  2. Genomics Unit, National Key Laboratory of Enzyme and Protein Technology, University of Science, Vietnam National University, Hanoi (VNU), 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

    Hanh-Dung Thai, Bich-Phuong Thi Nguyen, Van-Manh Nguyen & Van-Tuan Tran

  3. Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Science, Vietnam National University, Hanoi (VNU), 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

    Quang-Huy Nguyen

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  1. Hanh-Dung Thai
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Thai, HD., Nguyen, BP.T., Nguyen, VM. et al. Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae. World J Microbiol Biotechnol 37, 92 (2021). https://doi.org/10.1007/s11274-021-03060-z

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