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A newly constructed Agrobacterium-mediated transformation system based on the hisB auxotrophic marker for genetic manipulation in Aspergillus niger

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Abstract

The filamentous fungus Aspergillus niger is widely exploited as an industrial workhorse for producing enzymes and organic acids. So far, different genetic tools, including CRISPR/Cas9 genome editing strategies, have been developed for the engineering of A. niger. However, these tools usually require a suitable method for gene transfer into the fungal genome, like protoplast-mediated transformation (PMT) or Agrobacterium tumefaciens-mediated transformation (ATMT). Compared to PMT, ATMT is considered more advantageous because fungal spores can be used directly for genetic transformation instead of protoplasts. Although ATMT has been applied in many filamentous fungi, it remains less effective in A. niger. In the present study, we deleted the hisB gene and established an ATMT system for A. niger based on the histidine auxotrophic mechanism. Our results revealed that the ATMT system could achieve 300 transformants per 107 fungal spores under optimal transformation conditions. The ATMT efficiency in this work is 5 − 60 times higher than those of the previous ATMT studies in A. niger. The ATMT system was successfully applied to express the DsRed fluorescent protein-encoding gene from the Discosoma coral in A. niger. Furthermore, we showed that the ATMT system was efficient for gene targeting in A. niger. The deletion efficiency of the laeA regulatory gene using hisB as a selectable marker could reach 68 − 85% in A. niger strains. The ATMT system constructed in our work represents a promising genetic tool for heterologous expression and gene targeting in the industrially important fungus A. niger.

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All data for this study are included in the manuscript and its supplementary material file.

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Funding

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.04-2018.36. Hanh-Dung Thai was funded by Vingroup JSC and supported by the Master, PhD Scholarship Programme of Vingroup Innovation Foundation (VINIF), Institute of Big Data, code VINIF.2021.TS.076.

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Authors and Affiliations

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

    Hanh-Dung Thai, Loc Thi Binh Xuan Do, Xuan Thi Nguyen, Tao Xuan Vu, Huy Quang Nguyen & Van-Tuan Tran

  2. Faculty of Biology, University of Science, Vietnam National University, Hanoi (VNU), 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam

    Hanh-Dung Thai, Huyen Thi Thanh Tran, Huy Quang Nguyen & Van-Tuan Tran

  3. Center for Experimental Biology, National Center for Technological Progress, Ministry of Science and Technology, C6 Thanh Xuan Bac, Thanh Xuan, Hanoi, Viet Nam

    Tao Xuan Vu

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  1. Hanh-Dung Thai
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  2. Loc Thi Binh Xuan Do
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  3. Xuan Thi Nguyen
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  4. Tao Xuan Vu
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  5. Huyen Thi Thanh Tran
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  6. Huy Quang Nguyen
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  7. Van-Tuan Tran
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Contributions

VTT and HDT designed the study. HDT, LTBXD, XTN, TXV, and HTTT performed experiments. HDT, HQN, and VTT conducted the data analysis. VTT and HDT wrote the manuscript. VTT and HQN edited the final manuscript. All authors read and approved the final version of the manuscript.

Corresponding author

Correspondence to Van-Tuan Tran.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Communicated by Yusuf Akhter.

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Thai, HD., Do, L.T.B.X., Nguyen, X.T. et al. A newly constructed Agrobacterium-mediated transformation system based on the hisB auxotrophic marker for genetic manipulation in Aspergillus niger. Arch Microbiol 205, 183 (2023). https://doi.org/10.1007/s00203-023-03530-y

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