Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain<italic/> of <i>Aspergillus oryzae</i> RIB40

GAO Yuqing; ZHANG Haojie; ZHANG Danfeng; PAN Yutian; ZHANG Feng

doi:10.13386/j.issn1002-0306.2022040179

Volume 44 Issue 1

Dec. 2022

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Science and Technology of Food Industry > 2023 > 44(1): 200-207. > DOI: 10.13386/j.issn1002-0306.2022040179

GAO Yuqing, ZHANG Haojie, ZHANG Danfeng, et al. Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40[J]. Science and Technology of Food Industry, 2023, 44(1): 200−207. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022040179.

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Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40

1.
The Engineering Technological Center of Fungus Active Substances of Fujian Province, Minnan Normal University, Zhangzhou 363000, China
2.
College of Biological Sciences and Technology, Minnan Normal University, Zhangzhou 363000, China

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Received Date: April 17, 2022
Available Online: October 30, 2022

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Abstract

Abstract

Objective: An auxotrophic strain with high efficiency of homologous recombination was developed in Aspergillus oryzae RIB40. Methods: The AopyrG mutant of A. oryzae RIB40 was firstly obtained using a homologous recombination technique and the 5-fluoroorotic acid (5-FOA) selection for the uridine/uracil auxotroph. To disrupt the ∆Aoku70 gene, the Aoku70 gene of AopyrG mutant was replaced with A. fumigatus pyrG gene. Then, under the screening effect of 5-FOA, the AfpyrG gene of the ∆Aoku70 strain was deleted via genome self-cyclization. To test the utilization of the resulting strain, the mCherry gene, a red fluorescent protein gene, was integrated with the histone H2B gene in the ∆Aoku70∆AopyrG strain. The subcellular localization of red fluorescent protein was detected by laser confocal microscope. Results: An auxotrophic mutant ∆Aoku70∆AopyrG with high homologous recombination efficiency was established via disruption of the A. oryzae RIB40 genes Aoku70 and AopyrG. The red fluorescence was observed in the nucleus of the fungal cell, indicating the utility of the ∆Aoku70∆AopyrG strain. Conclusion: The ∆Aoku70∆AopyrG strain can be used as a recipient strain with high-efficiency homologous recombination and uridine/uracil auxotroph for future gene modification in A. oryzae RIB40.
- Aspergillus oryzae RIB40,
- uridine/uracil auxotroph,
- pyrG,
- ku70,
- fluorescence localization

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References

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