Abstract
Marine-derived Aspergillus terreus produces a variety of structurally novel secondary metabolites, most of which show unique biological activities. However, the lack of efficient genetic tools limits the discovery of new compounds, the elucidation of involved biosynthesis mechanism, as well as the strain engineering efforts. Therefore, in this study, we first established both an effective PEG-mediated chemical transformation system of protoplasts and an electroporation system of conidia in a marine-derived fungus A. terreus RA2905. To overcome the insensitivity of RA2905 to fungicides, the uracil auxotrophy strain (pyrG gene deletion mutant, ΔpyrG) was constructed using PEG-mediated transformation system, and using ΔpyrG as the genetic background, the methyltransferase gene laeA-overexpression transformants were further constructed through both PEG- and electroporation-mediated transformations, which showed enhanced terrein production. Besides, in this study, an efficient CRISPR/Cas9 genome-editing system was established for the first time in A. terreus, and a higher gene deletion efficiency of 71% for APSES transcription factor gene stuA could be achieved when using short homologous arms compared with conventional long homologous ones. In addition, using a non-integrative Cas9 plasmid, another efficient and marker-free genome-editing system was established, which allowing repeatable and unlimited genetic manipulation in A. terreus. Using the marker-free genome-editing system, we successfully developed the ΔpyrGΔku70 double-deletion mutant in RA2905, which could further improve gene deletion efficiency. In conclusion, efficient genetic manipulation systems along with a variety of functional mutants were developed in this study, which would significantly expedite both theoretical and applied researches in not only A. terreus but also other marine-derived filamentous fungi.
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Acknowledgements
We would like to thank Pro.f Chang-Yun Wang (Ocean University of China) for providing fungal strain, Dr. Jibin Sun (Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) and Dr. Guodong Liu (Shandong University) for providing plasmids. We also would like to thank a lot for Yao-Yao Zheng (Ocean University of China) for his excellent technical assistance.
Funding
This work was supported by Grants from Minjiang University (JAT190622, MYK19011 and MJY19019), China Ocean Mineral Resources R&D Association (DY135-B2-13), Project from Natural Science Foundation of Fujian Province (2021J011043, 2019J01766 and 2018N2001) and Program for Innovative Research Team in Science and Technology in Fujian Province University.
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GSY, XFC, and JMC conceived and designed the research work. GSY, YJH, XFC and HWZ performed the experiments and analyzed the data. GSY wrote the manuscript, and YJH, ZHW, XFC and JMC revised the manuscript.
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Yao, G., Chen, X., Han, Y. et al. Development of versatile and efficient genetic tools for the marine-derived fungus Aspergillus terreus RA2905. Curr Genet 68, 153–164 (2022). https://doi.org/10.1007/s00294-021-01218-8
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DOI: https://doi.org/10.1007/s00294-021-01218-8