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Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system

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Abstract

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.

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Funding

This work was supported by the National Natural Science Foundation of China (No. 31872623, 31071710), and National Key Research and Development Program of China (2023YFD1401400).

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Authors and Affiliations

  1. Department of Plant Pathology and MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian district, Beijing, 100193, China

    Siji Zhao, Ruya Yin, Mengwei Zhang, Ziqi Zhai, Zhen Shen, Yan Mou, Dan Xu, Ligang Zhou & Daowan Lai

Authors
  1. Siji Zhao
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  2. Ruya Yin
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  3. Mengwei Zhang
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  4. Ziqi Zhai
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  5. Zhen Shen
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  6. Yan Mou
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  7. Dan Xu
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  8. Ligang Zhou
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  9. Daowan Lai
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Contributions

Conceptualization, L.Z. and D.L.; methodology, S.Z., D.X., D.L.; software, S.Z., D.X.; formal analysis, S.Z., Z.S., D.L.; investigation, S.Z., R.Y., M.Z., Z.Z., Z.S and Y.M.; data curation, S.Z.; writing, S.Z., D.L.; supervision, L.Z. and D.L. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Daowan Lai.

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Zhao, S., Yin, R., Zhang, M. et al. Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system. World J Microbiol Biotechnol 40, 176 (2024). https://doi.org/10.1007/s11274-024-03988-y

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