2. Academic Validation
  3. Exploration of the pneumocandin biosynthetic gene cluster based on efficient CRISPR/Cas9 gene editing strategy in Glarea lozoyensis

Exploration of the pneumocandin biosynthetic gene cluster based on efficient CRISPR/Cas9 gene editing strategy in Glarea lozoyensis

  • Int J Biol Macromol. 2024 Nov;279(Pt 2):135220. doi: 10.1016/j.ijbiomac.2024.135220.
Kaili Jiang 1,  Yating Jin 1,  Pan Luo 1,  Xinxin Wang 1,  Yuanwei Zhang 1,  Tianqiong Shi 2,  Jingjing Chen 3,  Ping Song 4,  Ling Lu 5
Affiliations

Affiliations

  • 1 Jiangsu Key Laboratory for Pathogens and Ecosystems, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China.
  • 2 School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China.
  • 3 State Key Laboratory of Bioactive Substance and Function of Natural Medicines, NHC Key Laboratory of Biosynthesis of Natural Products, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China. Electronic address: chenjingjing@imm.ac.cn.
  • 4 School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China. Electronic address: songping@njnu.edu.cn.
  • 5 Jiangsu Key Laboratory for Pathogens and Ecosystems, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China. Electronic address: linglu@njnu.edu.cn.
PMID: 39233151 DOI: 10.1016/j.ijbiomac.2024.135220
Abstract

Pneumocandin B0 (PB0) is a Lipopeptide produced by the fungus Glarea lozoyensis. The existing challenges with the low-yield and the extended-fermentation cycle emphasize necessity for strain improvement. In this study, we optimized conditions to obtain high-quality protoplasts and screened effective selection markers, leading to the construction of three CRISPR/Cas9 gene editing systems. Utilizing a constitutive Cas9 expression recipient strain, combined with dual sgRNAs targeting, we achieved highly efficient editing of target genes. We successfully knocked out 10 genes within the pneumocandin putative biosynthetic gene cluster and analyzed their roles in PB0 production. Our findings reveal that 4 of 10 genes are directly involved in PB0 production. Specially, the deletion of gltrt or gl10050 resulted in reduced PB0 production, while the absence of glhyp or glhtyC led to the complete loss of PB0 biosynthesis. Notably, the deletion of glhyp caused the silencing of nearly all cluster genes, whereas overexpression of glhyp led to a 2.38-fold increase in PB0 production. Therefore, this study provides the first comprehensive exploration of the functions of 10 genes within the pneumocandin putative biosynthetic gene cluster. Our findings provide valuable technical strategies for constructing bioengineering strains with purposefully enhanced PB0 production.

Keywords

CRISPR/Cas9; Dual sgRNAs; Glarea lozoyensis; Pneumocandin B(0).

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