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

Pneumocandin B (PB) 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 PB production. Our findings reveal that 4 of 10 genes are directly involved in PB production. Specially, the deletion of gltrt or gl10050 resulted in reduced PB production, while the absence of glhyp or glhtyC led to the complete loss of PB 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 PB 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 PB production.