Metabolic engineering of Glarea lozoyensis for high-level production of pneumocandin B₀

Pneumocandin B₀ (PB₀) is a lipohexapeptide synthesized by Glarea lozoyensis and serves as the precursor for the widely used antifungal drug caspofungin acetate (Cancidas®). However, the low titer of PB₀ results in fermentation and purification costs during caspofungin production, limiting its widespread clinical application. Here, we engineered an efficient PB₀-producing strain of G. lozoyensis by systems metabolic engineering strategies, including multi-omics analysis and multilevel metabolic engineering. We overexpressed four rate-limiting enzymes: thioesterase GLHYD, two cytochrome P450s GLP450s, and chorismate synthase GLCS; knocked out two competing pathways responsible for producing 6-methylsalicylic acid and pyranidine E; and overexpressed the global transcriptional activator GLHYP. As a result, the PB₀ titer increased by 108.7 % to 2.63 g/L at the shake-flask level through combinatorial strategies. Our study provides valuable insights into achieving high-level production of PB₀ and offers general guidance for developing efficient fungal cell factories to produce polyketide synthase-non-ribosomal peptide synthetase hybrid metabolites.