Engineering of Glarea lozoyensis for Exclusive Production of the Pneumocandin B0 Precursor of the Antifungal Drug Caspofungin Acetate

摘要

Pneumocandins produced by the fungus Glarea lozoyensis are acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B_(0) is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B_(0) is a minor fermentation product, and its industrial production was achieved by a combination of extensive mutation and medium optimization. The pneumocandin biosynthetic gene cluster was previously elucidated by a whole-genome sequencing approach. Knowledge of the biosynthetic cluster suggested an alternative way to produce exclusively pneumocandin B_(0). Disruption of GLOXY4 , encoding a nonheme, α-ketoglutarate-dependent oxygenase, confirmed its involvement in l-leucine cyclization to form 4 S -methyl-l-proline. The absence of 4 S -methyl-l-proline abolishes pneumocandin A_(0) production, and 3 S -hydroxyl-l-proline occupies the hexapeptide core's position 6, resulting in exclusive production of pneumocandin B_(0). Retrospective analysis of the GLOXY4 gene in a previously isolated pneumocandin B_(0)-exclusive mutant (ATCC 74030) indicated that chemical mutagenesis disrupted the GLOXY4 gene function by introducing two amino acid mutations in GLOXY4. This one-step genetic manipulation can rationally engineer a high-yield production strain.