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

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, alpha-ketoglutarate-dependent oxygenase, confirmed its involvement in L-leucine cyclization to form 4S-methyl-L-proline. The absence of 4S-methyl-L-proline abolishes pneumocandin A(0) production, and 3S-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.