Engineering of Glarea lozoyensis for Exclusive Production of the Pneumocandin B 0 Precursor of the Antifungal Drug Caspofungin Acetate

ABSTRACT 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.