Engineering of Glarea lozoyensis for Exclusive Production of the Pneumocandin B0Precursor of the Antifungal Drug Caspofungin Acetate

Abstract

ABSTRACTPneumocandins produced by the fungusGlarea lozoyensisare acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B0is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B0is 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 B0. Disruption ofGLOXY4, encoding a nonheme, α-ketoglutarate-dependent oxygenase, confirmed its involvement inl-leucine cyclization to form 4S-methyl-l-proline. The absence of 4S-methyl-l-proline abolishes pneumocandin A0production, and 3S-hydroxyl-l-proline occupies the hexapeptide core's position 6, resulting in exclusive production of pneumocandin B0. Retrospective analysis of theGLOXY4gene in a previously isolated pneumocandin B0-exclusive mutant (ATCC 74030) indicated that chemical mutagenesis disrupted theGLOXY4gene function by introducing two amino acid mutations in GLOXY4. This one-step genetic manipulation can rationally engineer a high-yield production strain.