Engineered Fusion Enzyme-Mediated Non-Consecutive Cyclization-Glycosylation Enables Heterologous Synthesis of Antifungal Enfumafungin.

Enfumafungin-type antibiotics, represented by enfumafungin and fuscoatroside, constitute a distinct class of fungi-derived fernane-type triterpenoids renowned for their potent antifungal activity. Notably, ibrexafungerp, a semi-chemically synthesized analogue of enfumafungin, has recently received approval as a novel antifungal drug. Thus, reconstituting the heterologous biosynthesis of enfumafungin holds great significance, as it offers a promising route for high-level production. Herein, the Aspergillus oryzae S184 chassis is first optimized. By deleting ku80 gene and refining counter-selection procedure, site-specific gene integration and substantially shortened the time required for selection marker recycling are significantly enhanced. Subsequently, an artificial biosynthetic pathway potentially involved in enfumafungin biosynthesis is successfully reconstructed. Crucially, the native terpene cyclase (TC)-glycosyltransferase (GT) fusion enzyme, EfuA, involved in enfumafungin biosynthesis, lost its functionality in A. oryzae. Conversely, a designed fusion enzyme EfuA_(TC)FsoA_(GT), which combines the TC domain of EfuA with the GT domain of FsoA (involved in fuscoatroside biosynthesis), along with FsoD/E/F, efficiently produced the putative enfumafungin intermediate. The functional analysis further revealed that while the fusion of the TC and GT domains is critical for maintaining dual enzymatic activity, these fusion enzymes catalyze unconventional, non-consecutive terpene cyclization and glycosylation steps during the biosynthesis of enfumafungin-type antibiotics, differing from other canonical fusion enzymes.