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

The artificial biosynthetic pathway potentially involved in biosynthesis of enfumafungin is first reconstructed in an optimized Aspergillus oryzae chassis with the increased site‐specific integration efficiency and shortened time for marker recycling, which requires an artificial fusion enzyme EfuA(TC)FsoA(GT). Crucially, the fusion enzymes, involved in biosynthesis of enfumafungin‐type antibiotics, catalyze non‐consecutive cyclization and glycosylation, different from canonical fusion enzymes. Abstract 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. The artificial biosynthetic pathway potentially involved in biosynthesis of enfumafungin is first reconstructed in an optimized Aspergillus oryzae chassis with the increased site-specific integration efficiency and shortened time for marker recycling, which requires an artificial fusion enzyme EfuA (TC) FsoA (GT). Crucially, the fusion enzymes, involved in biosynthesis of enfumafungin-type antibiotics, catalyze non-consecutive cyclization and glycosylation, different from canonical fusion enzymes. Abstract 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. Advanced Science, Volume 12, Issue 44, November 27, 2025.