Prebassianins from Akanthomyces araneicola: Discovery, Biosynthesis, and a Rare OYE-SDR Fusion Enzyme.

Two new 4-hydroxy-2-pyridone alkaloids, prebassianins F (5) and G (6), featuring a rare 1,4-cyclohexanediol moiety, were isolated from rice cultures of Akanthomyces araneicola GY29011. Genome mining and heterologous expression of the corresponding biosynthetic gene cluster (pre) in Aspergillus nidulans afforded six additional new analogues (3-4 and 7-10), along with two known compounds (1-2). Combinatorial expression and substrate feeding experiments enabled the elucidation of the biosynthetic pathway and functional assignment of the tailoring enzymes. Notably, PreC was identified as a rare bifunctional fusion enzyme comprising old yellow enzyme (OYE) and short-chain dehydrogenase (SDR) domains. Truncation of PreC led to the accumulation of a key intermediate (10), supporting its role in sequential reductions. cDNA analysis confirmed that PreC is encoded as a single open reading frame containing four introns, including a rare GC-AG intron. Compounds 3 and 4 exhibited antibacterial activity against Bacillus subtilis (MIC = 16 μg/mL). These findings expand the structural diversity of 4-hydroxy-2-pyridone alkaloids and provide new insights into the enzymatic logic and evolution of fungal biosynthetic pathways.