Decoding StuA-Mediated Sporulation Networks in Aspergillus cristatus: Potential Molecular Targets for Fu Brick Tea Quality Optimization

• First comprehensive study of StuA in a probiotic fungus: Reveals A. cristatus-specific regulatory networks governing sporulation, linking StuA to golden flower (cleistothecia) formation critical for Fu brick tea quality. • 2.Species-specific rewiring of conserved pathways: Identifies divergent StuA targets (e.g., repression of WetA, activation of CpcA) and a novel binding motif (5′-CTCTCCG-3′), contrasting with A. nidulans models. • 3.Ribosome biogenesis as a central hub: Demonstrates StuA deletion disrupts ribosomal subunit assembly (40S/60S), downregulating key genes (NOP10, Pop1)—a mechanism unreported in aspergilli sporulation. • 4.Probiotic-adapted stress coordination: Uncovers StuA’s role in synchronizing peroxisomal metabolism (CatA/Gor1) and autophagy to optimize oxidative stress tolerance during tea fermentation. • 5.Industrial application targets: Validates CpcA and SI65_00479 as engineering targets to enhance off-flavor metabolites in fermented foods. Aspergillus cristatus , the dominant probiotic fungus in Chinese Fu Brick tea (FBT) fermentation, governs the formation of health-promoting "golden flowers" (cleistothecia). However, the molecular regulation of its sporulation—critical for fermentation efficiency and functional metabolite production—remains unclear. In this study, we characterized the role and regulatory network of the APSES transcription factor StuA in A. cristatus by the combination of RNA sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-Seq). The ∆stuA mutant completely lacks both ascospores and conidiospores. We also found that the ∆stuA mutant presented lower colony radial growth rates than did the wild type, along with distinct brown‒yellow pigment accumulation and abundant deep-brown extracellular secretion. These morphological deficiencies highlight the crucial role that StuA plays in the developmental processes of A. cristatus . Surprisingly, transcriptomic analysis revealed that downregulated genes in the ∆stuA mutant during both the sexual and asexual stages were significantly enriched in the ribosome metabolism and ribosome biogenesis pathways. Weighted gene coexpression network analysis (WGCNA) revealed that the central genes associated with StuA are enriched primarily in MAPK signalling, autophagy, and mitotic cell cycle regulation. DAP-seq identified 490 direct genomic targets of StuA, with motif analysis pinpointing its binding preference to 5′-CTCTCCG-3′. Key targets ( CpcA, Acml ) were validated via EMSA and yeast one-hybrid assays, revealing StuA’s control over oxidative stress adaptation and secondary metabolism.Our work provides the first mechanistic insight into StuA-mediated sporulation in a food-grade fungus, offering genetic targets to enhance probiotic efficacy and functional compound production in fermented foods.