Regulatory roles of Smt3, an ortholog of small ubiquitin-like modifier, in fungal development and the production of amylases and organic acids in Aspergillus niger.

The small ubiquitin-like modifier (SUMO) is pivotal to SUMOylation and exerts diverse roles in filamentous fungi, yet its roles in the production of carbohydrate-active enzymes (CAZymes) and organic acids remain unclear and few studies have addressed its functions in Aspergillus niger, a filamentous fungus with dual roles in food fermentation and food spoilage. This study identified a single SUMO homologue (Smt3) in Pu-erh tea-isolated A. niger RAF106 and characterized its functions via multi-phenotypic and transcriptional analyses. smt3 deletion caused defects in fungal growth, correlating with limited utilization of several carbon/nitrogen sources, mitochondrial dysfunction, and excessive autophagy. Moreover, Δsmt3 severely impaired conidiation, as evidenced by reduced conidial yields, underdeveloped conidiphores, and downregulated transcription of conidiation-related genes. Notably, smt3 deletion reduced the yields of amylases and organic acids (notably oxalic acid and malic acid). Mechanistically, Smt3 promoted amylase synthesis and secretion via activating the expression of the transcription factor amyR, endoplasmic reticulum chaperones (kar2 and lhs1), and secretory-related flbE, while it regulated the production of oxalic acid and malic acid by controlling the expression of core metabolic genes and restraining aberrant authophagy. Additionally, Δsmt3 displayed increased sensitivity to sorbitol, with unchanged fruit spoilage capability and stress tolerance to NaCl, Congo red, and H2O2. Collectively, Smt3 acts as an important regulator of asexual development, cellular tolerance to sorbitol, amylase production, and organic acid synthesis in A. niger, advancing the understanding of SUMO in fungal physiology and providing a novel target for biotechnological improvement of amylase and organic acid production in food industrial application.