Glycerol StabilizesYeast-like Morphology and PromotesLaccase Biosynthesis through Metabolic Remodeling in Aureobasidium melanogenum ZN

Native laccase production in polymorphic black yeasts is often limited by unstable cellular differentiation and insufficient metabolic coordination. Here, we developed a glucose–glycerol cofermentation strategy to improve laccase production in Aureobasidium melanogenum ZN by regulating cellular morphology and metabolism. The results showed that glycerol stabilized yeast-like growth and suppressed melanized differentiation, while the optimized combination of 2% glucose and 7.5% glycerol increased laccase activity and yield to 418.43 U·mL–1 and 65.67 U·g–1 dry weight (DW), respectively. The crude laccase showed an optimal activity at 75 °C and efficiently decolorized triphenylmethane dyes, especially malachite green, with partial activity retained under NaCl-containing and simulated wastewater-like conditions. Physiological and multiomics analyses linked glycerol-enhanced laccase production to yeast-like-state maintenance, carbon-source regulation, respiratory metabolism, redox adjustment, antioxidant responses, and reduced melanized differentiation. This study provides a morphology–metabolism-directed strategy for enhancing laccase production in native polymorphic fungi.