Enhanced β-glucan production in Aureobasidium pullulans via combinatorial ARTP mutagenesis, dual-dye screening, and...

β-Glucan is a valuable food additive and nutraceutical. Aureobasidium pullulans naturally produces extracellular soluble β-glucan, but its low fermentation titer hinders industrial use. To address this, we developed a high-yielding mutant strain (No.7) through combinatorial Atmospheric and Room Temperature Plasma (ARTP) mutagenesis coupled with a novel dual-dye screening strategy (Trypan blue/Congo red plates). Systematic medium optimization (Single-factor, Plackett-Burman, and Response Surface Methodology) yielded an optimal medium (g/L: glucose 90.0, yeast extract 25.0, peptone 20.0, ZnSO₄ 0.3, K₂HPO₄ 2.5, (NH₄)₂SO₄ 1.0). This significantly enhanced β-glucan production to 6.02 g/L in shake flasks (25.7 % increase vs. original medium), despite a 26.1 % reduction in biomass. Scalability was confirmed in a 5 L bioreactor, where the optimized medium boosted β-glucan (6.68 g/L, +19.1 %) and pullulan (39.67 g/L, +27.0 %) titers versus the original strain/medium. Comparative transcriptomics revealed profound metabolic reprogramming in mutant No.7 (4279 DEGs: 2215 up, 2064 down), with significant enrichment in core pathways (glycolysis, gluconeogenesis, TCA cycle, pentose phosphate, cell wall biosynthesis). Critically, this reprogramming elevated the activities and expression levels of key enzymes (β-GS, PGM) and intracellular metabolite pools (ATP, NADH). This work establishes mutant No.7 as a potent industrial β-glucan producer and presents an integrated strategy for strain improvement.