Efficient Extracellular Production of Highly Branched β-(1→3,1→6)-Glucan by Engineered Aureobasidium melanogenum

Abstract β-(1→3,1→6)-Glucan is valued for its immunomodulatory activity, but conventional production via fungal cell wall extraction suffers from complex procedures and low yields. Here, the mangrove-derived strain Aureobasidium melanogenum BZ-11 was engineered by sequentially disrupting the melanin and pullulan biosynthetic pathways, generating the mutant strain BZΔPΔA capable of producing pigment-free, pullulan-free extracellular β-glucan. β-Glucan featured a β-(1→3,1→6)-linked structure with a high molecular weight of (1.33 ± 0.24) × 106 g/mol and a high degree of branching of 57%, along with favorable storage and processing stability. In a 10 L fermentation, BZΔPΔA achieved an extracellular β-glucan titer of 20.33 ± 1.42 g/L. Direct ethanol precipitation and lyophilization from the culture supernatant yielded 94.83% β-glucan purity and 97.78% recovery, enabling simple purification without harsh extraction. This work establishes a scalable and efficient platform for producing structurally defined extracellular β-(1→3,1→6)-glucan, with strong potential for applications in functional foods, pharmaceuticals, and biomedical products.