Characterization of a fungal mixed-linkage glucan synthase

Mixed-linkage (1,3;1,4)-β-glucan (MLG) is a cell wall polysaccharide found in fungi, bacteria, and plants, yet the enzymatic machinery and structural determinants governing fungal MLG biosynthesis remain poorly understood. In Aspergillus fumigatus , TFT1 ( Af TFT1) has previously been implicated in MLG biosynthesis, but its direct biochemical activity remained unresolved. The structural characterization of A. fumigatus wall MLG revealed a distinct polymer profile. Heterologous expression of Af TFT1 in Komagataella phaffii enabled MLG production, generating an oligosaccharide profile that closely matched that of the native fungal polymer and demonstrating that Af TFT1 functions as an MLG synthase. Phylogenetic analysis placed Af TFT1 within a distinct glycosyltransferase family 2 (GT2) fungal lineage containing related candidate MLG synthases across diverse filamentous Ascomycota and separate from the major plant and bacterial synthase lineages. Structure-guided mutagenesis showed that individual substitutions within the transmembrane pore and switch motif altered the lichenase-derived oligosaccharide profile while retaining detectable MLG production, whereas replacement of the entire switch motif with the corresponding Hv CSLF6 sequence resulted in no detectable MLG production. Together, these findings establish Af TFT1 as a fungal MLG synthase and identify the switch motif and adjacent transmembrane region as important determinants of MLG synthase function and product fine structure, providing insight into the structural and evolutionary diversification of MLG biosynthesis.