β-(1,3;1,4)-Mixed linkage glucans (MLGs) are linear homopolysaccharides composed of β-1,4-linked glucose oligomers interconnected by β-1,3 linkages. While previous studies have characterised MLG synthases from Romboutsia ilealis and Clostridium ventriculi, the broader diversity of these enzymes remains underexplored. To expand the repertoire of bacterial MLG synthases, nine glycosyltransferase family 2 (GT2) candidates homologous to R. ilealis GT2 were identified and heterologously expressed in an engineered Saccharomyces cerevisiae strain. Two candidates, Clostridium nigeriense GT2 (CnGT2) and Clostridium cuniculi GT2 (CcGT2), catalysed the formation of MLG polymers, albeit at reduced levels relative to the reference enzyme. Structural analysis and mutagenesis identified the PilZ domain's D/NxSxxG motif as a critical regulator of synthase activity. These findings provide a structural basis for optimizing bacterial MLG synthases, facilitating the development of efficient microbial production platforms.