Towards the expression of a cellulose synthase-like F (CSLF) protein, responsible for the synthesis of mixed linkage β-glucan, in a microbial model system

M Bain, D Oehme, T Bacic and M Doblin

ARC Centre of Excellence in Plant Cell Walls, School of Biosciences, The University of Melbourne, Victoria 3010, Australia

(1,3)(1,4)-β-glucans, also known as mixed linkage glucans (MLGs), are major cell wall matrix polysaccharides in grasses with a distinct structure comprising both β-(1,3)- and β-(1,4)-glucosidic linkages. This unique structure makes MLG an important source of soluble dietary fibre conferring benefits to human health. The cellulose synthase-like F (CSLF) glycosyltransferases, of the CAZy GT2 family, are primarily responsible for the synthesis of MLG, with the major isoform CSLF6 localised and integral to the plasma membrane. However, despite the importance of MLG to both plant growth and human health, the molecular mechanism by which this polysaccharide is synthesised is not well understood. With the recent publication of the first crystal structures for a GT2 family member, a bacterial cellulose synthase (BcsA), new opportunities are created to draw parallels between related proteins which synthesise diverse polysaccharide products. The optimisation of an Escherichia coli expression host for the structural characterisation of CSLF6 would provide a powerful tool for exploring differences between GT2 proteins. We will present our latest findings directed towards the expression and isolation of catalytically active CSLF6 in E.coli.