(162f) Characterization of Cellulose Biopolymer Synthesizing Enzymes Reconstituted in Vitro

Cell walls are one of the most important constituent of plant cells composed predominantly of complex polysaccharides. One such critical plant cell wall polysaccharide component composed of b-1,4-linked glucose units is cellulose. This abundant biopolymer is processively synthesized by integral membrane proteins called cellulose synthases (CesAs). These CesAs are arranged in a rosette-shaped manner and forms a larger complex containing six trimers of individual CesA units. Together they form a cellulose synthase complex (CSC). These cellulose synthase complexes contain several different cellulose synthase isoforms involved in primary and secondary cell wall formation. To understand how they synthesize cellulose at an enzymatic level, we are heterologously expressing two putative CesAs in Pichia pastoris and reconstituting them into lipid carriers. Such a system will aid in understanding the structural and biochemical characteristics of this challenging enzyme involved in the synthesis of this essential biopolymer. The reconstituted enzyme polymerizes UDP-glucose to cellulose by releasing UDP as detected by luminescence assays. The synthesized cellulose will be characterized using microscopic and enzymatic deconstruction studies. Ultimately, this work will help advance our mechanistic and holistic understanding of both in vitro and in vivo cell wall synthesis that would accelerate the development of improved transgenic crops for bioenergy and bioproducts relevant applications.