A multifaceted bioengineering toolbox to enable in vivo and in vitro characterization of polysaccharide synthases
Creators: name: Jayachandran, Dharanidaran
Publisher: name: No Publisher Supplied
Year: 2024

description: Polysaccharides are a major class of natural polymers found abundantly across all forms of life and play a critical role as structural, metabolic, or functional components in biomolecular processes. For example, the most abundant renewable polymer on earth, viz., cellulose, act as storage reserves or cell walls in plants and microbes that could potentially be converted into biofuels or useful bioproducts. Similarly, hyaluronan or hyaluronic acid is a multifunctional high molecular weight polysaccharide found in eyes and synovial fluid in joints that provides support and anchorage for cells, cell adhesion, and proliferation and facilitates movement and migration. Both cellulose and hyaluronan are synthesized by membrane-integrated glycosyltransferases (GT), namely cellulose synthase (CesA) and hyaluronan synthase (Has), respectively. Although these enzymes have been purified and characterized to some extent, there are huge gaps in our mechanistic, structural, and holistic understanding of these systems. Hence, in this work, we were interested in using protein engineering approaches and reconstitution strategies to understand these enzymes' biochemical and structural features in vivo and in vitro. Firstly, we studied the dynamics of cellulose synthesis in Arabidopsis plant protoplast cell walls in vivo. We designed different tandem carbohydrate-binding modules and biochemically characterized them to find the most suitable probe for real-time visualization of regenerating plant cell walls. In vivo cell wall regeneration observed using these protein probes provided fundamental insights into the cellulose synthesis mechanism, movement, and alignment on the surface of the plant membrane. Using these key events, a model was proposed for the regeneration of cellulose in plant cell walls. Then, cellulose synthases involved in primary (CesA5 from Physcomitrella patens) and secondary (CesA8 from Populus tremula x tremuloides) cell wall synthesis were heterologously expressed in Pichia pastoris, followed by purification and reconstitution into liposomes to study their substrate utilization, kinetics, and product characterization using biochemical assays. However, the expression of GTs in cell-based systems suffered from low yields because of numerous factors making them less suitable for structural characterization and nanodisc reconstitution. Fortunately, the wheat-germ-based cell-free expression system offered an alternative to the expression of these GTs. The cell-free expression system gave a higher yield, involved lesser steps, and was biochemically active. Mutations in the conserved residues of bacterial hyaluronan synthase (from Streptococcus equisimilis) showed an impact on substrate binding and polymer synthesis. Overall, this work demonstrates the development of various protein engineering toolkits that could be used to study and unravel the mechanism underlying polysaccharide synthesis with bioenergy and biomedical applications both in vivo and in vitro.