Directing the Expression of Thermostable Glycohydrolases in planta and Designer Glycopeptide Engineering Technology for Sustainable Sugar Beet Post-Harvest Processing

Sugar beets are targeted for expanded industrial sugar production beyond traditional growing regions to meet national needs for advanced biofuels, renewable chemical feedstocks, and for conversion to value-added biobased products.  We are investigating biochemical and molecular technologies for controlled sugar beet root cell wall modification and hydrolysis to provide economic and environmental benefits for sustainable beet-biomass processing.  We are assembling a toolbox with thermostable glycohydrolases, including a Citrus thermally-tolerant pectin methylesterase, a Geobacillus thermostable endo-arabinanase, and the Acidothermus cellulolyticus E1 endoglucanase, to evaluate effective action on beet pulp processing and demonstrate bioproduction systems.  Our goal is to establish their suitability to rationally manipulate the structural and functional properties of structural polysaccharides in situ with their direct expression in sugar beet tap-roots.  

Our experimental results showed that pectin methylesterase action dramatically reduces water binding in beet pulp through improved calcium binding.  Recombinant arabinanase produced by yeast was able to generate functional oligosaccharides from sugar beet pulp, specifically, feruloylated arabino-oligosaccharides (which may promote healthful colon functioning through prebiotic, anti-inflammatory, and mucosal immuno-modulatory activities).  We also leveraged hydroxyproline-O-glycosylation – a process unique to plant cell wall glycoproteins – as an innovative technology for de novo design and engineering of novel glycopeptides that can function as a molecular carrier for in planta-expressed cell wall-modifying enzymes (e.g., endoarabinanase), efficiently targeting enzymes to the cell wall matrix and conferring improved stability to the enzymes.  Our results demonstrate proof-of-principle for ultimate recombinant enzyme expression in sugar beet roots, which will provide a novel output trait for biotech beets.