Engineering algae polysaccharide sulfotransferases for biotechnology

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The proposal combines carbohydrate chemistry, enzymology, bioinformatics and plant biology in a multi-disciplinary project focused on biotechnological adaptation of OSTs:1. Enzymatic activity of algal OSTs. (enzymology and polysaccharide modification) We will use the PAPS/PAP sensor to define activity and substrate specificity for the recombinant algae OSTs in sulfation of different classes of plant polysaccharides. The saccharide product of the best performing enzymes will analysed by NMR spectroscopy to identify the site(s) of sulfation, that is the acceptor site selectivity of the enzymes. Sulfated polysaccharides will be tested for their rheology, surface activity and interaction with metal ions and water retention. 2. Structural analysis and engineering of OSTs. (protein engineering and structural biology) We will solve structures of the selected OST(s) with high enzymatic activity by X-ray crystallography. We will introduce a range of mutations into the acceptor site to modify the specificity and activity of the enzymes, aiming to adapt them for the sulfation of heterologous polysaccharides. The mutants will be tested in HTS screens based on the Eu PAPS/PAP sensor and the equipment in GeneMill, currently employed for HTS of large (1500) compound libraries for HSSTs inhibitors.3. Search for new STs. (plant biology and genome bioinformatics) We will expand the phylogenetic analysis across the Tree of Life to search for organisms that are likely to have STs suitable for biotechnology, with a focus on OSTs from the plant Kingdom. This will establish the relationships of, e.g., the known polysaccharide STs, including our algal ones, and identify putative novel polysaccharide STs. We will sequence a small number of the most likely candidates to validate our predictions and if time allows express these and undertake a limited and focused analysis based on the strategy outlined in (1) and (2).