Engineering algae polysaccharide sulfotransferases for biotechnology

该提案将碳水化合物化学、酶学、生物信息学和植物生物学结合在一个多学科项目中，重点研究OSTs的生物技术适应性。藻类ost的酶活性。我们将使用PAPS/PAP传感器来确定重组藻类OSTs在不同种类植物多糖硫酸酸化中的活性和底物特异性。性能最好的酶的糖产物将通过核磁共振波谱分析来确定硫酸化位点，即酶的受体位点选择性。硫酸盐酸化多糖将测试其流变性、表面活性、与金属离子的相互作用和保水性。2. ost的结构分析与工程。（蛋白质工程与结构生物学）我们将通过x射线晶体学来解析选择的高酶活性OST(s)的结构。我们将在受体位点引入一系列突变来改变酶的特异性和活性，旨在使它们适应异源多糖的硫酸化。突变体将在基于Eu PAPS/PAP传感器和GeneMill设备的HTS筛选中进行测试，该设备目前用于大型（1500）HSSTs抑制剂化合物库的HTS。搜寻新的STs。（植物生物学和基因组生物信息学）我们将扩展整个生命之树的系统发育分析，以寻找可能具有适合生物技术的STs的生物，重点是来自植物界的STs。这将建立关系，例如，已知的多糖STs，包括我们的藻类STs，并确定假定的新的多糖STs。我们将对少数最有可能的候选者进行排序，以验证我们的预测。如果时间允许，我们将这些候选者表达出来，并根据(1)和(2)中概述的策略进行有限的重点分析。

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).