Engineering algae polysaccharide sulfotransferases for biotechnology

用于生物技术的工程藻类多糖磺基转移酶

• 批准号: 2438684
• 金额: --
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2438684
• 关键词: Engineering; algae; polysaccharide; sulfotransferases; biotechnology

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).

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，重点是OST的生物技术适应：1。藻类OST的酶活性。（酶学和多糖修饰）我们将使用PAPS/PAP传感器来确定重组藻类OST在不同种类的植物多糖的硫酸化中的活性和底物特异性。将通过NMR光谱分析最佳性能酶的糖产物以鉴定硫酸化位点，即酶的受体位点选择性。将测试硫酸化多糖的流变性、表面活性以及与金属离子的相互作用和保水性。2. OST的结构分析和工程。（蛋白质工程和结构生物学）我们将通过X射线晶体学解析所选择的具有高酶活性的OST的结构。我们将在受体位点引入一系列突变以改变酶的特异性和活性，旨在使其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中的设备的HTS筛选中进行测试，所述设备目前用于HSST突变体的大型（1500）化合物文库的HTS。寻找新的ST。（植物生物学和基因组生物信息学）我们将扩大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ST的生物，重点是来自植物王国的OST。这将建立关系，例如，已知的多糖ST，包括我们的藻类，并确定推定的新的多糖ST。我们将对少数最有可能的候选人进行排序，以验证我们的预测，如果时间允许，我们将表达这些候选人，并根据（1）和（2）中概述的策略进行有限和集中的分析。