改造葡聚糖蔗糖酶用于天然产物糖基化的研究

Glycosylation of natural products could improve their solubility, stability, bioactivity and so on. Glycosyltransferases (GTs), which catalyze the attachment of a sugar moiety to an aglycone, are key enzymes for the biosynthesis of many valuable natural products. Most GTs catalyze the attachment of a sugar from an NDP-activated sugar donor to an aglycone acceptor, forming a glycosidic bond. Because NDP-sugars are generally quite expensive and unstable, the application of GTs is limited. Glucansucrases catalyze the split of sucrose into glucose and fructose as well as transferring the glucose moiety to the non-reducing end of saccharides. In this project directed evolution strategy as well as high-throughput screening method will be applied to engineer the acceptor substrate specificities of glucansucrase GTF-D from Streptococcus mutans, with the aim of making it able to catalyze the attachment of glucosyl moiety from sucrose to the aglycones of a series of natural products. The engineered GTF-D mutants will combine the donor substrate specificity of glucansucrase and the acceptor substrate specificity of GTs, and they will not only play an important role in studies of natural products glycosylation but also lower the cost of in vitro biosynthesis of glycosylated natural products comparing with GTs. Furthermore, the mutants will be able to introduce α-glycosides on the aglycones of natural products which are rarely found in nature. The formation of glycosylated natural products with α-glucosyl configuration will improve the natural-product diversification and open the door to the study of the bioactivity and other properties of α-glucosylated natural products.

天然产物的糖基化修饰对其稳定性、水溶性、生物活性等多种特性具有重要意义。自然界中，催化天然产物糖基化的关键酶是糖基转移酶，但由于此类酶所需的活化糖基供体（如NDP-糖基等）昂贵且不稳定，限制了糖基转移酶的体外应用。葡聚糖蔗糖酶可催化蔗糖水解，并将其葡萄糖基团转移至受体底物寡糖的末端生成葡聚糖。本研究将采用定向进化与高通量筛选法，对来自Streptococcus mutans的葡聚糖蔗糖酶GTF-D的受体底物特异性进行改造，使其成为能以廉价的蔗糖作为糖基供体，对一系列天然产物骨架进行糖基化修饰的广谱性转移酶。改造后的突变酶将集成葡聚糖蔗糖酶的供体底物特异性和糖基转移酶的受体底物特异性，在天然产物糖基化修饰研究或体外糖基化天然产物大量合成中发挥重要作用。同时，该突变酶能合成自然界少见的α-葡萄糖基化天然产物，为扩充天然产物结构库和研究α-葡萄糖基化天然产物的生物活性及其它特性奠定了基础。

通过定向进化法和高通量筛选，改造葡聚糖蔗糖酶GTF-D的受体底物结合口袋，获得以蔗糖为底物向多种天然产物骨架的酚羟基上转移葡萄糖基的酶突变体，其糖基受体底物谱从寡糖扩充到包括黄酮类化合物等的天然产物分子，拓展葡聚糖蔗糖酶的底物特异性，并就酶学性质将突变体蛋白与野生型酶进行比较。通过分子建模的方式，分析葡聚糖蔗糖酶突变体与天然产物受体的结合，深入认识该酶结构与功能的关系。利用突变体蛋白催化天然产物，获得几种自然界不存在的α-葡萄糖基化天然产物，对其进行分离纯化及结构鉴定，并测定水溶性、抗氧化活性等方面的性质。所获得的新型的α-糖基化天然产物将丰富现有天然产物结构库，为研究其性质、功能奠定基础。本研究项目依照计划顺利执行，取得预期研究结果，发表SCI文章3篇，培养硕士研究生一名。