Functional analysis of small molecule glycosyltransferases from plants by aglycone libraries

天然产物糖基化是植物次生代谢产物的重要修饰。在植物中，糖基化参与调节激素稳态、外源生物解毒以及次生化合物的生物合成、储存和功能。普遍存在的糖基转移酶（GT）家族的一个特定亚类催化这些反应。虽然这些酶已经被深入研究了几十年，但到目前为止，只有少数酶在植物中被鉴定出来。相比之下，植物基因组计划揭示了该家族中意想不到的复杂性，并且首次高通量筛选（HTS）揭示了酶的底物混杂性，这阻碍了单基因的功能分析和它们在植物中的功能鉴定。最近，我们开发了一种新的方法来鉴定GTs的体内底物，即使这些底物不是市售的。该方法使用天然糖苷水解产生的生理苷元文库进行底物筛选，并允许公正地发现由未知功能基因编码的酶活性。该创新方法将用于苹果、草莓、覆盆子、葡萄和番茄的GTs功能表征的HTS优化和应用。候选基因将从基因组序列和相关转录组数据中选择，重组蛋白将异种表达，并从叶片和果实组织中产生苷元文库。各种给体底物将被合成和新的糖苷结构鉴定。最后，将通过反向遗传方法在植物中验证GT功能。该项目将揭示新型GTs在生产小分子糖苷中的生理作用，并将使GTs在生产食品添加剂的生物技术过程中得到应用。

Glycosylation of natural products is a crucial modification of plant secondary metabolites. In plants, glycosylation is involved, among others, in the regulation of hormone homeostasis, detoxification of xenobiotics and the biosynthesis, storage and function of secondary compounds. A specific subclass of the ubiquitous glycosyltransferase (GT) family catalyzes these reactions. Although these enzymes have been studied intensively for decades, to date only a handful have been characterized in plants. In contrast, plant genome projects have uncovered unexpected complexity within this family and first high-throughput screens (HTS) have revealed substrate promiscuity of the enzymes that is hindering the functional analysis of single genes and the identification of their in planta functions. Recently, we have developed a novel method for the identification of in vivo substrates of GTs even if the substrates are not commercially available. This method uses a physiologic aglycone library produced by hydrolysis of natural glycosides for the substrate screening and allows unbiased discovery of enzymatic activities encoded by genes of unknown function. The innovative method will be optimized for and used in a HTS for the functional characterization of GTs from apple, strawberry, raspberry, grape and tomato. Candidate genes will be selected from genome sequences and related transcriptome data, recombinant proteins will be heterologously expressed and aglycone libraries will be produced from leaf and fruit tissue. Various donor substrates will be synthesized and novel glycosides structurally identified. Finally, GT functions will be verified in planta by reverse genetic approaches. The project will uncover the physiological roles of novel GTs in the production of small molecule glycosides and will enable the application of the GTs in biotechnological processes for the production of food additives.