Functional analysis of small molecule glycosyltransferases from plants by aglycone libraries

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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.