Functional characterization of terpene glycosyltransferases from Vitis vinifera

萜类化合物是天然产物的主要类别之一，广泛应用于从保健和制药到食品和化妆品中的色素、香料和芳香化合物等多种用途。生物合成通过甲伐酸或1-脱氧-D-木糖-5-磷酸途径进行。在植物中，萜类化合物进一步被糖装饰，这些糖与活性基团OH和/或COOH相连。在葡萄(Vitis Vinifera)和葡萄酒中，单萜对品种风味的重要性得到了广泛的认可，这些化合物中的主要部分是非挥发性的、不具香气活性的萜苷。虽然这种可溶于水的部分是香气的宝贵来源，但人们对葡萄中催化松油醇糖基化的基因及其编码的酶知之甚少。最近对拟南芥糖基转移酶(GT)基因的功能分析得出了27个序列，其编码的蛋白质可使多种萜类化合物糖基化。我们从已发表的葡萄基因组数据库中提取了67个同源的推定的GT序列，并将其分为8个亚组。潜在的VvGT基因在不同葡萄品种中的空间和时间表达水平将被确定，并将与不同组织中萜类糖苷的水平进行比较，以缩小可能的萜类VvGT的数量。亚组中最有希望的成员将以异源方式表达，并具有许多潜在的萜烯醇底物的生化特征。重组蛋白的立体选择性和区域选择性将被确定为各种萜类化合物。这一结果可用于育种项目，以选择具有低萜烯GT活性的基因型，以及可能具有较高香气活性和游离萜烯醇水平的基因型，并开发生物技术过程来替代化学合成。

Terpenoids represent one of the major classes of natural products and are used in many applications, from health care and pharmaceutical uses to colour, flavour and fragrance compounds in food and cosmetics. Biosynthesis occurs through either the mevalonic acid or the 1-deoxy-D-xylulose 5-phosphate pathway. In plants, terpenoids are further decorated with sugars which are linked to the active groups OH and/or COOH. In grapes (Vitis vinifera) and wines, where the importance of monoterpenes on varietal flavour is widely recognized a major fraction of theses compounds is present as non-volatile, aroma-inactive terpene glycosides. Although this water-soluble fraction is a precious source of aroma, little is known about the genes and their encoded enzymes catalyzing the glycosylation of terpenols in grapes. A recent functional analysis of Arabidopsis thaliana glycosyltransferase (GT) genes has yielded 27 sequences whose encoded proteins glucosylate a diversity of terpenes. We have extracted 67 homologous, putative GT sequences from the published Vitis vinifera genome database which cluster into 8 subgroups. Spatial and temporal expression levels of the potential VvGT genes will be determined in different grapes varieties and will be compared with the levels of terpene glycosides in different tissues to narrow down the number of putative terpene VvGTs. The most promising members of the subgroups will be heterologously expressed and biochemically characterized with a number of potential terpenol substrates. Stereo- and regioselectivity of the recombinant proteins will be determined for a variety of terpenoids. The results can be applied in breeding programs to select for genotypes with low terpene GT activities and, presumably, higher levels of aroma-active, free terpenols and to develop biotechnological processes as alternative to chemical synthesis.