Functional characterization of terpene glycosyltransferases from Vitis vinifera

葡萄萜烯糖基转移酶的功能表征

• 批准号: 170114906
• 负责人: Professor Dr. Ernst H. Rühl
• 金额: --
• 依托单位: Hochschule Geisenheim University
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/170114906?language=en
• 关键词: Functional; characterization; terpene; glycosyltransferases; Vitis

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.

萜类化合物是天然产品的主要类别之一，被广泛应用于许多领域，从保健和制药用途到食品和化妆品中的着色、调味和芳香化合物。生物合成通过甲羟戊酸或1-脱氧-d - 5-磷酸木糖途径进行。在植物中，萜类化合物被糖进一步修饰，这些糖与活性基团OH和/或COOH相连。在葡萄和葡萄酒中，单萜烯对品种风味的重要性被广泛认识，这些化合物的主要部分以非挥发性、无芳香活性的萜烯苷的形式存在。虽然这种水溶性部分是一种珍贵的香气来源，但人们对葡萄中催化萜烯醇糖基化的基因及其编码酶知之甚少。最近对拟南芥糖基转移酶（GT）基因的功能分析已经获得了27个序列，这些序列编码的蛋白质葡萄糖化了多种萜烯。我们从已发表的葡萄基因组数据库中提取了67个同源的推测的GT序列，这些序列分为8个亚群。将测定不同葡萄品种中潜在VvGT基因的时空表达水平，并将其与不同组织中萜烯苷的水平进行比较，以缩小推定的萜烯VvGT的数量。亚群中最有希望的成员将是异种表达和生物化学特征与一些潜在的萜酚底物。将测定重组蛋白对多种萜类化合物的立体选择性和区域选择性。这些结果可以应用于育种计划，以选择具有低萜烯GT活性的基因型，并且可能具有更高水平的芳香活性，游离萜烯醇，并开发生物技术工艺作为化学合成的替代品。