Genome-wide characterization of glycosyltransferases involved in C-glycosylation and the metabolism of small molecules in the tea plant (Camellia sinensis)

用来泡茶的茶树的叶和叶芽是生物活性植物化学物质的丰富来源，这些化学物质经常以O-和C-糖结合物的形式出现。C-糖苷表现出与相应O-糖苷相似的生物活性，但同时表现出显著提高的体内清除稳定性，这使得它们在药物化学方面具有吸引力。尿苷二磷酸-糖依赖的糖基转移酶(UGTS)通常通过催化活化的核苷酸供体转移到一系列受体苷元形成糖苷来介导植物代谢物的糖基化。虽然糖苷前体决定了茶树的表现和成品茶的品质，但涉及小分子糖基化，特别是茶树中C-糖基化的基因和酶的研究很少。直到最近，中国的合作伙伴才公布了中华绒毛虫变种的基因组序列。这为茶叶基因组和茶叶质量的进化提供了洞察力。由于德国和中国的合作伙伴在小分子糖苷的生物技术生产和植物UGT的表征方面分别拥有丰富的经验，正如几份联合出版物所证明的那样，现在有可能确定茶树低分子糖苷生物合成所涉及的基因和酶的特征，并促进其生物技术生产。我们在茶叶基因组数据库中鉴定出298个UGT家族1基因。在目前的项目中，中国和德国的研究小组希望通过生物信息学分析来研究基因组扩展机制，并从茶树中分离出所有的UGT家族1基因。我们计划在宿主中表达重组蛋白，通过高通量筛选鉴定潜在的供体和受体底物，并通过在转基因植物系统中的过度表达和下调来研究选定的候选蛋白的生物学功能。UGT的活性将通过体外实验和生物转化实验进行分析。候选基因的功能特征将在拟南芥、烟草和茶树的植物中进行，并通过比较转录组和代谢物图谱分析进行。重点将放在参与C-糖基化、芳香化合物代谢和植物防御的UGT上。这些结果将为改善茶叶风味和茶树防御性以及生物技术生产具有更好的抗水解稳定性的生物活性碳苷奠定基础。

Leaves and leaf buds of the tea plant (Camellia sinensis) used to make tea are a rich source of bioactive phytochemicals that frequently occur as O- and C-glycoconjugates. C-glycosides show biological activity similar to the respective O-glycoside, but display at the same time a substantially improved stability against in vivo clearance, which make them attractive for medicinal chemistry. Uridine diphosphate-sugar (UDP)-dependent glycosyltransferases (UGTs) usually mediate glycosylation of plant metabolites by catalysing the transfer of an activated nucleotide sugar donor to a range of acceptor aglycones to form glycosides. Although glycoside precursors determine tea plant performance as well as the quality of the product tea, the genes and enzymes involved in the glycosylation of small molecules, especially the C- glycosylation in the tea plant, are rarely studied. Only recently, the Chinese partners have published the genome sequence of C. sinensis var. sinensis, which provided insights into the evolution of the tea genome and tea quality. Since the German and Chinese partners have extensive experience in the biotechnological production of small molecule glucosides and the characterization of plant UGTs, respectively, as demonstrated by several joint publications, it is now possible to characterize the genes and enzymes involved in the biosynthesis of low-molecular glycosides in tea plants and to facilitate their biotechnological production. We identified 298 UGT family 1 genes in the tea genome database. In the current project, the Chinese and German groups would like to investigate the genome expansion mechanisms by bioinformatics analysis and isolate all UGT family 1 genes from the tea plant. It is planned to express the recombinant proteins in host organisms, identify potential donor and acceptor substrates by high-throughput screens and investigate the biological function of selected candidates by overexpression and downregulation in transgenic plant systems. The activity of the UGTs will be analysed by both in vitro assays and biotransformation experiments. Functional characterization of candidates will be performed in planta in Arabidopsis thaliana, Nicotiana benthamiana and Camellia sinensis as well as by comparative transcriptome and metabolite profiling analyses. The focus will be on UGTs involved in the C-glycosylation, the metabolism of aroma chemicals and plant defence. These results will provide the foundation for improvement of tea flavour and tea plant defence, as well as the biotechnological production of bioactive C-glycosides with improved stability against hydrolysis.