Formation of ellagic acid precursors in Fragaria

草莓（Fragaria x ananassa）含有大量的生物活性多酚，特别是鞣花酸/鞣花单宁。最近的重大发现有力地表明鞣花酸/鞣花单宁对多种人类疾病，包括心血管疾病、神经退行性疾病和激素癌症，如前列腺癌和乳腺癌，提供有效的保护。鞣花单宁对细菌、酵母菌和病毒具有很强的抗菌活性，因此可以作为抵御多种微生物感染的天然屏障。虽然这些化合物的生物学意义已被广泛承认，但其生物合成途径的遗传背景几乎是未知的。我们正计划克隆和异种表达编码可能的没食子酸酯的基因：udp -葡萄糖葡萄糖基转移酶（GT）和没食子酸酯酰基转移酶（AT），它们被认为参与鞣花单宁前体的形成。候选基因将根据生物化学特征（羟基）苯甲酸酯gt和酰基葡萄糖依赖性ATs的序列信息，结合下一代测序（NGS）和定量PCR的表达分析进行鉴定。或者，我们将分离和纯化鞣花单宁途径酶从f.x ananassa通过活性引导测定。将对这些蛋白进行测序，并从最近发表的F. vesca基因组序列中提取相应的开放阅读框。据推测，这两个Fragaria物种有共同的祖先。重组蛋白将在大肠杆菌或酿酒酵母中表达并进行功能表征。这些数据可用于开发高鞣花酸/鞣花单宁含量的分子标记，从而缩短育种周期，培育具有额外健康效益的品种。研究结果可用于鞣花酸衍生物的生物技术生产。

Strawberry (Fragaria x ananassa) contains high amounts of bioactive polyphenols, in particular ellagic acid/ellagitannins. Recent significant findings strongly suggest that ellagic acid/ellagitannins provide effective protection against diverse human diseases including cardiovascular diseases, neurodegenerative diseases, and hormonal cancers like prostate and breast cancer. Ellagitannins possess strong antimicrobial activity towards bacteria, yeasts and viruses and thus serve as natural defence against diverse range of microbial infections. Although the biological significance of these compounds is widely acknowledged the genetic background of their biosynthetic pathway is almost unknown.We are planning to clone and heterologously express genes coding for putative gallate:UDP-glucose glucosyltransferases (GT) and gallate acyltransferases (AT) assumed to be involved in the formation of ellagitannin precursors. Candidate genes will be identified on the basis of sequence information of biochemically characterized (hydroxyl)benzoate GTs and acyl-glucose dependent ATs in combination with expression analysis performed by next-generation sequencing (NGS) and quantitative PCR. Alternatively, we will isolate and purify ellagitannin-pathway enzymes from F. x ananassa by activity guided assays. The proteins will be sequenced and the corresponding open reading frames extracted from the recently published genome sequence of F. vesca. It is supposed that both Fragaria species share a common ancestor. Recombinant proteins will be expressed in Escherichia coli or Saccharomyces cerevisiae and functionally characterized. The data can be used to develop molecular markers for high ellagic acid/ellagitannin content that will shorten the breeding period towards cultivars with additional health benefits. Besides, the result can be applied for the biotechnological production of ellagic acid derivatives.