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/鞣质单宁。最近的重要发现有力地表明，鞣花酸/鞣花单宁可有效预防各种人类疾病，包括心血管疾病、神经退行性疾病以及前列腺癌和乳腺癌等激素性癌症。鞣皮单宁对细菌、酵母菌和病毒具有很强的抗菌活性，因此对各种微生物感染具有天然防御作用。虽然这些化合物的生物学意义已被广泛认识，但它们的生物合成途径的遗传背景几乎是未知的。我们正计划克隆并异源表达推测为没食子酸的编码基因：UDP-葡萄糖葡萄糖转移酶(GT)和没食子酸酰基转移酶(AT)，它们被认为参与印胶单宁前体的形成。候选基因将根据生化特征的(羟基)苯甲酸GTS和酰基葡萄糖依赖的ATS的序列信息，结合下一代测序(NGS)和定量PCR进行的表达分析来确定。或者，我们将通过活性指导分析来分离和纯化来自F.x ananassa的纤维素单宁途径酶。这些蛋白质将被测序，并从最近公布的F.Vesca基因组序列中提取相应的开放阅读框架。据推测，这两个Fragaria物种拥有共同的祖先。重组蛋白将在大肠杆菌或酿酒酵母中表达，并进行功能鉴定。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，培育出对健康有额外好处的品种。此外，研究结果还可应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.