Functional analysis of small molecule glycosyltransferases from plants by aglycone libraries

通过苷元文库对植物小分子糖基转移酶进行功能分析

• 批准号: 312032787
• 负责人: Professor Dr. Wilfried Schwab
• 金额: --
• 依托单位: Professur für Biotechnologie der Naturstoffe (BiNa)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/312032787?language=en
• 关键词: Functional; analysis; small; molecule; glycosyltransferases

Glycosylation of natural products is a crucial modification of plant secondary metabolites. In plants, glycosylation is involved, among others, in the regulation of hormone homeostasis, detoxification of xenobiotics and the biosynthesis, storage and function of secondary compounds. A specific subclass of the ubiquitous glycosyltransferase (GT) family catalyzes these reactions. Although these enzymes have been studied intensively for decades, to date only a handful have been characterized in plants. In contrast, plant genome projects have uncovered unexpected complexity within this family and first high-throughput screens (HTS) have revealed substrate promiscuity of the enzymes that is hindering the functional analysis of single genes and the identification of their in planta functions. Recently, we have developed a novel method for the identification of in vivo substrates of GTs even if the substrates are not commercially available. This method uses a physiologic aglycone library produced by hydrolysis of natural glycosides for the substrate screening and allows unbiased discovery of enzymatic activities encoded by genes of unknown function. The innovative method will be optimized for and used in a HTS for the functional characterization of GTs from apple, strawberry, raspberry, grape and tomato. Candidate genes will be selected from genome sequences and related transcriptome data, recombinant proteins will be heterologously expressed and aglycone libraries will be produced from leaf and fruit tissue. Various donor substrates will be synthesized and novel glycosides structurally identified. Finally, GT functions will be verified in planta by reverse genetic approaches. The project will uncover the physiological roles of novel GTs in the production of small molecule glycosides and will enable the application of the GTs in biotechnological processes for the production of food additives.

天然产物的糖基化修饰是植物次生代谢产物的重要修饰方法。在植物中，糖基化尤其涉及调节激素稳态、外源物质的解毒以及次生化合物的生物合成、储存和功能。普遍存在的糖基转移酶（GT）家族的一个特定亚类催化这些反应。尽管这些酶已经被深入研究了几十年，但迄今为止，只有少数酶在植物中得到了表征。相比之下，植物基因组计划已经发现了这个家族中意想不到的复杂性，并且第一个高通量筛选（HTS）已经揭示了酶的底物混杂性，这阻碍了单个基因的功能分析和它们在植物中功能的鉴定。最近，我们已经开发了一种新的方法，用于识别在体内的底物的GT，即使基板是没有商业上可获得的。该方法使用由天然糖苷水解产生的生理糖苷配基文库用于底物筛选，并且允许无偏地发现由未知功能的基因编码的酶活性。该创新方法将被优化并用于HTS中，用于对来自苹果、草莓、树莓、葡萄和番茄的GT进行功能表征。候选基因将从基因组序列和相关转录组数据中选择，重组蛋白将异源表达，糖苷配基文库将从叶和果实组织中产生。将合成各种供体底物，并在结构上鉴定新型糖苷。最后，GT功能将在植物中通过反向遗传方法进行验证。该项目将揭示新型GT在小分子糖苷生产中的生理作用，并将使GT在食品添加剂生产的生物技术过程中得到应用。

项目成果

Novel biotechnological glucosylation of high-impact aroma chemicals, 3(2H)- and 2(5H)-furanones

• DOI: 10.1038/s41598-019-47514-9
• 发表时间: 2019-07-29
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Effenberger, Isabelle;Hoffmann, Thomas;Schwab, Wilfried
• 通讯作者: Schwab, Wilfried

Glucosylation of the phytoalexin N-feruloyl tyramine modulates the levels of pathogen-responsive metabolites in Nicotiana benthamiana.

• DOI: 10.1111/tpj.14420
• 发表时间: 2019-10
• 期刊: The Plant journal : for cell and molecular biology
• 作者: Guangxin Sun;M. Strebl;M. Merz;Robert Blamberg;F. Huang;Kate McGraphery;T. Hoffmann;W. Schwab-

Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases

• DOI: 10.3390/ijms21062208
• 发表时间: 2020-03-01
• 期刊: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
• 影响因子: 5.6
• 作者: McGraphery, Kate;Schwab, Wilfried
• 通讯作者: Schwab, Wilfried

Six Uridine-Diphosphate Glycosyltransferases Catalyze the Glycosylation of Bioactive C13-Apocarotenols1

六种尿苷二磷酸糖基转移酶催化生物活性 C13-脱胡萝卜素的糖基化1

• DOI: 10.1104/pp.20.00953
• 发表时间: 2020
• 期刊: Plant Physiology
• 影响因子: 7.4
• 作者: Guangxin;Putkaradze;Natalia;Bohnacker;Joncyk;Hoffmann;Thomas;Bernhardt;Härtl;Schwab;Wilfried
• 通讯作者: Wilfried