Suche nach neuartigen pilzlichen Prenyltransferasen der DMATS-Superfamilie durch funktionelle Genomik und zielgerichtete Mutagenese

通过功能基因组学和定点诱变寻找 DMATS 超家族的新型真菌异戊二烯基转移酶

• 批准号: 191073366
• 负责人: Professor Dr. Shu-Ming Li
• 金额: --
• 依托单位: Institut für Pharmazeutische Biologie und Biotechnologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/191073366?language=en
• 关键词: Suche; nach; neuartigen; pilzlichen; Prenyltransferasen

The aim of this proposal is to get new prenyltransferases of the DMATS superfamily, which are involved in the biosynthesis of secondary metabolites in ascomycota and catalyze prenylation of tryptophan, tyrosine, cyclic dipeptides or analogues thereof. For this purpose, 22 putative prenyltransferases are planned to be cloned from Neosartorya fischeri, Aspergillus nidulans, A. terreus, A. oryzae and Chaetomium globosum. The overproduced recombinant fusion proteins will be incubated with about 100 potential aromatic substrates in the presence of dimethylallyl or geranyl diphosphate. The enzyme products will be isolated on HPLC and identified by NMR and MS analyses.Based on the structure information of two indole prenyltransferases FgaPT2 and FtmPT1, site-directed mutagenesis will be also used to create enzyme derivatives with new features such as changed substrate specificity, prenylation pattern and position as well as reaction efficiency. Furthermore, structures of more prenyltransferases should be solved and used as basis for site-directed mutagenesis to create new enzyme derivatives.In addition to ion dependence and kinetic parameters, the biochemical characterization of the obtained prenyltransferases should be focused on the substrate specificity and their application as tools for production of prenylated indole and tyrosine derivatives, which was successfully used in our group.

这项建议的目的是获得新的DMATS超家族的异丙烯基转移酶，它们参与子囊菌次生代谢产物的生物合成，并催化色氨酸、酪氨酸、环二肽或其类似物的异戊烯基化。为此，我们计划克隆出22个可能的异戊烯基基转移酶，它们分别来自Neosartorya facheri，Aspergillusnidulans，A.terreus，A.oryzae和Chaetomium lobosum。过量生产的重组融合蛋白将与大约100个潜在的芳香族底物在二甲基烯丙基或香叶基二磷酸存在下孵育。根据两种吲哚戊烯基转移酶FgaPT2和FtmPT1的结构信息，还将利用定点突变技术来创造具有改变底物专一性、预烯基化模式和位置以及反应效率等新特征的酶衍生物。此外，还需要解决更多的戊烯基转移酶的结构，并将其作为定点突变的基础，以创造新的酶衍生物。除了离子依赖性和动力学参数外，所获得的戊烯基转移酶的生化表征应侧重于底物专一性及其作为生产戊烯基化吲哚和酪氨酸衍生物的工具的应用，这已在本课题组成功使用。