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

本发明的目的是获得新的DMATS超家族的异戊二烯基转移酶，其参与子囊菌门中次级代谢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二烯化。为此目的，计划从费氏奈氏酵母、构巢曲霉、A. terreus，刺毛菊A.和球毛壳（Chaetomium globosum）。将过量产生的重组融合蛋白与约100种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。基于两种吲哚异戊烯基转移酶FgaPT 2和FtmPT 1的结构信息，还将使用定点突变来产生具有新特征的酶衍生物，例如改变底物特异性、异戊烯基化模式和位置以及反应效率。此外，更多的异戊烯基转移酶的结构应该被解决，并作为基础的定点突变，创造新的酶衍生物，除了离子依赖性和动力学参数，所获得的异戊烯基转移酶的生化特性应集中在底物特异性和它们的应用作为工具，生产异戊烯基吲哚和酪氨酸衍生物，这是在我们的小组中成功地使用。

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.