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

点击翻译按钮获取中文摘要

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.