Cloning and characterization of cytochrome P450 oxidoreductase type from Maytenus ilicifolia and their effect on increasing the production of quinonamethide triterpenes by heterologous expression in Saccharomyces ceravisiae
Among the secondary metabolites produced by higher plants, the terpenoids attract special attention based on its important biological potential. The quinonemethide triterpenes are considered a special class of secondary metabolites mainly related to biological, structural and biosynthetic point of views. Chemical studies of Celastraceae species demonstrated that such class of metabolites shows accumulation in the root bark of seedelings in low concentration. The quinonemethides are considered chemical markers of Celastraceae species and show a wide range of biological activities, including antitumor, anti-inflammatory, antioxidant and antimalarial. Biosynthetic studies performed by our research group have demonstrated the role of friedelin as a quinonemethide triterpene's precursor, the compartimentalization of the pathways involved and the identification and characterization of cyclases (friedelin synthase) and oxidoreductases (citocrome P450 (CPRs)). These are the key enzymes on the oxidosqualene cyclization steps to friedelin and oxidation of friedelin to quinonemethide triterpenes. These results supported later studies of metabolic engeneering aiming to increase the friedelin production. To achieve that, the friedelin synthase genes (cyclase) extracted from Maytenus ilicifolia species were cloned, identified and expressed in a heterologous system, Saccharomyces cerevisiae. After complementary site-directed mutations studies, the production of friedelin in S. cerevisiae was a succes. In order to improve the metabolic engineering studies to increase the quinonemethide triterpenes production, this project aims to clone and characterize genes of oxidoreductase enzymes from the cytochrome P450 type (CPR) followed by its expression in a genetically modified heterologous system (S. cerevisiae).
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