Untersuchungen zum Biosyntheseweg von Flavonolignanen in der Mariendistel Silybum marianum

Due to its active natural compounds, milk thistle (Silybum marianum) is particularly important for medicinal therapy (Valková 2020). Extracts of this plant are commonly used for the treatment of liver and gall bladder disorders for over 2000 years (Delmas et al. 2020). In addition, the main active ingredient silymarin shows natural anti-inflammatory activity (Corchete 2008), as well as potential inhibitory activities on various cancers (Won et al. 2018). Silymarin is a flavanonollignan mixture of several regioisomers and diastereomers. They are formed by oxidative coupling of a flavonoid (taxifolin) and a phenylpropanoid (coniferyl alcohol) moiety. The reaction putatively is a radical coupling (Bernards 2010), which requires radical formation first, possibly catalyzed by a laccase or a peroxidase (POD) (Poppe and Petersen 2016, Bijak 2017). Thereafter, dirigent proteins (DIR) presumably mediate the regiospecific and stereospecific coupling that leads to the formation of the individual regioisomers and diastereomers (Pickel and Schaller 2013). Biosynthesis of both precursor units takes place with the help of several enzymes of phenolic metabolism. Phenylalanine ammonia-lyase (PAL) is the first key enzyme in the biosynthesis of phenylpropanes, catalyzing a non-oxidative deamination of phenylalanine to trans-cinnamic acid. Chalcone synthase (CHS) is another important enzyme in the flavonoid biosynthetic pathway, synthesizing naringenin chalcone. Taxifolin is formed via further reaction steps. Cinnamoyl-CoA reductase (CCR) is the first enzyme that directs phenylpropanoid metabolites into the monolignol pathway. It catalyzes the conversion of 4 coumaroyl-, feruloyl and sinapoyl-CoA to 4-coumaraldehyde, coniferaldehyde, and sinapaldehyde with the help of NADPH. After that, coniferylalcohol dehydrogenase (CAD) forms coniferylalcohol using NADPH. These enzymes as well as the coupling mechanism itself have not been studied in milk thistle (Silybum marianum) yet. In this work, it was further more aimed at expressing the secretory class III peroxidase as well as two dirigent proteins from milk thistle. Due to the presence of signal peptides and several glycosylation sites, no successful expression of the genes could be recorded despite of several attempts of method optimization. However, PAL, CHS, CCR and CAD were successfully expressed heterologously in Escherichia coli. Milk thistle contains two PAL isoforms with a molecular mass between 75 and 100 kDa. Protein characterization revealed enzymatic affinities for L- and D-phenylalanine and L-tyrosine. Both PALs show a temperature optimum at 60 °C and a pH optimum at 8.5 and 9.5. Km-values for L-phenylalanine could be determined at 12 and 35.2 µM. D-phenylalanine had a Km at 146 µM and 236 µM and L-tyrosine at 2.4 and 2.6 mM. CHS possesses a protein mass between 63 and 75 kDa. The enzyme shows a pH-optimum at pH 7.7. The temperature optimum could be determined to be 40 °C. 4-Coumaroyl-CoA had a Km-value of 1.48 µM and malonyl-CoA a Km of 12 µM. Additionally, a CAD was characterized in this thesis. A protein mass of approximately 40 kDa could be determined via gel electrophoresis. Protein biochemical characterizations revealed an optimal enzyme reaction at a pH of 9.1 as well as a temperature optimum at 55 °C. Km-values were pointed out to be at 25 µM for coniferyl alcohol and 3.2 µM for the cofactor NADP. The last characterized enzyme was CCR. This monomeric enzyme has a molecular weight between 35 kDa and 48 kDa and shows substrate affinity to feruloyl-CoA, followed by sinapoyl-CoA, 4-coumaroyl-CoA and caffeoyl-CoA. The pH-optimum was found out to be at pH 6.5 and the temperature optimum ranged from 29 °C to 34 °C. The Km-value is 15 µM for feruloyl-CoA, 21.3 µM for sinapoyl-CoA, 23 µM for NADPH and 68 µM for NADH. In summary, several key enzymes from phenol metabolism were identified and expressed heterologously in this work. In addition, relevant parameters have been obtained, providing important insights and knowledge into the in vitro nature of selected key enzymes. These results provide a solid basis for further studies of the plant phenylpropanoid biosynthetic pathway in Silybum marianum.