Gewinnung und Modifikation von Flavonoiden in Mikroorganismen

Flavonoids are an important class of secondary metabolites that is becoming increasingly significant for the food and drug industry. The major objective of this work was to optimize several systems for biotechnological flavonoid production. One of the goals was to establish a method for the generation of flavonoid glucuronides. The human UDP-glucuronic acid transferase UGT1A1 is known to accept various flavonoids (Tukey and Strassburg 2000) and seemed therefore a suitable candidate for biotechnological applications. In an in vitro assay, (commercial) UGT1A1 has been used to catalyze the glucuronidation of kaempferol, apigenin, genistein and daidzein. In addition, UGT1A1 produced glucuronides with a pseudosubstrate octylgallate and the chalcone xanthohumol. The attempted heterologous expression of UGT1A1 in Saccharomyces cerevisiae and Nicotiana benthamiana didn’t result in detectable glucuronidation activity. Successful expression was achieved after choosing Pichia pastoris as a host. The UGT1A1 activity was measurable but insufficient to support large-scale synthesis. Optimization of expression procedure is necessary to increase the efficiency of the system. Isoflavones are derived from flavanones in a two-step reaction (Steele et al. 1999, Akashi et al. 2005). Co-expression of the two involved enzymes, IFS1 and HIDH, in one baker’s yeast strain, isolation of the proteins followed by an in vitro reaction afforded radiolabelled genistein from (2S)- [4a,6,8- 14C]naringenin as starting point. In biotransformation experiments with unlabelled naringenin, acidification of the yeast cell broth prior to product extraction was necessary to retrieve genistein. A number of radiolabelled flavonoids were enzymatically synthesized using [14C]-2-Malonyl-CoA and pC-CoA as starter molecules. Naringenin, eriodictyol, kaempferol, dihydroquercetin, and dihydrokaempferol were produced in high yield and purity. Optimization of the biotransformation methods was conducted utilizing the S. cerevisiae strain harbouring a construct for the expression of parsley FNS I that was demonstrated to be active previously (Martens et al. 2001). To explore the catalytic capacities of this 2-ODD, its substrate specificity was determined with a variety of flavonoids. Hesperetin, eriodictyol, homoeriodictyol, pinocembrin and liquiritigenin were accepted as substrates in addition to naringenin. Variation of biotransformation procedure enhanced the turnover efficiency. Besides, using acetone as solvent for the substrate (NAR) and increasing the cultivation temperature to 37 °C were beneficial for the overall yields. Maximal relative turnover of 85 % was measured after longer incubation at 30 °C, with naringenin supplied in DMSO as solvent. Purification using HSCCC afforded 98% pure apigenin. Summarizing, the method established here allows for a relatively simple and efficient production of flavones. The findings and conclusions of the optimization process are applicable to further relevant enzymes, such as P450-monooxygenases. Hydrolysis of glucose-conjugated flavonoids (NAR-7-O-gluc) by transformed and wild-type yeast was observed in the course of biotransformation studies. It implied baker’s yeast possesses endogenous glucosidases capable of cleaving the sugar off at least some flavonoid glucosides, thus limiting the range of potential biotransformation substrates, e.g. rendering the production or processing of glucosides impossible. In order to identify the responsible enzyme(s) and if possible, knock them out, ten putative O-glucosidases (BGL2, EXG1, SPR1, YIR007W, SUC2,YGR287C, YJL216C, YIL172C, DSE 2 und CWH41), were selected, expressed in yeast and their catalytic properties analyzed. While three of the studied enzymes were α-glucosidases (YGR287C, YIL172C and YJL216C) and three other proteins (EXG1, SPR1 and YIR007W) demonstrated β-glucosidase activity, preferring 7-O- and 4’-O glucosides of flavanones, flavones, flavonols and isoflavones. Similar regiospecificity has been reported for the human cytosolic β-glucosidase (hCBG, Berrin et al. 2002, 2003). Residues whose alteration negatively affected hCBG’s affinity for flavonoid glucosides could be tentatively identified in glucosidases studied here. Subsequent site-directed mutagenesis of corresponding moieties revealed deleterious effect on their activity with flavonoid glucosides. Subsequent tests with yeast strains deficient in respective glucosidase genes disclosed one of the mutants, EXG1 (YO5210), was unable to catabolise the fed substrate NAR-7-glc. After being transformed with expression constructs for FNSI and FHT, the YO5210 strain was again found to be incapable of cleaving the naringenin glucoside in a biotransformation experiment. Hence, a yeast system suitable for the future production and manipulation of flavonoid glucosides has been identified.