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Contents 1 Organisms 1.1 Organisms used in our LabWork 1.1.1 Escherichia coli 1.1.2 Saccharomyces cerevisiae 1.2 Organisms from which genes were derived 1.2.1 Sulfolobus acidocaldarius 1.2.2 Gibberella fujikuroi

Organisms

Organisms used in our LabWork

Escherichia coli

E.coli is a well known model organism for molecular biology. It is a gram negative bacteria whose generation time is about 30 minutes. We used E.coli to amplify the plasmids that we isolated from yeast after transformation via homologue recombination.

Saccharomyces cerevisiae

S.cerevisiae is our main organism. We want to produce steviol in yeast and we did the transformations in this organism. In addition we are able to do the transformations via gap repair which is possible in yeast because of its ability for homologue recombination with only about 40 bp. Furthermore S.cerevisiae has got an ER which is needed because some enzymes for steviol production are localized in the ER, S.cerevisiae is an established food additive producer and is closer related to Stevia than E.coli for example.

Organisms from which genes were derived

Sulfolobus acidocaldarius

Sulfolobus acidocaldarius(Brock et al.) is a hyperthermophile (temperature optimum above 80°C) and acidophile archea. It feeds upon elemetary sulfur, hydrogen sulfid or Iron(II) Oxidation and can be found in volcanic springs.

We used the GGPP synthase from this organism.

Gibberella fujikuroi

Gibberella fujikuroi (Sawada) Wollenw., (Syn.: Fusarium spec., Oospora spec.) is filamentous fungus from the same phyllum (Ascomycota) as Saccharomyces cerevisiae. It is a rice pathogene and the causator of the bakanae disease which leads to infertile plants which are not able to produce an edible seed. Gibberella fujikuroi does so by producing compounds from the class of gibberellines. These are common phytohormons. The surplus of gibberellines causes extraodinary growth of the plants. The additional growth is due to hypertrophy which means that the cells increase in size (not in number). The increased cell size makes the tissue prone for mechanical strains and the plants often collapse when they reach a certain hight.

Gibberellines are produced by the same precurser as steviol: ent-kaurenoic acid. While the steviol synthase introduces a hydroxy function at the position 13 the gibberline specific pathway is entered by substitution with a hydroxy function at the position 16.

Gibberelline metabolism is not a common feature of fungal organisms. But for Gibberella only few funguses are able to synthesize them. Most of them are phytophathogenes and it is likly that the ability of producing gibberellines is a covergent evolution and a fitment on the phyto pathogenity.

The synthesis of ent-kaurene is conducted by an enzyme which possesses both the capability of the cyclation of geranylgeranyl pyrophosphate to ent-copalyl pyrophosphate and the cyclation of the previous compount to ent-kaurene. Because of this difference and the closer relation of Giberella to Saccharomyces we plan to use this enzyme in our synthetic pathway instead of using a plant enzyme. We are also planning to use the ent-kaurene oxidase because of the closer relation of the organisms.