Team:Frankfurt/Project/Description

Stevia - the object of desire!

The Stevia plant produces several sweeteners known as Steviolglycosides which have only recently been admitted as a foodadditive in the European Union. The iGEM-Team Frankfurt 2013 searches for ways to transfer the pathway of the plant into Saccharomyces cerevisiae in order to enable stevia production with both, lower effort and lower costs. Several of known problems with carbohydrate sweeteners like diabetes or caries could be overcome by the Steviolglycosides which are produced by Stevia rebaudiana. We're building up on results gained from last year's competition which gave us the possibility to transfer a mevalonate plasmid into yeast to increase the production of a steviol-precursor: geranylgeranyl-pyrophosphate (GGPP). This year we're trying to proof the increased production of geranylgeranyl-pyrophosphate in yeast via gas chromatography coupled with mass spectrometry. For this reason, the necessary enzymes (encoded on the mevalonate plasmid) are expressed in yeast and the produced geranylgeranyl-pyrophosphate is isolated and purified.

Besides, we are trying to optimize the purification of our first product (GGPP). Moreover, we want to assemble a second plasmid that contains three genes encoding for enzymes for the synthesis of steviol from geranylgeranyl-pyrophosphate. These enzymes are not originally found in yeast. A bifunctional cyclase is derived from Gibberella fujikuroi. The bifunctional cyclase catalyzes the cyclization of GGPP to ent-kaurene in two steps. The ent-kaurene oxidase (as well from Gibberella fujikuroi) catalyzes the reaction from ent-kaurene to ent-kaurenoic acid. Last but not least, a ent-kaurenoic acid hydroxylase for the catalyzation of our desired product steviol is needed. The hydroxylase is derived from Stevia rebaudiana. For the assembly of the genes two methods are pursued. First of all, we are trying to establish Gibson Assembly for the construction of our steviol plasmid. Therefore, we first have to work with Escherichia coli before we can transfer the prepared plasmid into yeast. Our second possibility is to work with gap repair directly in yeast. Moreover, biobricks of all genes should be constructed, so that the biobricks for steviol production can be used in other organisms or can also be used for other purpose. Finally, the aim is to co-transform the mevalonate plasmid and the steviol plasmid into yeast to enable the sustainable production of steviol in yeast. The introduction of several uracil glycosil transferases (UGTs) from Stevia rebaudiana should catalyze glycosylations for the production of rebaudiosides out of steviol. The most suitable UGT has to be found.