It s gonna be sweet. Team Frankfurt
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1 It s gonna be sweet Team Frankfurt
2 Advantages of Stevia Stevia = composite of several steviol glycosides anticariogenic antidiabetic no impact on blood sugar no delay of satiety few calories ph stable, heat stable protective effect on vitamin C sweet diterpenes have certain advantages compared to both sugar and other sweeteners 2
3 Rebaudioside A Rebaudioside A = steviol glycoside of the stevia plant best taste highest sweetness (300 saccharose) least bitterness of all steviol glycosides extraction: stevia plants are dried and a composition (including rebaudioside A) of steviol glycosides are isolated 3
4 Saccharomyces cerevisiae a model organism Why do we use S.cerevisiae? two required enzymes are localized in the ER established food additive producer closer related to Stevia than E.coli is 4
5 Metabolic pathway 5
6 Metabolic pathway 6
7 Metabolic pathway 7
8 Metabolic pathway 8
9 Gap repair homologue recombination in yeast Homologous Sequences: At least 40 bp long Free ends will be recombined Similiar to double strand break repair 9
10 Gap repair example from our project Required Components linearized plasmid DNA fragments containing appropriate homology to the neighbouring fragments In vivo assembly in yeast 10
11 Yeast BioBrick Assembly (RFC proposal) Aim: Getting biobricks compatible to gap repair cloning construction of two standard primers for gene amplification Primers contain homologue overlaps to terminator/promoter-fragments Gap repair cloning is possible 11
12 Yeast BioBrick Assembly (RFC proposal) Aim: Variability of the terminator/promoter-fragment construction of standard primers that bind prefix of promoter and have a homologue overlap to the respective terminator possibility to combine every promoter every terminator 12
13 Results of the project Assembly of the mevalonate pathway overexpression plasmid and transformation in yeast Assembly of the steviol synthesis plasmid Creation of BioBricks of genes HMG-CoA-R: BBa_K FPPS: BBa_K Bifunctional Cyclase: BBa_K KO: BBa_K Geranylgeranyl pyrophosphate detection Yeast BioBrick Assembly (new BioBrick RFC proposal) Human practice & Ethics 13
14 Acknowledgements Team Frankfurt 2012 thanks : Martin, Tamara, Corinna, Virginia, Charlotte, Andre, Marco Sebastian, Christian, Leonard, Wagner, Jana (from left to right) Prof. E. Boles (instructor, lab support) Boles group (lab support) Prof. J. Soppa (lab space) Prof. K. Fendler (financial support) Prof. M. Schubert-Zsilavecs (university support) Prof. B. Tudzynski (provision of plasmids) Prof. H. Bode (analytical support) Prof. H. Schwalbe (recomendation) Christina Wagner (financial management) Prof. E. Bamberg (financial support) 14
15 References Pictures: Slide 1: CC-BY Ethel Aardvark: Slide 2: Slide 4: Key References: J.E. Brandle, P.G. Telmer, Steviol glycoside biosynthesis. Phytochemistry. 68, Tania V. Humphrey, Alex S. Richman, Rima Menassa, Jim E. Brandle, Spatial organisation of four enzymes from Stevia rebaudiana that are involved in steviol glycoside synthesis. Plant Molecular Biology. 61, Pia Dahm, Metabolic engineering der Taxolbiosynthese in Saccharomyces cerevisiae. K. A. Donald, R. Y. Hampton, I. B. Fritz, Effects of overproduction of the catalytic domain of 3-hydroxy- 3methylglutaryl coenzyme A reductase on squalen synthesis in Saccharomyces cerevisiae. Applied and Environmental Microbiology. 63 (9), Kenro Tokuhiro, Masayoshi Muramatsu, Chikara Ohto, Toshiya Kawaguchi, Shusei Obata, Nobuhiko Muramoto, Masana Hirai, Haruo Takahashi, Akihiko Kondo, Eiji Sakuradani, Sakayu Shimizu, Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae. Applied and Evironmental Microbiology. 75 (17), DOI: /AEM Juan Rico, Ester Pardo, Margarita Orejas, Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-Methylglutaryl Coenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae. Applied and Environmental Biology. 76 (19), Udo Kienle, Stevia rebaudiana Natürliche Süße im Behördendschungel. Journal Culinaire No. 5. Shao-bing Hua, Mengsheng Qiu, Eva Chan, Li Zhu, Ying Luo, Minimum Length of Sequence Homology Required for in Vivo Cloning by Homologous Recombination in Yeast. Plasmid. 38 (2), Tomonobu Toyomasu, Hiroshi Kawaide, Atsuko Ishizaki, Shoko Shinoda, Minoru Otsuka, Wataru Mitsuhashi, Takeshi Sassa, Cloning of a Full-length cdna Encoding ent-kaurene Synthase from Gibberella fujikuroi: Functional Analysis of a Bifunctional Diterpene Cyclase. Bioscience, Biotechnology and Biochemistry. 64 (3),
Products 1 and 3 are commercially available, products 2 and 4 are experimental and not commercially available.
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