Advantages of Stevia Stevia = composite of several steviol - PowerPoint PPT Presentation

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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

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

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

Metabolic pathway 5

Metabolic pathway 6

Metabolic pathway 7

Metabolic pathway 8

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

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

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

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

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_K849000 • • FPPS: BBa_K849001 • Bifunctional Cyclase: BBa_K849003 • KO: BBa_K849004 Geranylgeranyl pyrophosphate detection Yeast BioBrick Assembly (new BioBrick RFC proposal) Human practice & Ethics 13

Acknowledgements Team Frankfurt 2012 thanks : 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) Martin, Tamara, Corinna, Virginia, Charlotte, Andre, Marco Christina Wagner (financial management) Sebastian, Christian, Leonard, Wagner, Jana (from left to right) Prof. E. Bamberg (financial support) 14

References Pictures: Slide 1: CC-BY Ethel Aardvark: http://en.wikipedia.org/wiki/File:Stevia_rebaudiana_foliage.jpg. 04.10.2012 Slide 2: http://www.sugarbeater.com/joomla/images/stories/white%20powder%20stevia.jpg. 04.10.2012 Slide 4: http://130.91.8.212/images/S_cerevisiae.jpg. 04.10.2012 http://de.wikipedia.org/wiki/Datei:Nucleus_ER.svg. 04.10.2012 Key References: J.E. Brandle, P.G. Telmer, 2007. Steviol glycoside biosynthesis. Phytochemistry . 68, 1855 – 1863. • • Tania V. Humphrey, Alex S. Richman, Rima Menassa, Jim E. Brandle, 2011. Spatial organisation of four enzymes from Stevia rebaudiana that are involved in steviol glycoside synthesis. Plant Molecular Biology . 61, 47 – 62. Pia Dahm, 2011. Metabolic engineering der Taxolbiosynthese in Saccharomyces cerevisiae. • • K. A. Donald, R. Y. Hampton, I. B. Fritz, 1997. 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), 3341. • Kenro Tokuhiro, Masayoshi Muramatsu, Chikara Ohto, Toshiya Kawaguchi, Shusei Obata, Nobuhiko Muramoto, Masana Hirai, Haruo Takahashi, Akihiko Kondo, Eiji Sakuradani, Sakayu Shimizu, 2009. Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae . Applied and Evironmental Microbiology. 75 (17), 5536. DOI: 10.1128/AEM.00277-09. Juan Rico, Ester Pardo, Margarita Orejas, 2010. 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), 6449. • Udo Kienle, 2007. Stevia rebaudiana – Natürliche Süße im Behördendschungel. Journal Culinaire No. 5. • Shao-bing Hua, Mengsheng Qiu, Eva Chan, Li Zhu, Ying Luo, 1997. Minimum Length of Sequence Homology Required for in Vivo Cloning by Homologous Recombination in Yeast. Plasmid. 38 (2), 91-96. • Tomonobu Toyomasu, Hiroshi Kawaide, Atsuko Ishizaki, Shoko Shinoda, Minoru Otsuka, Wataru Mitsuhashi, Takeshi Sassa, 2000. 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), 660- 664. 15