It‘s gonna be sweet

Team Frankfurt

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

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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

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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

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Metabolic pathway

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Metabolic pathway

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Metabolic pathway

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Metabolic pathway

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Gap repair – homologue recombination in yeast

Homologous Sequences:

At least 40 bp long

Free ends will be

recombined

Similiar to double strand

break repair

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Gap repair – example from our project

Required Components

linearized plasmid

DNA fragments containing

appropriate homology to the

neighbouring fragments

In vivo assembly in yeast

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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

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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

Aim:

Variability of the terminator/promoter-fragment

Yeast BioBrick Assembly (RFC proposal)

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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

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Acknowledgements

Martin, Tamara, Corinna, Virginia, Charlotte, Andre, Marco Sebastian, Christian, Leonard, Wagner, Jana (from left to right)

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)

Christina Wagner (financial management)

Prof. E. Bamberg (financial support)

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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.

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