Team:TU Munich

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Revision as of 15:49, 26 September 2012


TUM-Brew: iGEM's first and finest SynBio Beer


TUM12 AE120612001.jpg

The TU Munich iGEM Team engineers Saccharomyces cerevisiae, also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.

As an example, for iGEM’s first “SynBio Beer” the compounds xanthohumol (anticancerogenic), limonene (limeflavor), caffeine (CNS-stimulant) as well as the thaumatin (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.

The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector pTUM100, which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.

Constitutive, ethanol-inducible and light-switchable promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest SynBio Beer.

Vision


We, TU Munich’s iGEM team 2012, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.

Overview


  • 878 Documented experiments in our labjournal
  • 923 Plasmids
  • 49 Annotated biobricks
  • 107 Days in the lab
  • 283 Sequencing orders
  • 14 Subprojects
  • 19 Students
  • Over 10.000.000.000 systems of differential equations solved

Outside the Lab


Vector Design


What is the use of DNA sequences coding for valuable enzymes without the possibility to express them and analyze the protein activity?

As we planned to detect the enzymes from our biosynthetic pathways via Strep-tag II, a RFC25 compatible backbone was necessary. As no such backbone was availabe for yeast in the PartsRegistry, an important task at the beginning of our project was the design of an expression vector for yeast which is compatible to the iGEM cloning principles and standards.

Ethanol Inducible Promoter


The KlADH4-promoter from the yeast Kluyveromyces lactis regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in K. lactis also occur in S. cerevisiae [Mazzoni et al., 2000]. This is why we are confident that this promoter maintains its unique characteristics when transformed into S. cerevisiae.

Light Switchable Promoter


The idea behind a lightswitchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelengths.

This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction cheap, fast, precise and also compatible to the bavarian purity law.

Constitutive Promoter


To control expression of the enzymes and proteins from our biosynthetic pathways a variety of promoters is essential. Since gyle from beer is a complex medium, the controlled induction of protein expression proves to be a difficult task.

Therefore constitutive promoters are the ideal solution for that problem. They offer simplicity, and therefore are the first choice for the introduction of new biosynthetic pathways in yeast in complex media.

In order to finetune biosynthetic pathways with two or more enzymes, as in our with caffeine pathway, we want to use 3 constitutive promoters ADH1, TEF1 and TEF2 to account for the turnover rates of the different enzymes.

Thaumatin


Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (Thaumatococcus daniellii). It is said to be 2.000 to 100.000 times sweeter than sucrose on molar basis, but the sweetness builds slow and lasts long. It has been approved as a sweetener by the European Union (E957).

Our aim is to have S. cerevisiae secrete functional Thaumatin by expressing Preprothaumatin – a principle which has been proven by Edens et al. in 1984.

Integration


As we can't obey the letter of the German Purity Law (there is a zero tolerance policy concerning transgenic ingredients), we try our best to meet the spirit. Thus, it is unacceptable for us to work with antibiotics to keep up the selective environment. Since we can't work with auxotrophies in beer either, we have to make sure the yeasts don't get rid of the biobricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve genome integration.

Limonene


Limonene is a cyclic terpene and a major constituent of several citrus oils. D-Limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.

We will produce the flavoring substance limonene by expressing limonene synthase in S. cerevisiae, which naturally synthesizes the educt geranyl pyrophosphate.

Caffeine


Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants. The molecule acts as a competitive antagonist on adenosine receptors and therefore increases indirectly neurotransmitter concentrations resulting in warding of drowsiness and restoring of alertness.

The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''.

Xanthohumol


Xanthohumol is known as a putative cancer chemopreventive agent, due to its antioxidant activities. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in S. cerevisiae.

The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of phenylalanine and followed by four further enzymatic reactions.

Human Practice


Our project envisions genetically modified organisms in the scope of food production. In Germany genetic engineering is a highly sensitive topic. Keeping in mind that Germany has a long tradition of brewing beer it is even more important to inform the general public about synthetic biology and our project.

RFC


iGEM’s core idea is standardization: genetic elements are modified to easily combine them. A central element of this idea is the Parts Registry.

Yet over time our team has come to the conclusion that iGEM’s core idea, standardization, is not fully implemented in the Parts Registry. Accessing the Registry frequently to plan a project using BioBricks, one very quickly realizes that the part descriptions are often unstructured, hence we came up with a new RFC for a standardized annotation of parts.

Modeling


To be able to predict the behavior of a given biological system, one has to create a mathematical model of the system. The model is usually generated according to the Law of Mass Action and then simplified by assuming certain reactions to be fast. This model then could e.g. facilitate optimizations of bio-synthetic pathways by regulating the relative expression levels of the involved enzymes.

The mathematical framework of Bayesian Inference that we introduced in iGEM, perfectly fits into the setting of BioBricks: Priors are small parts that are added to your simulation, and either you improve them by performing inference yourself and adding the resulting marginal distribution of the parameter as new, improved prior.

Beer Brewing


Contrary to popular opinion the chief ingredient of beer is not YPD but gyle, a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, S. cerevisiae, suggests that it is used in the beer brewing process, the yeast strains generally employed in brewing process have strongly adapted to gyle, as they are reutilized in every succeeding brewing cycle. Hence some investigation on how our yeast performs in gyle was necessary.

Reference


  • [1] Synthesis and processing of the plant protein thaumatin in yeast, Luppo Edens, Isaäc Bom, Adrianus M. Ledeboer, Jan Maat, Marjolein Y. Toonen, Chris Visser, C. Theo Verrips. Cell Volume 37, Issue 2, June 1984, Pages 629–633
  • [2] C.L Miranda, J.F Stevens, V Ivanov, M McCall, B Frei, M.L Deinzer, D.R Buhler, Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro, J. Agric. Food Chem., 48 (2000), pp. 3876–3884
  • [3] Insertion of Coffein, Römpp Online. Version 3.19
  • [4] Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin in Kluyveromyces lactis. Michele Saliola, Cristina Mazzoni, Nicola Solimando, Alessandra Crisà, Claudio Falcone, Gerard Jung, and Reinhard Fleer. Appl Environ Microbiol. 1999 January; 65(1): 53–60.
  • [5] Development of a genetic switch based on a photosensitive protein signals the beginning of light-based biological engineering. Andrew R. Mendelsohn Nature Biotechnology, Vol. 20, October 2002, Pages 985-987
  • [6] Molecular analysis of UAS, a cis element cotaining stress response elements responsible for ethanol induction in the KlADH4 of Kluyveromyces lactis. Cristina Mazzoni, Francesca Santori, Michele Saliola and Claudio Falcone Research in Microbiology, Vol. 151, 2000, Pages 19-28


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