Team:TU Munich/Project/Overview

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Contents

Overview

Motivation

Yeast Basics

Vector Design

To be able to test and quantify the expression of desired enzymes in yeast we designed an expression vector which is compatible to the iGEM RFC25 standard based on the commercially available pYES2 vector from Invitrogen.

Genome Integration

Promoter

By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make S. cerevisiae dynamically respond to concentration changes in its medium as well as to external stimuli. An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively.

Ethanol Inducible Promoter

The KlADH4-Promoter from the yeast Kluyveromyces lactis regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way.

The upstream activating sequence of the promoter contains the consensus sequence of the of the RAP1 (repressor activator protein 1) binding site as well as 5 stress response elements. Those 6 site are essential to the ethanol inducibility of the promoter and their respective transcription factors are present in K. lactis as well as S. cerevisiae.(4,6)

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

Ingredients

Xanthohumol

Xanthohumol is known as a putative cancer chemopreventive agent, due to its antioxidant activities (Miranda et al., 2000).(2) 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.

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.

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.(1)

Caffeine

Caffeine is a purine-alkaloid and its biosynthesis is known from coffee plants and tea plants.(3) It can block specific receptors in the hypothalamus in a competitive manner, which leads to decreased neurotransmitter-release and therefore decreased neuron activity.

The biosynthetic pathway of caffeine starts with xanthosine, which is a natural component of the purine-metabolism of all organism and involves a total of 3 enzymes.


References

  • [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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