Template:Team:TU Munich/Overview

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.

Ethanol Inducible Promoter

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

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

When all enzymes are expressed under control of promoters with the same strength, the enzyme with the lowest kinetic rates will cause a bottleneck in the pathway. Hence a multitude of promoters of different strengths is needed.

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)

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.(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.

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.