Presentation of Research Results in our internal Wiki

General Remarks

1. Prof. Skerra recommended to stick to a consistent structure when presenting research results.

• Short summary of what you are about to say (e.g. "AlcR is a simple two component system from aspergilus nidulans which can be induced with ethanol")
• Give detailed information, such as: nucleotide and/or amino acid sequence, Link to PDB-entry, ...
• Last but not least, Your personal view & comments

Please make sure to update your entries to match this structure

2. There are some general arguments in favor of our project idea. One of them is, that the application of genetically modified yeast should be legal, because beer is filtered anyway, so no GMOs would be released from the closed system.

Promoters

Ethanol-inducible Promoters

1. KlADH4-Promoter

The information given in our wiki was presented. The main question discussed was whether this system works specifically (similar to the lac-operon) or unspecifically (similar to a general stress response). If the system works specifically, it is unlikely that the KlADH4 promoter is going to work in S. cerevisiae, because in this case, an unknown specific transcription factor is most likely involved which is not present in s. cerevisiae. If the system works unspecific, we thought that only general stress factors and transcription factors are involved. In this case, chances are that KlADH4 can work in s. cerevisiae, because the necessary factors are most likely present. However, we don't know for sure which mechanism is the correct one (specific or unspecific). Therefore further literature research is necessary for this system.

1. AlcR-Promoter

The information given in our wiki was presented. The system might be a good candidate, because it seems to be well characterized. Because we do not know many details about this system, further literature research is necessary for this system.

1. Methanol-Inducible Promoter from Pichia pastoris, and methanol inducible systems from prokarya, such as Methylococcus capsulatis (BATH)

During the discussion about ethanol-inducible systems, two additional suggestions were made. The yeast pichia pastoris is known for its ability to express recombinant proteins upon methanol induction. Maybe this system can be adapted to respond to ethanol - apparently it is based on a specific methanol binding transcription factor. Further literature research is necessary. Also, there is a variety of bacteria which can grow with alcohols (e.g. methanol) as carbon source (Methylococcus capsulatis (BATH)). Maybe they can also provide a system for S. cerevisiae. Further literature research is necessary.

Inducible Yeast Promoters

1. Chemical Induction: Last year, the iGEM team [British Columbia 2011] created the BioBrick BBa_K517000. This is a Galactose-inducible yeast-promoter which worked for them.
2. There is a light-switchable promoter system for yeast which has been shown to work for example in a PhD-thesis. See Project Ideas for details. The fact that short light pulses instead of continuous radiation is sufficient to induce expression is an advantage, because light can destroy flavors.

Biosynthesis of small molecules

Resveratrol

There are two approaches for the synthesis of resveratrol, but both require the intermediate Coumaryl-CoA. The 2008 iGEM-Team from [iGEM Rice 2008] tried to submit the resveratrol synthesis pathway in yeast to the registry, but their BioBricks are not functional (for example, they still include forbidden restriction sites). However, the basic functionality of their construct has been shown to work by a different research lab. It would be a good idea to use the DNA they submitted to the registry and make it functional (e.g. removing the forbidden restriction sites...), because this is a great way to improve an already existing BioBrick, which is one of the things iGEM judges really like to see. Volker has already contacted them and got a friendly response.

Xanthohumol

Xanthohumol has never been produced in yeast. However, attempts have been made to alter the hops plant to increase its production of xanthohumol, which shows that beer with increased concentration of xanthohumol is a great idea. Two of the required genes have been well characterized, the other two haven't. The biosynthesis of xanthohumol also includes the intermediate Coumaryl-CoA.

Raspberry Ketone

The synthesis of Raspberry Ketone also includes Coumaryl-CoA.

Caffeine

Caffeine has never been produced in yeast. The required substrate for the synthesis occurs naturally in yeast, because it is a part of the nucleic acid metabolism. Two genes are required for synthesis of caffeine. Caffeine has been shown to inhibit growth of bacteria and yeasts at concentrations similar to that in coffee.

Aspirin

No enzymes are known which produce aspirin.

Nicotine

We decided against the production of nicotine for moral reasons. Our beer should be a healthy one.

Strawberry flavor

Strawberry flavor is not one single substance but a mixture of several hundreds. However, peach-flavor has been produced in yeast already. For interview teams: Ask Prof. Schwab about Furaneol (Dimethylfuraneol)!

Colors and pigments

In addition to the information given in our wiki, please look at [iGem 2011 Uppsala].

Beneficial Peptides and Proteins

The general question concerning Peptides and Proteins is how to ensure their secretion in yeast.

Knottins

Knottins do not fit our requirements well.

Thaumatin

The protein thaumatin is a natural sweetener. Its production should be easy, because only one gene is required.

Lactoglobulin

the digestion of ß-Lactoglobulin exhibits a broad variety of physiological active peptides. How many genes are required?

Discussion & Next steps

Decision on structure of project

We decided to focus on the following modules in our project:

1. Promoters: Design a library containing three types of promoters: ethanol inducible (for EtOH-Sensor), light switchable and chemically inducible (for control of biosynthesis).
2. Biosynthesis: The main focus will be on products derived from Coumaryl-CoA, because it is an intermediate for many interesting molecules. In addition to this, we also want to try to establish caffeine-synthesis, because only two genes are required.
3. Proteins: The secretion of proteins is a good idea, because it will give us a third field of research. This module is promising, because it is likely to lead to easy successes (only one gene required for the production of thaumatin...)

Next Steps

Everybody should assign themselves to one of the following topics:

I) Detailed literature research

For one desired product/construct, find out exactly...

• How many genes are required?
• Which genes (including nucleotide sequence)?
• How do we get the physical DNA (ask a research lab to send it to us, extract from organisms via PCR, synthesis...)?

Please post your results in the wiki

II) "Taskforce Vector Design"

This group should focus on how to transform yeast. Which vectors are available, what techniques are necessary,...? For example, look at the 2011 iGEM Teams John Hopkins and Britsh Columbia. They worked with yeast and got decent results.

Interviews

Interviews with experts will take place on Friday, March 9th. If you are interested in helping, please contact Lara Kuntz.

Miscellaneous

Does anybody know a student from "Brauereiwesen"? It might be good to have one on the team!

Overview

The main topics of the meeting were the interviews and the project structure. We agreed that everybody should research his/her topic until the next meeting in two weeks in order to get into the lab as soon as possible (beginning of April). Additionally, we should meet during a weekend (e.g. 15th of April) to work together for a whole day and to address issues such as fundraising and human practice.

We agreed on the following project structure:

Project structure

Promoter and regulation systems

1. Ethanol-inducible

2. Light-switchable

3. Chemically inducible

4. Constitutive active

Biosynthesis systems

1. Coumaryl-pathway

2. Limonene

3. Caffeine

4. Thaumatin

Interviews

1. Dr. Zankow, Beverage Oriented Biotechnology

Security issues

• In general, brewing beer with genetically modified yeast should not be a problem as far as security is concerned. If we manage to brew a beer with our yeast it should be safe for us to drink it because the filtration process is very efficient. However, we should not let other people who are not part of the team drink the beer.
• Dr. Zankow remarked that the filtration process might filter out resveratrol and that we should check how much resveratrol is still in the beer after filtration and whether it is functioning.

Alternative Project ideas suggested by Dr. Zankow:

• Removal of mycotoxins that are found in the grain
• Beer for a special group of patients (e.g. Celiac disease, Gout)
• nonalcoholic beer: since nonalcoholic beer does not taste as “satisfying” as normal beer, breweries are searching for something that adds taste to nonalcoholic beer. Dr. Zankow suggested Lactic acid

We are allowed to use the equipment of the chair to brew the beer.

2. Prof. Schwab, Biotechnology of Natural Products

He also made several suggestions for possible syntheses

• Eugenol (clove-like aroma)
• Limonene
• According to him, strawberry aroma is very difficult to make

Prof. Schwab can give use information on several pathways, e.g. for limonene

3. Prof. Hoffmann

• He was quite critical about resveratrol since only 1 % of the substance is actually functional in the body. Therefore a large amount of resveratrol is necessary to produce a positive effect.

He has mass spectrometers that we are allowed to use, protocols for analyzing beer are available and we can do as many analyses as we want to. Only 5 µl are needed for one analysis.

More detailed information on the interviews are to be posted soon by the interviewers.

Fundraising

• Lara talked to Prof. Herrmann and he is willing to give us money. We should send a specification of costs to his secretary.
• Another possible source could be the excellence cluster.

Human Practice

We should use the contacts from the team from last year (e.g. Spiegel). We briefly talked about other institutions that could help us to get some publicity:

• Carl von Linde Akademie
• Galileo (as Prosieben is based in Munich)
• Deutschlandfunk (contact person: Andreas Lang)
• Quarks&Co (contact person: Ranga Yogeshwar)

TO DO

• research the topic you have chosen until the next meeting in 2 weeks! (enzymes, sequences, sources etc.)