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Team:Valencia Biocampus/Yeast
From 2012.igem.org
Yeast Subteam
THE IDEA
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.
To do this, we are going to use two gene constructions:
• The ADH2 promoter is fused to the YAP1 protein coding sequence. The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptative response. It is stored in the citoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus acting as a transcription factor. The ADH2 promoter is activated in the absence of glucose.
Thus, complete disappearence of glucose triggers the production of YAP1 in the citoplasm, and YAP1 concentration increases if the lack of glucose continues. Note that we are working with a delta-yap1 mutant.
• The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence. The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors (YAP1 and SKN7 in the oxidative form). Both only bind to the promoter if H2O2 has been previously added to the culture medium.
INDUSTRIAL APPLICATIONS
The main industrial application of our talking yeast is to know the moment at which they started fermentation. This is possible because we can calculate the time passed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: “How long have you been fermenting?” And the cells will answer telling us how long it has been since all the glucose was consumed. One possible answer could be “I have been fermenting for 1 hour”.
There is also a second possible application, but it needs a more detailed explanation:
There are several factors affecting yeast ethanol production during
alcoholic fermentation. Saccharomyces cerevisiae seems to have adapted
all along its evolution to optimize its growing rate in environments
that are rich in easily assimilable nutrients, such as sugars and amino
acids.
There are two important characteristics responsible of S. cerevisiae
adaptation to this particular niche. One of them is its capability of
metabolizing glucose and fructose through both the respiration and the
fermentation pathways. The second one is its ability to grow in aerobic
and anaerobic conditions. All of this makes this species exhibit some
metabolic peculiarities, such as the Pasteur and the Crabtree effects.
The Crabtree effect, described in S. cerevisiae and in a few other yeast species, must be taken into account when ethanol production
is being studied or modified during fermentation.
This effect causes that, even under high concentration of
oxygen and with a relatively low amount of glucose, a considerable
fraction of the consumed sugar is used to produce ethanol through the
fermentative pathway. That is why in every industrial process which
requires fermentation it is necessary to provide the optimum amount of
glucose that permits its direct consumption through the fermentative
pathway. We present here one way of monitoring the fate of glucose in the
medium. By adding a small amount of
hydrogen peroxide as a chemical input, we plan to ask our culture: “Is there any glucose left?”, and the culture would answer -according to the amount of
glucose already present in the medium- what concentration would be
necessary to obtain a higher amount of ethanol through the Crabtree
effect.
=== '''MOLECULAR MECHANISMS''' ===
Click on each plasmid to learn how our constructions work!
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