Team:Valencia Biocampus/Yeast

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

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 which are rich in easily assimilable nutrients, such as sugars and amino acids.

There are two important characteristics which take part 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 that it can grow in aerobic and anaerobic conditions. All of this makes this species present some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in S. cerevisiae and in a few other yeasts, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even in 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. One way of monitoring if the glucose concentration in the medium is correct could be using our yeasts. Adding the proper amount of hydrogen peroxide we could ask our culture: “What glucose concentration do you need?”, 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!

Each construction was carried in diferent plasmids for their expression in yeast:
YEplac181 carried Fermentative response construction. To see further information about the vector, click here.
YEp352 carried Oxidative response construction. To see further information about the vector, click here.

EXPERIMENTAL OUTLINE

- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication.
- We were supplied the Yeplac181 and Yep352 yeast vectors by our laboratory.
- We carried out four transformations of E. coli strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the Transformation Protocol Using Heat Shock .
- We obtained several transformants of E. coli in four plates and took some colonies of each DNA (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks.
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the Mini-prep Protocol.
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the Protocol for Gel Extraction
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the Digestion Protocol .
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the ligation Protocol.
- The day after that we transformed E. coli with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection.
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks.
- The day after we extracted the plasmid DNA. See the Mini-prep Protocol.
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.
- We introduced the first of the DNA recombinant plasmids in the yeast. See the Yeast transformation protocol.
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol here.
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the Yeast transformation protocol.
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the Yeast induction protocol .