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Original organism: Kluyveromyces lactis

K. lactis is a crabtree-negative yeast, which means it does not produce ethanol under aerobic conditions. S. cerevisiae, on the other hand, is crabtree-positive. Another interesting fact about K. lactis is its ability to grow on ethanol as the only carbon source (Breunig et al., 1999, [http://www.ncbi.nlm.nih.gov/pubmed?term=10862884]).

KlADH4

Nucleotide sequence of the KlADH4 promoter region responsive to ethanol induction (UAS_E). The five STREs (shadowed boxes), the putative HSEs, the RapI and the YapI binding sites are also indicated. Nucleotides matching with the HSE consensus sequence are underlined. Picture taken from: Mazzoni et al., 2000

The K. lactis gene KlADH4 encodes a mitochondrial alcohol dehydrogenase which can be specifically induced by ethanol and is insensitive to glucose repression. A small region in the KlADH4 promoter, called UAS_E, renders the expression of a gene ethanol-dependent (Mazzoni et al., 2000 [http://www.ncbi.nlm.nih.gov/pubmed?term=10724480]). UAS_E is a cis-element of the KlADH4 promoter spanning from -953 to -741. When inserted into the promoters of heterologous genes, these genes become ethanol-inducible in K lactis.

The sequence contains (Mazzoni et al. 2000):

• a putative binding site for Yap1p between -906/-900, a transcription factor involved in stress response in S. cerevisiae and K. lactis
• consensus sequence of the binding site of Rap1 protein. RAP1 (repressor activator protein 1) is an essential gene present in both S. cerevisiae and K. lactis and acts positively or negatively on the expression of many genes. Rap1 binding sites have been found in promoters of glycolytic genes where they play a positive role in transcription.
• Five STREs (stress response elements). STREs have been found in the promoters of many S. cerevisiae genes, where they are present in two or more copies in both orientations
• Two HSEs (heat shock elements). HSEs have been found in combination with STREs in many stress-responsive genes of S. cerevisiae.

Despite the presence of the STREs, KlADH4 is not a stress responsive gene but specifically induced by alcohol (Mazzoni et al., 2000).

Although there is evidence that the KlADH4 promoter can be used for ethanol dependent production of recombinant proteins in K. lactis ([http://www.ncbi.nlm.nih.gov/pubmed/9872759 Saliola et al. 1999]), we did not find any literature which indicated that this alcohol-inducible promoter has been used in other organisms such as S. cerevisiae.

Mazzoni et. al (2000) annotated binding sites for two transcription factors (Rap1 and Yap1) as well as some small cis-elements (HSEs and STREs) in the UAS_E-region of the KlADH4-promoter. All of these factors are not only present in K. lactis but also in S. cerevisiae (Kuge & Jones, 1994 [http://www.ncbi.nlm.nih.gov/pubmed?term=8313910], Shore & Nasmyth, 1987 [http://www.ncbi.nlm.nih.gov/pubmed?term=3315231], Schueller et al., 1994 [http://www.ncbi.nlm.nih.gov/pubmed?term=7523111], Ruis & Schueller, 1995 [http://www.ncbi.nlm.nih.gov/pubmed?term=8526890]).

S. cerevisiae is a very potent brewer and quickly produces alcohol. This made negative controls during the characterization of the KlADH4-promoter difficult.

We isolated genomic DNA from K. lactis using the LiOAc-SDS-method ([http://www.ncbi.nlm.nih.gov/pubmed?term=21548894 Looke et al. 2011]) and used this gDNA as template for PCR. The following gel picture documents the success of this:

lane 1: Fermentas GeneRuler 100bp; lane 2: empty; lane 3: PCR-Product (KlADH4-promoter); lane 4: Fermentas GeneRuler 1kb

We also sequenced the construct for verification (data not shown) and found some single point mutations compared to the published genome sequence of K. lactis. These are not in any conserved regions and should have no influence on the promoters functionality.

The Part was entered into the Partsregistry as BBa_K801020

First experiment using over-night pre-cultures

In a first experiment, the transformed yeast cells were picked grown in a pre-culture (SC-U Medium, 30 °C, 180 rpm) over night and transferred into SC-U Medium with different concentrations of ethanol (0%, 4%, 8%, 10%, v/v). The eGFP-fluorescence and the OD600 were measured at t = 0h, 3h, 18h, 21h, 24h. Also, the ethanol concentration of the cultures was measured using an Alchohol-Dehydrogenase-Assay.

For the evaluation of the experimental data, the measured fluorescence was divided by the respecitve OD600, to normalize the fluorescence to the respecitve cell count. This was done to take the intrinsic auto-fluorescence in account. The results are shown in picture 1.

Picture 1: First characterization experiment of the KlADH4-promoter (& eGFP) in S. cerevisiae. After an overnight pre culture, the transformed yeast cells were transferred into SC-U Media with different ethanol concentration and the eGFP-fluorescence and the OD600 were measured at different times.

The promoter is generally functional in S. cerevisiae, which can be seen by the fact that eGFP is expressed.

The fact that the expression of eGFP is low in the cultures with 8% and 10% (v/v) ethanol can be explained with the fact that the viability of the yeast cells is dramatically decreased at these high ethanol concentrations. This can also be seen by the growth curves (picture 2).

Picture 2: Growth curves of S. cerevisiae INVSc1 in SC-U Medium with different ethanol concentrations. At 8% and 10% Ethanol (v/v) the growth of the yeast cells is dramatically reduced.

At first glance, the fact that there is a significant signal in the culture with 0% ethanol added looks as if the promoter is constitutive and not specifically induced by ethanol. However, the cells could also be induced by the ethanol produced by the yeast themselves during the over night pre-culture. Because of this ambiguity, further experiments were performed.

Second experiment: Characterization of the KlADH4-promoter without over-night pre-cultures

The goal of this series of experiments was to avoid pre-cultures in which the cells would produce alcohol and hence be induced before the actual measuring series. Therefore, three measures were taken:

• Use of baffled flasks to increas oxygen transfer into the medium, thus lowering fermentation
• Transfer of a large amount of transformed yeast cells into SC-U Medium with different ethanol concentrations immediately after transformation and start measuring the eGFP-expression. The transformed cells were not plated on selective plates and thus, picking of clones and a pre-culture became unneccesary. (Experiment 2.1)
• Cultivate a control culture of transformed yeast in SC-U Medium with Glycerol as sole carbon source. Glycerol is a non-fermentable carbon source for S. cerevisiae (Feldmann, 2005). (Experiment 2.2)

Result of experiment 2.1: cells transferred into liquid medium immediately after transformation

Figure 3: Fluorescence per cell of yeast cells transformed with a plasmid carrying BBa_K801020 in front of an eGFP-gene. The different alcohol concentrations are indicated

Due to the fact that the cultures examined here also contain untransformed yeast cells, the OD600-value is falsely high. The untransformed yeast cells are not viable (due to uracil auxotrophy and cultivation in SC-U, a medium lacking uracil) and cannot express eGFP, but still they disperse light and thus increase the measured OD. This renders the data shown in Fig. 3 uninterpretable. However, the cultures which were used for these measurements expressed eGFP - enough to produce fluorescence visible to the naked eye (Fig. 4).

Figure 4: Fluorescent yeast cells carrying a plasmid with the KlADH4-promoter controlling eGFP expression. In each picture, the flask to the left shows a culture in which eGFP-expression is repressed by using the GAL1-Promoter and a medium containing glucose. A: Culture 1: 0 % Ethanol added at t = 0. B: Culture 2: 2 % Ethanol added at t = 0. C: Culture 3: 4 % Ethanol added at t = 0

Result of experiment 2.2: negative culture grown in SC-U glycerol

The aim of this experiment was to keep the ethanol production as low as possible by the use of glycerol as sole carbon source. As shown in Fig. 5, the Ethanol concentration in the culture remained very low (less that 0.21 Vol.-%). Also, the "Fluorescence per cell" does not increase over time - the yeast cells do not express eGFP.

Figure 5: Fluorescence per cell and ethanol produced of transformed yeast cells in SC-U Medium with glycerol as sole carbon source

To verify the fact that the cells did not express eGFP, excitation and emission spectra were recorded at t = 24 h. For comparison, reference spectra of a culture expressing eGFP is also shown (Fig. 6). The characteristic peaks of eGFP (excitation: 488 nm, emission: 509 nm) are not visible in the culture grown on glycerol as sole carbon source (ethanol concentration: 0,2 Vol.-%).

Figure 6

The fact that no expression of eGFP is detected in this culture (ethanol concentration: 0.2 Vol.-%) suggests that the part is ethanol inducible in S. cerevisiae, too. The lowest measured alcohol concentration in a culture expressing eGFP was 0.9 Vol.-%. Further experiments are being done to verify this first statement.