CONTRIBUTION

Characterization of yeast promoters

Motivated by the central role of promoters in the synthetic biology toolbox for gene expression regulation, we set out to quantitatively characterize a set of yeast promoters. Quantitative information of the activities of different promoters sets the basis for optimization of engineered biological systems. As yeast cell factories can use different carbon sources and growth conditions, we investigated the effect of carbon source on the activities of these promoters. We meticulously reviewed the iGEM part registry and selected three constitutive promoters, namely, pADH1, pURA3, and pCYC1 (Table 1). We used enhanced green fluorescent protein (EGFP) as a reporter to characterize these promoters. We transformed yeast cells with the integration vectors containing EGFP placed under the control of the chosen promoters. EGFP fluorescence of the yeast cells containing these expression cassettes was measured in 96-well plates using a fluorescence plate reader. The fluorescence intensity of the reporter protein served as an indicator of promoter activity.

pADH1 promoter

The pADH1 promoter governs the ADH1 gene's function. This gene encodes for the enzyme alcohol dehydrogenase, essential for converting acetaldehyde to ethanol in fermentation. Besides, Adh1 has additional functions such as methylglyoxal reduction, assisting in NADH oxidation, and producing fusel alcohol by decomposing amino acids (Bennetzen & Hall, 1982). Researchers and biotechnologists frequently use the pADH1 promoter in yeast to promote the expression of foreign proteins.

pCYC1 promoter

The pCYC1 promoter drives the expression of CYC1, which yields the iso-1 variant of cytochrome c. The protein, Cyc1, plays a pivotal role in the mitochondrial respiratory chain by aiding electron transport. With its heme group, it facilitates electron transfer across respiratory complexes, ending with oxygen. Cyc1 is predominantly located in the inner mitochondrial membrane. In oxygen-rich cell environments, the iso-1 type accounts for 95% of total cytochrome c (Sherman, 2005; Sherman et al., 1966). pCYC1 is a weaker promoter compared to pADH1.

pURA3 promoter

The pURA3 promoter controls the URA3 gene. This gene is responsible for producing the enzyme orotidine 5-phosphate decarboxylase (ODCase), which is crucial for the synthesis of pyrimidine ribonucleotides (Umezu et al., 1971).

Plasmid construction

The promoters were PCR-amplified from the yeast genome using primers that contained SacI (forward primer) and BamHI (reverse primer) restriction sites in their 5’-overhangs. After PCR and restriction digestion, the DNA fragments containing the promoters were ligated into SacI/BamHI-restricted pRS304-based vector carrying EGFP coding sequence and tCYC1 terminator (Table 2).

Yeast strain construction

Prior to yeast transformation, the integration plasmids were restricted with HindIII to linearise the plasmids for homologous recombination into the TRP1 locus in the yeast genome. The restricted plasmids were used to transform the S. cerevisiae DOM90 strain. Transformants were selected for Trp⁺ phenotype on tryptophan-dropout synthetic media (CSM-TRP) agar plates containing 2% glucose. All yeast strains generated and used for promoter characterization are listed in Table 3.

EGFP fluorescence measurements

Before conducting fluorescence measurements, yeast seed cultures were cultivated in complete synthetic media (CSM) containing 2% (m/v ratio) raffinose until the cultures reached an optical density (OD₆₀₀) ranging from 1 to 2. Subsequently, the yeast cultures were diluted to an OD₆₀₀ of 0.3, and various carbon sources, including glucose, raffinose, galactose, or glycerol, were added into the cultures to achieve a 2% (m/v) concentration of the respective carbon source. After 6 hours of growth, 200 μl of the cell suspension was carefully transferred into designated wells on 96-well plates for subsequent fluorescence measurements.

To measure EGFP fluorescence, a BioTek Synergy Mx Microplate Reader equipped with a 458 nm wavelength LED for GFP excitation was utilized. The emitted fluorescence was measured at a wavelength of 528 nm.

Results

In this study, we assessed the level of gene expression driven by the promoters pADH1, pCYC1, and pURA3 in different growth conditions by employing a fluorescent protein as a reporter. The promoter-containing constructs were integrated into the trp1-1 locus in the yeast genome, and the EGFP reporter protein fluorescence was quantified in a 96-well plate. To establish a baseline of background fluorescence in the culture, we measured the fluorescence in a control strain, DOM90, which does not express any fluorescent proteins.

Compared to the background fluorescence of DOM90, yeast strains with EGFP under the control of pCYC1 displayed a 1.16-fold increase in EGFP fluorescence when using glucose as a carbon source. The pCYC1-driven EGFP fluorescence levels were indistinguishable from the DOM90 background fluorescence in other carbon sources. In the case of the pADH1 promoter, there was a 4.42-fold increase in EGFP fluorescence when grown with glucose, a 2.62-fold increase with both raffinose and galactose, and a 1.3-fold with raffinose or glycerol. The results from pADH1-induced EGFP expression in glycerol are in compliance with prior findings which noted the suppression of ADH1 transcription during non-fermentative sugar consumption (Denis et al., 1983). For yeast strains with EGFP regulated by the pURA3 promoter, a 1.17-fold increase was observed with glucose as the carbon source, and a 2.6-fold increase in EGFP fluorescence was noted with raffinose and galactose. In contrast, using either raffinose or glycerol alone as carbon sources resulted in fluorescence levels comparable to those of the DOM90 background. (Fig. 1).

Figure 1. pCYC1, pADH1, pURA3 lead to different expression levels of EGFP. Bars indicate the mean fluorescence intensity in arbitrary units (AU) measured in pCYC1-EGFP, pADH1-EGFP, and pURA3-EGFP strains or in DOM90 negative control strain measured in a plate reader. Error bars show standard deviation.

In our study, we examined three yeast promoters sourced from the iGEM part registry. Our results showed that EGFP under pADH1 promoter drove the highest fluorescence levels on all carbon sources, while the pCYC1 promoter was the weakest.

We evaluated the expression levels in the presence of different carbon sources. For the constructs harboring the pADH1 promoter, strong gene expression was observed when glucose was used as the carbon source, moderate expression was detected in the presence of both raffinose and galactose, and weak expression was noted with either raffinose or glycerol alone. Our results show that pCYC1 promoter should be used when a very weak gene expression is desired. On the other hand, for constructs with the pURA3 promoter, moderate gene expression was evident when using raffinose and galactose together, while weak expression was observed when glucose was the sole carbon source. This information augments the quantitative knowledge on the activities of yeast promoters and aids in enhancing the precision of synthetic biology applications.