- Technical Notes
- Open Access
Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris
© Delic et al; licensee BioMed Central Ltd. 2013
- Received: 9 November 2012
- Accepted: 23 January 2013
- Published: 24 January 2013
Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production.
Here we present five novel repressible Pichia pastoris promoters of different strength: P SER1 , P MET3 , P THR1 , P PIS1 and P THI11 . eGFP was expressed under the control of each of these promoters and its fluorescence could be successfully decreased in liquid culture by adding different supplements. We also expressed the essential genes with different native promoter strength, ERO1 and PDI1, under the control of two of the novel promoters. In our experiments, a clear down-regulation of both repressible promoters on transcriptional level could be achieved. Compared to the transcript levels of these two genes when expressed under the control of their native promoters, only ERO1 was significantly down-regulated.
Our results show that all of the novel promoters can be used for repression of genes in liquid culture. We also came to the conclusion that the choice of the repressible promoter is of particular importance. For a successful repression experiment it is crucial that the native promoter of a gene and the repressible promoter in its non-repressed state are of similar strength.
- Repressible promoter
- Pichia pastoris
- Promoter exchange
The methylotrophic yeast Pichia pastoris is a favored microorganism for the production of heterologous proteins in biotechnology. It has gained its popularity, because this yeast is easy to manipulate, can grow to high cell densities on cheap cultivation media and is well suited for the production of secreted proteins [1, 2]. Recently P. pastoris has also gained increasing interest as a model for cell biological studies of higher eukaryotic cells [3–5].
For biotechnological processes as well as for fundamental research, controlling of gene expression is required. In the industrial production of proteins inducible or repressible promoters are desirable, as they enable the separation of the cell growth phase from the protein production phase. Inducible/repressible promoters are also a useful tool for studying the function of essential genes, where the cells would not survive a complete knock-out.
Studies on repressible promoters are quite rare in yeast, and have mostly been performed with Saccharomyces cerevisiae. There, the most prominent candidates are the promoters of the genes MET3 (repressed by methionine), PHO5 (down-regulated by inorganic phosphate), CUP1 (responsive to Cu2+ ions), and the GAL genes (active in presence of galactose, inactive with glucose) . However, the commonly used regulable promoters in P. pastoris are those of the methanol utilization pathway, which enable high expression levels. These promoters are well suited for recombinant protein production, but not for studying gene function in cell biology. The best known is the alcohol oxidase 1 (AOX1) promoter which is activated on methanol and repressed on glucose . As the switch from glucose to methanol as carbon source leads to massive physiological changes in the cell, this class of promoters is unfavorable for cell biological research. Therefore the demand is rising for alternatives. Recently, we discovered novel P. pastoris promoters by cultivating strains on different carbon sources (glycerol, glucose and methanol). Among them, especially P THI11 has been shown to be independent from regulation by the tested carbon sources, but was repressed in presence of thiamine . Based on literature search in S. cerevisiae[9–12] and own microarray experiments in P. pastoris we identified other potential repressible promoters (P PIS1 , P THR1 , P SER1 and P MET3 ), which all could be of interest for studying physiology of the cell.
Determination of promoter activities with eGFP as reporter protein
Gene functions related to the promoters used in this work (from the Saccharomyces Genome Database)
Protein involved in synthesis of the thiamine precursor hydroxymethylpyrimidine (HMP)
Repressible with addition of thiamine
Conserved protein required for threonine biosynthesis (homoserine kinase)
Repressible with addition of L-threonine, L-valine, L-leucine and L-isoleucine
ATP sulfurylase, catalyzes the primary step of intracellular sulfate activation, essential for assimilatory reduction of sulfate to sulfide, involved in methionine metabolism
Repressible with addition of L-methionine
3-phosphoserine aminotransferase, catalyzes the formation of phosphoserine from 3-phosphohydroxypyruvate, required for serine and glycine biosynthesis
Repressible with addition of L-serine
Phosphatidylinositol synthase, required for biosynthesis of phosphatidylinositol, which is a precursor for polyphosphoinositides, sphingolipids, and glycolipid anchors for some of the plasma membrane proteins
Repressible with addition of zink
The P. pastoris promoter P MET3 responded to addition of L-methionine and the initial fluorescence was fully down-regulated in the presence of this amino acid. The ability of the MET3 promoter to be repressed by addition of methionine was already shown for S. cerevisiae and Ashbia gossypii[11, 12]. Dünkler and Wendland have shown that this promoter was almost fully inactive during a period of 5 h, when the medium was supplemented with 2.5 mM methionine. Usually, the MET3 promoter was used for short-term studies on cell physiology in S. cerevisiae as shown in Dünkler and Wendland . We used P MET3 for the regulation of the promoter expression in liquid culture cultivations for a period of ~20 h with 10 mM methionine. The higher methionine concentration is possibly the explanation for the longer repression period of this promoter.
Addition of L-threonine in order to reduce eGFP fluorescence when expressed under the control of the P THR1 promoter resulted only in a slight decrease of the signal. As the L-threonine biosynthesis pathway branches into the synthesis of L-isoleucine, L-valine and L-leucine, we added these three amino acids to the culture and combined them in the following cultivations with L-threonine. The highest repression of this promoter is achieved, when all four amino acids are present in the cultivation medium, resulting in a residual fluorescence of ~0% (Figure 1).
Contrary to published data for the S. cerevisiae PIS1 promoter , the P. pastoris P PIS1 promoter responded only slightly to addition of inositol to the culture. As reported for S. cerevisiae[9, 10], we could achieve a down-regulation of ~49% of the P PIS1 promoter by supplementing the medium with zinc sulfate (Figure 1).
The results above show that all tested P. pastoris promoters are repressible to a certain level under defined cultivation conditions. Under repressing conditions, residual fluorescence of the reporter protein eGFP could be detected only in case of P SER1 and P PIS1 , which means that the other tested promoters were almost fully repressed under these conditions. In this study we tested only one concentration of all supplements. We do not rule out that an increased concentration of the amino acids, zinc sulfate, or other additional components would not cause different regulation of the chosen promoters. Also different cultivation conditions or cultivation time could influence the efficiency of down-regulation.
Comparison of promoter strength
Example: Expression analysis of different essential genes under the control of novel repressible promoters
Exemplarily, we combined two of the tested promoters with genes that are involved in oxidative protein folding, ERO1 and PDI1. Their deletions have been shown to affect the viability of the yeast S. cerevisiae. These two genes exhibit promoters of different strength, ERO1 having a weaker native promoter (~14% of the strong GAP promoter) than PDI1 (~35% of the GAP promoter) in P. pastoris ( and own unpublished data).
From these results we can conclude that not only the strength of the repressible promoter in its non-repressed state, but also in its repressed state is of great importance. This should be taken into account when a repressible promoter is chosen for down-regulation of the transcription of a gene of interest. In this case both, the repressible and the native promoter of this gene should be of comparable strength.
Repressible promoters are an attractive tool for studying effects of conditionally down-regulated expression of genes that cannot be deleted. These promoters are mostly easy to handle and can be regulated with addition of few supplements. Here we present five repressible P. pastoris promoters of different strength. Each of the analyzed promoters can be successfully down-regulated or completely turned off in long term cultivations in liquid culture. The promoter strength ranged from a very weak promoter P SER1 to a strong promoter P THI11 with comparable expression levels to the strong P. pastoris P GAP promoter.
In our experiments, we succeeded to down-regulate the transcriptional expression of two essential genes, ERO1 and PDI1, when they were expressed under the control of two different repressible promoters P THR1 and P THI11 . Remarkably, only ERO1 was really down-regulated below its native transcriptional level, when the results of expression levels were compared to those of the wild type strain. These experiments demonstrated clearly how important the choice of the repressible promoter is, as the residual expression levels of these two genes after down-regulation differed immensely. The repressible promoter should be comparable in its non-repressed state to the native promoter of a gene of interest.
We could also observe a significant transcript level down-regulation of the promoters P MET3 and P SER1 when we cultivated the strains in complex medium (modified YPD medium), but not of the promoter P THR1 (data not shown).
Primers used for cloning of the expression vectors
P MET _fw (BstX I):
P MET _rv (Sbf I):
P THR _fw (BstX I):
P THR _rv (Sbf I):
P SER _fw (BstX I):
P SER _rv (Sbf I):
P PIS _fw (BstX I):
P PIS _rv (Sbf I):
P THI _fw (BstX I):
P THI _rv (Sbf I):
P ERO _fw (Asc I):
P ERO _rv (Apa I):
P PDI _fw (Asc I):
P PDI _rv (Apa I):
M2 minimal medium contained per liter: 20 g of glucose, 20 g of citric acid, 3.15 g of (NH4)2HPO4, 0.03 g of CaCl2.2H2O, 0.8 g of KCl, 0.5 g of MgSO4.7H2O, 2 mL of biotin (0.2 g L-1), 1.5 mL of trace salts stock solution. The pH was set to 5.0 with 5 M KOH solution. Trace salts stock solution contained per liter: 6.0 g of CuSO4.5H2O, 0.08 g of NaI, 3.0 g of MnSO4.H2O, 0.2 g of Na2MoO4.2H2O, 0.02 g of H3BO3, 0.5 g of CoCl2, 20.0 g of ZnCl2, 5.0 g of FeSO4.7H2O, and 5.0 mL of H2SO4 (95-98% w/w).
All analyzed clones were grown over night in 5 mL M2 medium as pre-culture. Main cultures were inoculated to an OD600 = 0.1 and incubated in 100 mL shake flasks at 28°C with 170 rpm (rotations per minute). Fluorescence of the cells was measured after 24 h of cultivation. All clones were supplemented two more times (after ~12 h) with the respective components (concentration as described below).
For comparing the non-repressed and repressed state of a promoter, cultivations of same clones in the M2 medium with and without supplements were performed. For repressing the promoters following concentrations of supplements were added to the medium: for P THI11 10 mM of thiamine hydrochloride (Merck), for P SER1 10 mM of L-serine (Serva), for P MET3 10 mM of L-methionine (Serva), for P THR1 10 mM of L-threonine (Serva), L-leucine (Serva), L-isoleucine (Serva) and L-valine (Serva), and for P PIS1 100 μM inositol or 1.5 μM zinc sulfate (Merck).
For selection on plates, 2% agar was added to the M2 medium and the pH was set to ~ 7.
Fluorescence measurement with flow cytometry
1 mL cells of an OD600 of 0.4 were harvested by centrifugation and resuspended in 2 mL PBS. Flow cytometry analysis was performed using a FACS Canto (Becton Dickinson) with these settings: excitation wavelength at 488 nm, emission wavelength at 530 nm (green filter, 525–550 nm). Measured fluorescence was referred to the cell size.
Isolation of total RNA from P. pastoris, reverse transcription of mRNA and determination of transcript levels by quantitative real time PCR
Real time PCR primers for ERO1 and PDI1
Amplicon size (bp)
The authors thank Michaela Reichinger and Roland Prielhofer. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT – Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG, and by the Austrian Centre of Industrial Biotechnology (ACIB GmbH).
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