Team:Aachen/Description
Salt water is causing problems all over the world, leading to a loss of diversity in ecosystems and freshwater scarcity. A main reason for this pollution is industrial wastewater.
We tackled this problem by using salt accumulating microorganisms engineered through synthetic biology.
Our project is based on Saccharomyces cerevisiae. Read more about why we chose this organism in our Design section.
We engineered different ion transporters to increase the NaCl uptake into the prevacuolar compartments and the vacuole of S.cerevisiae.
We started by knocking out the native systems responsible for the extrusion of sodium to the extracellular room.
Additionally, we overexpressed the native sodium hydrogen antiporter Nhx1 that, together with the chloride channel Gef1, is responsible for the sequestration of NaCl in the prevacuolar compartments.
As part of their salt tolerance mechanism plants like Arabidopsis thaliana are able to sequestrate ions in their vacuole. We used this system to increase the salt accumulation in S.cerevisiae.
We integrated an optimised variant of the Arabidopsis sodium hydrogen antiporter into the vacuolar membrane of S.cerevisiae, enabling salt sequestration in that compartment.
AtNHXS1 uses H+ protons to transport sodium against its concentration gradient. By integrating the Arabidopsis pyrophosphatase AVP1 into the vacuolar membrane of yeast we acidify the vacuole and thus increase the efficiency of transporters using such a proton gradient.
Following our Integrated Human Practices we expanded our project from NaCl to also accumulate other salts. For this reason we integrated Arabidopsis channels for the import of sulphate (AtSultr1;2) and potassium (AtAKT1).
To get an idea of the efficiency of our designed system we modelled the kinetics and interactions between the tranporters.
We proved that S.cerevisiae does survive high salt concentrations and furthermore does not burst when the intracellular salt concentration is significantly higher than the extracellular one.
We proved that our designed yeast accumulates more salt from the environment than the wildtype organism.
In cooperation with General Electric we developed a membrane system to guarantee that no organisms are released to the environment in our application.
A sustainable application of our “SALT VAULT” that we evolved together with representatives from industry, research and public, is one of the results of the integration of our Human Practices.
While developing our mutante and our application we took care of minimizing negative environmental impacts. Therefore, we created an organism carrying no antibiotic resistances and developed systems to prevent environmental exposure.
On the African continent and in the Middle East, the supply of fresh water is a major problem. But also in Germany there is a contamination of rivers and groundwater by municipalities, agriculture and, most of all, industry. The sewage is contaminated with various pollutants. But in particular, industrial wastewater often contains large amounts of different salts.
"The Werra between the Hessian potash territory and its confluence with the Fulda is currently the most heavily polluted river in Germany" Prof. Dr. Braukmann
The Werra, a German river, is considered one of the dirtiest in Central Europe. The reason for this is an extremely high salt load. The value for chloride is about 5000% higher than that of a comparable flow without an extreme introduction of sewage. The amounts of magnesium and potassium are also far above the normal value.
The reason for the load is the introduction of lye from the mining industry. At the Werra, potash salts are extracted, which are exported for fertilizer production worldwide. A waste product of this potash salt recovery is sodium chloride, however magnesium salts and others as well. These waste products are either disposed of on huge hills, pressed into the ground or led into the Werra as the already mentioned lye.
Fig.2: The ion concentration in the Werra (green) compared to a similar river without salt introduction from potash industry (red).
This leads to a chloride concentration in the Werra that is over 2000 mg / L high. This increased chloride content, together with other increased ion concentrations, leads to catastrophic damage to the ecosystem. Before the destruction through the introduction of industrial waste water without prior purification, the macrozoobenthos of the Werra was composed of 60-100 species.
Nowadays, the environment is dominated by only one species. The boosted river cancer, a brackish water species from North America, is a Neozoe (unnaturally introduced species) and became the dominant living being. In addition, the New Zealand dwarf snail, also a Neozoe, occurs frequently. There is only a small amount of mosquitos, fly larvae and some other species from the originally occurring species in the body of water with an extremely small share of the individual species of the ecosystem.
To tackle the significant damage salt stress causes in rivers like the Werra, but also to find a solution for ion burden in industrial process wasters, we developed "SALT VAULT".
The aim of our "SALT VAULT" system is to absorb as many sodium and chloride ions as possible to reduce the salt content in water. The system developed by us is based on the yeast Saccharomyces cerevisiae, which is not only a model organism, but also has a vacuole, which is of central importance for our project (details of why we chose this organism, see the >> design section).
S. cerevisiae has a large number of ion transporters and channels involved in the transport of sodium, potassium and chloride ions. These proteins are controlled by various regulatory mechanisms and work together to maintain the ion homeostasis in the cytosol of the yeast when the external salt concentrations vary, which is of central importance for metabolism.
There are multiple transport systems in the plasma membrane that lead to an influx of sodium, but there are no specific sodium importers in the plasma membrane [8]. The influx is only significant when sodium has much higher extracellular concentration than potassium [9,8] which, in our application, is the case:
- Phosphate influx into the cytoplasm is coupled to sodium and carried out by the cotransporter Pho89, contributing to sodium import [8,10].
- Low affinity nonspecific cation importers in the plasma membrane do not discriminate between K+ and Na+, leading to an import of both [11].
- Trk1, actually a potassium importer, transports Na+ in addition to K+, although with lower affinity [12].
Sodium ions play an important role for the function of macromolecules [8]. On the other hand, an increased intracellular concentration can lead to toxic effects because sodium ions can disturb the correct function of specific cellular pathways [9]. The most important transporters forming this system are shown in Figure 4.
Fig.4: ENA1 and NHA1 form the native efflux system responsible for the extrusion of sodium from the cytosol. NHX1 and GEF1 lead to sequestration of sodium and chloride in the prevacuolar compartments confering salt tolerance while also reducing toxic effects of sodium in the cytosol.
When there is an increased concentration of sodium chloride in the environment, S. cerevisiae reacts essentially in two ways to the entrance of sodium (and chloride) into the cytosol:
1. An efflux system consisting of two proteins encoded by the ENA1 and NHA1 genes transports sodium back into the extracellular space [15–18]:
Because the natural abundance of sodium ions in the environment is higher than the relatively low intracellular concentration, most eukaryotic organisms developed mechanisms to remove sodium ions from the cytosol. Especially at high external sodium concentrations, Saccharomyces cerevisiae relies on the ability to extrude the cation from the cytosol to maintain a low intracellular concentration. To achieve this, yeast developed two extrusion mechanisms.
The H+/Na+ antiporter, encoded by the Nha1 gene, can extrude Na+, Li+ and K+ using the proton gradient across the plasma membrane. However, the primary pathway for the extrusion of sodium ions is the plasma membrane P-type Na+ -ATPase encoded by Ena1 (for exnus natru) [17,18]. The ENA1 ATPase couples the hydrolysis of ATP to the transport of cations against the electrochemical gradients out of the cell [19]. This is because in yeast, the ENA1 gene plays an important role in salt tolerance. It encodes the most important protein for the extrusion of Na+ and Li+ out of the cell under saline stress conditions [20,18]. In response to saline or alkaline pH stress, the expression of ENA1 increases significantly, amongst other things, through the activation of calcineurin [21–23,17].
Fig.5: The two proteins responsible for the efflux of sodium as a stress response are highlighted in pink. ENA1 is a primary active sodium ATPase while NHA1 is a secondary active antiporter relying on the proton gradient over the plasma membrane
Because the Δena1 mutant lacks most of the sodium efflux ability, the intracellular sodium content is increased when compared to that in the wild-type [24]. This organism then heavely relies on the internal detoxification system to survive salt stress.
2. Nhx1 transports sodium into prevacuolar compartments [25,26]. The GEF1 channel causes the follow-up of chloride ions:
This system is described with the sequestration model, characterizing the sequestration of sodium in the prevacuolar compartments and thereby lowering the toxic cytosolic concentration in the Δena1 mutant [25,27]. Nhx1 is a Na+/H+ exchanger and mediates the sequestration of sodium into the prevacuolar compartments, but is not contributing to the efflux of Na+ to the extracellular room [26]. It has been shown that NHX1 is localized exclusively in unique endosomal/prevacuolar compartments [25]. Through enhancing the sequestration of sodium in the endosomal membrane system, it was possible to restore the salt tolerance of the Δena1 mutant [24]. This is due to the reduced salt concentration in the cytosol but increased concentration in the prevacuolar compartments. Responsible for that is Nhx1 that uses the proton gradient over the membrane of this acidic compartments generated by the vacuolar ATPase, to sequestrate sodium [26].
Fig.5 b: 2D Topology of the GEF1 chloride channel
Thus, and due to other mechanisms, the organism is able to maintain a healthy ratio of sodium and potassium in the cytosol, even if the environmental conditions strongly fluctuate. If a hyperosmotic shock occurs, S.cerevisiae can reduce its intracellular water potential by importing these ions into the compartments via NHX1 and GEF1 and consequentially survive, making the organism capable of a certain natural salt tolerance [26].
In order to achieve our goal of accumulating as much sodium chloride as possible in the cell, we have modified some of these channels to prevent the salt from leaving the cell. At the same time, it is to be ensured that as much salt as possible is stored in the prevacuolar compartments.
The changes we made in the yeast native channel system are shown in Figure 6. They can be subdivided into two areas:
- The destruction of the two most important efflux mechanisms ENA1 and NHA1, so that as little salt as possible leaves the cell.
- Overexpression of NHX1 to maximize the sodium (and therefore chloride) uptake into the prevacuolar compartments.
Fig.6: We knocked out the two efflux mechanisms ENA1 and NHA1 and overexpressed NHX1 to increase the sequestration of ions in the intracellular compartments.
These mutations were performed individually as well as in various combinations to identify the most efficient mutant (>> results). To learn more about the methodology we used for these changes, check out our Workflow and Protocols pages.
But while working on and modifying the native transport system of S. cerevisiae, we have also integrated a new system which offers further possibilities of increasing the salt intake. For this purpose, we used a combination of ion transporters that are found in the plant Arabidopsis thaliana. The system differs from that in S. cerevisiae, since plants react comparable but not similar to salt stress.
AtNHX1
Plant vacuolar membrane Na+/H+ antiporter genes play an important role in cellular ion homeostasis, especially the sequestration of Na+ in the vacuole to confer salt stress resistance in plants [28–30]. Vacuolar membrane-bound and H+ translocating enzyme H+ adenosine triphosphatase (ATPase) and H+ inorganic pyrophosphatase (PPiase) generate an electrochemical potential across the membrane through a proton gradient [31,32] that is used by the Na+/H+ antiporters to move Na+ against its electrochemical potential and accumulate it inside the vacuole [31].
In Arabidopsis thaliana the most abundant Na+/H+ antiporter AtNHX1 is bound to the vacuolar membrane [33] . It mediates the import of not only Na+, but also K+ into the vacuole and with that contributes to the accumulation of cations significantly [28,29]. AtNHX1 is a channel which is homologous to the yeast NHX1 gene that encodes a Na+/H+ antiporter in the prevacuolar compartment [34]. When AtNHX1 is knocked out in Arabidopsis thaliana the plant loses its ability to sequestrate sodium and thus is more sensitive to salt stress [35].
Fig.7:The Arabidopsis thaliana proton sodium antiporter is integrated into the vacuolar membrane of S.cerevisiae in addition to the alteration in the native system of the organism.
Overexpression of AtNHX1 led to an increased vacuolar Na+/H+ transport rate that was higher than the relative increase in AtNHX1 protein abundance and increased accumulation of sodium in the vacuole of Arabidopsis thaliana. Through that, the salt tolerance was increased, confirming AtNHX1 plays an important role in salt stress response [28,29,36]. We decided to use this channel and integrate it into the vacuolar membrane of S.cerevisiae. But while overexpression of AtNHX1 increased its Na+/H+ transport rate, the still relatively low Vmax of this transporter is limiting the application in terms of fast hyperaccumulation of sodium in the vacuole [30].
Through DNA shuffling, it was possible to generate a mutated channel - called AtNHXS1 - that showed an about two-fold increased Na+/H+ Vmax while the K+/H+ exchange activity was not significantly altered when compared to the native form of AtNHX1. The channel is still localized in the vacuolar membrane but the plant cell accumulated more sodium than the native strain with AtNHX1 [30]. AtNHXS1 consists only of 4 transmembrane helices, the C-terminus and the 5 nearest transmembrane helices of AtNHX1 where deleted, resulting in the increase in Na+/K+ selectivity ratio described previously [30].
AtNHXS1 also shows two mutations (L29P and S158P) in its amino acid sequence that might be responsible for the increased sodium transport activity and thus enable increased sodium sequestration in the vacuole (Figure 8) [30]. We integrated this synthetic transporter into the vacuolar membrane to use the yeast´s vacuole as an additional compartment for salt storage with higher volume than the prevacuolar compartments (Figure 7).
Fig.8: Comparison of AtNHXS1 and AtNHX1. AtNHXS1 was generated through DNA shuffeling and lost 5 transmembrane helices and the regulatory C-terminus. This mutant of AtNHX1 is more efficient and is not negatively regulated, resulting in higher sequestration.
AVP1
To increase the efficiency of this channel we will additionally integrate the protein encoded by AVP1 into the vacuolar membrane of S.cerevisiae.
Cation antiporters like AtNHX1 or NHX1 mediate the transport of solutes from the cytosol into the vacuolar lumen against their concentration gradient using the proton gradient over the vacuolar membrane (or prevacuolar compartment membrane in case of NHX1) [37]. In yeast, this proton gradient is generated by the membrane bound H+-ATPase that uses ATP to translocate protons into the vacuole and thus acidifies this compartment [38]. In principle, by increasing the availability of protons through an enhanced expression of the vacuolar H+-ATPase the sequestration of ions in the vacuole should increase [39]. But the overexpression of vacuolar H+-ATPase would be difficult because the yeast vacuolar H+-ATPase is a multi-subunit protein [40]. Each of the subunits would have to be overexpressed exactly at the right level at the same time to get an increased activity of the complex protein [41].
Fig.9: AVP1 uses pyrophosphate to pump protons into the vacuole thus causing acidification of this compartment. The proton gradient can be used by secondary active transporters like AtNHXS1 to pump ions into the vacuole against their concentration gradient.
In contrast to yeast, the proton electrochemical gradient across the vacuolar membrane (tonoplast) of Arabidopsis thaliana is generated by two proteins, the plant vacuolar H+-ATPase and AVP1, the vacuolar membrane bound H+-pyrophosphatase, which is encoded by only a single gene [42]. AVP1, as a vacuolar H+-pyrophosphatase, hydrolyses cytosolic inorganic pyrophosphate (PPi) to orthophosphate (Pi) to use the derived energy to actively pump protons into the vacuole (Figure 9) [43–45]. This process not only reduces the cytosolic PPi concentration, but more importantly acidifies the vacuole [46,47]. The difference in electrochemical potential for protons across the tonoplast, generated by the vacuolar H+-ATPase and AVP1, can then be used by vacuolar transporters, e.g. AtNHX1, to pump ions into the vacuole [46,48,49].
Resulting from that, there will also be a compensatory transport of anions to maintain electroneutrality [50]. Through expression of the Arabidopsis thaliana H+-PPi- ase AVP1 in yeast it is possible to increase the translocation of protons and anions over the tonoplast and thus acidify the vacuole [44]. This increased proton-influx leads to more cytosolic Na+ being sequestered inside the vacuole via the NHX1 exchangers and in general leads to a higher solute accumulation through H+-syn- and antiporters [40,39,43]. This shows that the vacuole acidification through AVP1 plays a key role in facilitating vacuolar Na+ sequestration [51] and increases the concentration of cations in the vacuole [39].
Fig.10: AVP1, together with AtNHXS1 leads to an increased sequestration of sodium in the vacuole. This system is added to the altered native one
We have integrated the gene AVP1 together with AtNHXS1 into the yeasts vacuolar membrane (Figure 10). Together with the changes we carried out in the native transporter system of S. cerevisiae this can be described as an expanded and optimized sequestration model to maximize the salt concentration in the yeast and remove it from the wastewater. You can compare the salt uptake of different combinations of transporters we integrated into S.cerevisiae in our results page. We also compared the salt tolerance of the different mutants because this is a good indicator for the functionality of our laboratory work and model. Before we integrated the different combinations of channels we created a mathematical model of our system to determine the maximal amount of salt our system can take up.
Following our intensive Integrated Human Practice work, we realized that contamination of wastewater through salts is not restricted to Na+ and Cl- but that ions like K+ and SO4- lead to problems, too. Read more about the negative effects of these ions in our integrated HP section. Potassium contamination is discussed in the section about the Werra while details about problems with sulphate can be found in the interviews at the municipal and chemical wastewater treatment plants.
Following the input we got from these cooperations we decided to identify possible channels for alteration to increase the accumulation of potassium and sulphate.
AtAKT1
We identified AtAKT1 as a promising gene to increase the potassium accumulation. AtAKT1 is a potassium channel originally from Arabidopsis thaliana root tissue. It mediates potassium uptake over a wide range of potassium concentration in the extracellular room [52–55]. While there are multiple other families of transporters involved in potassium uptake, AtAKT1 specifically is a high affinity transporter in the plasma membrane [56–58]. Since sodium has a negative effect on the efficiency of AtAKT1 [59], we did not combine a sodium accumulating mutant with this channel, but rather created a separate mutant called NAKT. Therefore, we integrated the channel in the plasma membrane of a mutant with ENA1 and NHA already knocked out. We did this, because the genes not only mediate efflux of sodium but also transport potassium back into the extracellular room.
Compared to the system we created to accumulate sodium inside a cell, the AtAKT1 system does not struggle with the toxicity of the accumulated ions, since potassium is a nutrient and indispensable for many physiologic functions and does not inhibit metabolism [8]. We were able to accumulate potassium in this mutant. Details can be found on our results page.
Fig.11: 2D topological structure (left) and function (right) of AtAKT1. AtAKT1 uses ATP to actively import potassium into the cytosol. CYCLN and ANKY are intracellular regulation domains.
AtSultr1;2
Since we want to increase the uptake of sulfate into the vacuoles of Saccharomyces cerevisiae, we decided to integrate the AtSultr1;2-gen. The gene encodes for a high-affinity sulfate transporter that naturally occurs in Arabidopsis thaliana and is expressed at epidermis and cortex of its roots [60].
AtSultr1;2 belongs to a family of sulfate transporters and due to studies with mutants the AtSultr1;2 seems to be the most important one. The mutant having a AtSultr1;1-knockout showed up a phenotype similar to the wildtype while the AtSultr1;2-mutant showed differences in the phenotype compared to the wildtype relating to root length, growth and sulphate uptake. This confirms the assumption that AtSultr1;2 plays a major role in the sulfate uptake system [61] [62].
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