Abstract

Water scarcity is one of the biggest challenges facing mankind in the 21st century. As of today, 2 billion people (1 in 4) worldwide lack safe drinking water. Our reservoirs are at an all-time low, but an even greater issue is the poor quality of the remaining water.

The current and predominant agricultural model is responsible for leaving us with poor quality water. Industrial livestock farming with its excessive excrement from macrofarms and industrial agriculture, with its massive use of fertilizers are poisoning this most precious resource. It is estimated that in the period 2016-2021 the municipalities and county councils of Catalonia, at least, have made an investment valued at 19.101.169,8 €.

At iGEM Barcelona-UB we’re committed to fighting against water pollution, and we have developed AlgaGenix.

AlgaGenix plans to improve the microalgae model organism Chlamydomonas reinhardtii through synthetic biology so that it removes nitrates from polluted waters and turns them into cytokinin-rich biomass –which can later be used as a fertilizer– while improving water quality. This way, we are cleaning polluted water whilst producing value-added products, embracing the circular economy.

The genetic modifications will be conducted through MoClo and consist in the overexpression of 4 endogenous genes aimed at enhancing nitrate uptake and cytokinin biosynthesis, as well as the introduction of an exogenous Glutamine Synthetase from Synechocystis sp. PCC6803 to avoid the endogenous regulation of its homologous protein in C. reinhardtii and to ensure the link between the uptake and the production parts of the pathway.

THE WATER CRISIS

Water is the most abundant resource worldwide, and is also the most used. We not only need water to live, but to keep our economy afloat, our industries running and, most importantly, our ecosystems thriving.

Due to climate change, and myriad other factors, water in Catalonia and many other parts of the world has never been so scarce. Our reservoirs are at an all-time low, but an even greater issue is the poor quality of the remaining water.

Agriculture and livestock farming have surged as the most water-polluting activities in the region. In fact, nitrate pollution alone mars around 36% of Catalonia’s underground reservoirs, affecting nearly 40% of Catalonia’s population, which is close to 3 million people.

For the past three years, there has been no substantial rains to refill the water reservoirs of Catalonia. This has led to a “state of exceptionality” which exerts water-usage limitations that, as of now, have practically forbidden the agricultural sector from watering crops, which means an immense decrease in crop-production, and have vastly reduced water-usage in many other sectors, even affecting human consumption.

As previously told, non-polluted water reservoirs in Catalonia are scarce, and this drought seems far from receding. Additionally, the remaining water is at high risk of eutrophication, so it is evident that we need an imminent solution to prevent further contamination and mass eutrophication, as well as to ensure water availability, which will help mitigate the effects Climate Change is having on the region.

The main pollutants in our reservoirs are nitrates, which derive from a wide variety of activities such as farming, agriculture, industry and sewerage, and build up to create a significant strain on our resources. Despite the fact that methods to remove these pollutants from the water exist –as many water-related companies have tried to implement new measures to battle the crisis– the problem persists, which demonstrates the need for new methods.

This is why opening the door to new organisms can provide a myriad of new opportunities, and since the water issue is exerting an enormous pressure on our environment and regular human activities, we are running against the clock.

Our team members come from different corners all around Catalonia, and every one of us is experiencing this new reality differently. However, this allows us to evaluate the needs of each region and tailor a more accurate and realistic approach to solving the problem. Isolated mountain towns could run out of their sole safe water sources in wells and aquifers; the canal water with which we irrigate our crops could harm them instead of nurture them; the water our families drink could cause severe disease or the food industry could be severely affected: we see the many facets this issue has, and we understand the impact it is having in our homes, our communities and our society.

We know that by fixing this issue, or at the very least by reducing its magnitude, millions of people would benefit, and wide areas of our land could begin to heal and thrive, mending the harm done by long exposure to these pollutants

Our Solution

At iGEM Barcelona-UB we’re committed to fighting against water pollution, and we have developed AlgaGenix.

Cytokinins are of special interest because they are phytohormones, natural stimulants that induce cell division and thus enhance plant growth. They are also very difficult to chemically synthesize, making them very expensive and valuable compounds. In this way, we would be converting a waste product into a high added-value product, which has a very beneficial effect on crops. Also, we would not have to add new fertilizers to the field and worsen the situation in which we find ourselves.

The genetic modifications will be conducted through the Modular Cloning kit (MoClo) and consist in the overexpression of 4 endogenous genes aimed at enhancing nitrate uptake and cytokinin biosynthesis, as well as the introduction of an exogenous Glutamine Synthetase from Synechocystis sp. PCC6803 to avoid the endogenous regulation of its homologous protein in C. reinhardtii and to ensure the link between the uptake and the production parts of the pathway. (more info in the engineering page)

Nitrate assimilation is an apparently simple process in photosynthetic eukaryotes. The process involves two transport and two reduction steps to produce ammonium in the chloroplast, the main site of ammonium incorporation into carbon skeletons, and takes place by the glutamine synthetase/glutamate synthase cycle.
The transport steps consist of the entry of nitrate into the cell and nitrite into the chloroplast. The first reduction step from nitrate to nitrite occurs in the cytosol and is catalyzed by the NR (Nitrate Reductase) enzyme, and the second reduction step from nitrite to ammonium in the chloroplast is catalyzed by NiR (Nitrite Reductase).

The key master in the regulation of these transporters is the Nit-2 gene. Nit-2 is a nitrate-inducible gene that encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including NR and NiR. But such expression is strongly inhibited in the presence of ammonia, therefore repressing NR and NiR.

To bypass these inhibitions, we expressed NR and NiR genes under a constitutive promoter, PPSAD, which was not affected by ammonia, enhancing the nitrate assimilation pathway.

But nitrogen incorporation in the cell is only the starting point, though. Once inside, nitrogen compounds are transformed into NH4+, which is in turn introduced into the metabolism through a sequence of enzymes. The most important one is glutamine synthetase (GS), which is subject to exquisite regulation at multiple levels, among them gene expression regulation to control GS abundance, as well as feedback inhibition and covalent modifications to control enzyme activity.

However, we could bypass this regulation by introducing a GS which was regulated differently.
Enterobacteria’s GS is well characterized, especially that of E. coli, where the partial inhibition due to negative feedback is through reversible adenylylation. In cyanobacteria, like Synechocystis sp. PCC 6803 (a cyanobacteria model organism), GS is regulated through different systems to that of E. coli, some of which involve the interaction with two small proteins called inactivating factors (IF17 and IF7) that inhibit GS linearly with their abundance, as well as specific riboswitches that refine the activity of those IFs.
With this in mind, we decided to introduce C.reinhardtii’s GS and Synechocystis’ GS both under the same constitutive promoter, PPSAD.
Synechocystis’ GS would need both IFs to be completely inactivatedi in vivo, and after reviewing literature and performing BLAST alignments, we confirmed that C.reinhardtii did not express the IFs.

But as well you know, our project does not end here, in simply absorbing more. We wanted to convert nitrates into valuable and useful products of high interest.

Although the specific details of cytokinin biosynthesis in microalgae are not yet fully understood, the general pathway is similar to that observed in higher plants.

It involves many enzymes and precursors, but the key enzymes of the pathway are IPT (isopentenyl transferase) and LOG (LONELY GUY).

Previous research in Chlorella shows that overexpression of these key enzymes translates in increased biosynthesis of cytokinins. So we co-expressed LOG and IPT under the same constitutive promoter, PPSAD.

Our algae is designed to fit in a bioreactor right within highly-polluted nitrate waters, from where our engineered Chlamydomonas will transform said nitrates into biological matter, enriched with cytokinins. (more info in the project implementation page)

Then, the algae are lysed and killed, and after lyophilization, the final solid product can be applied onto fields as a natural fertilizer.

This is why we believe that modifying the relatively unexplored genus of microalgae opens a new window of possibilities, especially taking into account their particularly efficient absorption and production rates. This way, we will be able to avoid some of the limitations of current water-filtering genetically-modified microorganisms and present a sustainable and efficient alternative.

On top of that, we are implementing 2 Sustainable Development Goals, by cleaning polluted waters from contaminants and then converting them into valuable products, promoting a circular economy.

Related Works

There are different iGEM teams that have tried to solve this problem previously, such as China-FAFU 2022 (https://2022.igem.wiki/china-fafu/) that also uses algae to absorb these pollutants.

But other teams have done similar work with bacteria like UMaryland 2022 (https://2022.igem.wiki/umaryland/description).

Also some teams worked about phosphates and nitrates absorption with different organisms: Dusseldorf 2020 (https://2020.igem.org/Team:Duesseldorf/Description) or UIUC_Illinois 2022 (https://2022.igem.wiki/uiuc-illinois/).

Other work with microalgae has been done too: Sorbonne U Paris 2020 (https://2020.igem.org/Team:Sorbonne_U_Paris/Plant).

Other References

Sanz-Luque E, Chamizo-Ampudia A, Llamas A, Galvan A, Fernandez E. Understanding nitrate assimilation and its regulation in microalgae. Front Plant Sci. 2015 Oct 26;6:899. doi: 10.3389/fpls.2015.00899. PMID: 26579149; PMCID: PMC4620153.

Navarro, M. C. D., Prieto, R. H., Fernández, E., & Galván, A. (1996). Constitutive expression of nitrate reductase changes the regulation of nitrate and nitrite transporters in Chlamydomonas reinhardtii. Plant Journal, 9(6), 819-827. https://doi.org/10.1046/j.1365-313x.1996.9060819.x

MÉRIDA, A., et al., 1991. Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium. Available at: < https://journals.asm.org/doi/abs/10.1128/jb.173.13.4095-4100.1991 >