The Netherlands has been facing a pressing nitrogen crisis for several years. This crisis is largely attributed to the agriculture sector, with over 80% of ammonia (a nitrogenous compound) emissions coming from manure [1] and chemical fertilizers [2].

The over-use of fertilizers has a detrimental effect on the environment through the deposition of excess nitrogen oxides and ammonia in the ground, excessively enriching the environment with nutrients promoting uncontrolled plant and algal growth, or eutrophication, a form of nutrient imbalance [3] that negatively impacts the local biodiversity. This highlights the need of the hour: tackle the nitrogen crisis without negatively affecting food production, which still depends highly on fertilizers.

The Nitrogen Action Programme, introduced by the Dutch government in 2015, aimed to reduce nitrogen deposition, particularly in agriculture due to fertilizer use and ammonia emissions. However, in 2019, the Council of State deemed the programme insufficient, highlighting that nitrogen emissions were not just affecting rural ecosystems but also impacting urban development. As a result, new residential construction projects were halted until nitrogen emissions could be adequately compensated for, exacerbating the already critical housing shortage in the Netherlands [1]. This demonstrates how agricultural nitrogen management has far-reaching effects beyond the environment, directly influencing urban issues like the housing crisis, thereby emphasizing the urgency of addressing both challenges in tandem.

To combat global hunger and feed a growing population, an increase in global food production is crucial. This can be at least partially addressed through increasing crop yields, for which fertilizers are needed. Production of fertilizer is possible due to the Haber-Bosch process, where elemental nitrogen is converted into ammonia. Over-fertilization and its direct and indirect impact on the environment make agriculture the second leading contributor to short-term increases in global surface temperature [4].

In 2022, Dutch agriculture lost 74% (312,000 tons) of the nitrogen it spread as manure and synthetic fertilizer to the air and soil. Synthetic fertilizer production alone is also the cause of nearly 2% of global CO₂ emissions [5]. In addition to water pollution by leakage of nitrate, air pollution due to the conversion of nitrates to N₂O leads to a global greenhouse effect equivalent to 10% of that caused by the increase in atmospheric CO₂ [6]. For staple crops like cereals and maize, up to 40% of a farm’s operating cost is spent purchasing fertilizer [4]. Rising prices for fertilizer have been one of the problems leading to farmers' protests in Europe, and efforts to reduce nitrogen emissions in the Netherlands have been met with its own wave of protests [7].

Being a team from the Netherlands, we have actively followed the unfolding of the nitrogen crisis and seen the farmer's protests on the news. While nitrogen deposition is incredibly harmful to the environment, the Dutch agriculture sector is a big driving factor behind its economy, with agricultural exports being worth 124 billion euros in 2023 alone [8].

The Netherlands is also considered one of the front runners in terms of food and agriculture technology. Given the leadership of the Netherlands in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 [9] and Stony-Brook 2023 [10] that have tackled similar challenges, alongside a recent publication in Nature in April 2024 [11].

We are motivated by the vision of making the first step of what could be one of the biggest contributions to sustainable agriculture in the near future. We believe that the use of the nitroplast's capabilities could lead to more eco-friendly farming practices and help address some of the pressing challenges associated with current fertilization techniques, both in the Netherlands where there is a major nitrogen crisis, and globally where a growing demand for feed crops clashes with a need to reduce greenhouse emissions. Our project aims to harness the power of this organelle to create a more sustainable and efficient approach to crop cultivation, ultimately benefiting both the environment and the agricultural industry.

The Nature publication by Coale et al. examines UCYN-A, which evolved from a cyanobacterial species capable of converting N₂ into organic nitrogenous compounds, and its relationship with the marine alga Braadurosphaera bigelowii. It has already been established that UCYN-A and B. bigelowii have a symbiotic relationship, where B. bigelowii functions as a so-called host, and has taken up the UCYN-A bacteria into its cell in a process known as endosymbiosis. The symbiont, UCYN-A, fixes nitrogen for the host whereas B. bigelowii supplies organic carbon and a conducive living environment. This paper proved that UCYN-A is not simply a symbiont, but has instead evolved into an organelle for the eukaryotic alga for nitrogen fixation, and is now called the "nitroplast" [11].

The discovery of the nitroplast captured our interest - we had considered a project on nitrogen fixation before but failed to see a way in which we could innovate or propose new solutions to the problems previous teams faced. All diazotrophs (bacteria and archaea that fix atmospheric N₂) use the enzyme nitrogenase to fix nitrogen, but the expression of this enzyme presents great difficulties: it is irreversibly damaged by reacting with oxygen, while at the same time catalyzing an energetically demanding reaction. Due to this, diazotrophs have evolved very complex mechanisms to couple nitrogen fixation with respiration and/or photosynthesis, which so far has been beyond reach in terms of reproduction by synthetic biologists. The nitroplast solves this problem, acting as a fully contained compartment within a eukaryote where nitrogen fixation takes place, utilizing several years of evolutionary optimization.

Symbiotic relationships between diazotrophs and plants already exist in nature, specifically in crops - legumes have a relationship with rhizobia (bacteria living around the plant root), as do some grass species with other nitrogen-fixers. However, most other crops do not have anything of the sort. Besides transgenic nitrogenase expression, the other main avenue currently being explored in nitrogen fixation is the engineering of external symbiosis between diazotrophs and other plants. However, this poses a challenge in replicating fragile extracellular signaling pathways and physical conditions that are dependent on the plant species' roots, as well as potential containment issues.

Replicating endosymbiosis, while more ambitious than root-bacteria symbiosis, ensures by design that cell and organelle will work tightly together, preventing the difficulties associated with either root-dependence or nitrogenase expression. Our ideal long-term goal would be to introduce this organelle into crops. By doing this, it may be possible to reduce the reliance on synthetic fertilizers, thereby lowering environmental impact of their production and use, and enhancing sustainability in agriculture. This potential for positive change inspired our group to explore this innovative solution further.

One promising approach to balance the need for fertilizer and the welfare of the environment, is the development of plants that can fix atmospheric nitrogen independently. This innovation would not only reduce the need for synthetic fertilizers and manure but also help mitigate climate change and the nitrogen crisis. To this end, we first need to better study the nitroplast, how it interacts with the host organism and how it could be potentially introduced into other cells.

It has been discovered that, to ensure the endosymbiotic relationship, several proteins that are essential to UCYN-A are expressed in the host, B. bigelowii, and imported into the symbiont, similar to chloroplasts and mitochondria, though to a lesser extent [11]. Many of these proteins possess specialized localization peptides that direct their cellular function. In UCYN-A, these peptides are usually a C-terminal extension and are known as the “uTP” (UCYN-A Transit Peptide), although not yet identified [11]. Our first aim was to employ bioinformatics analyses to identify the characteristic motifs required for a protein to be imported by UCYN-A. For this, we made use of host (B. bigelowii) and nitroplast (UCYN-A) genome data as well as the proteomics data published in [11]. We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2.

To understand the functioning of the UCYN-A import mechanism, we attempted to identify the proteins involved in translocating host-encoded proteins into UCYN-A. First, we located genes in the host genome that are potentially involved in the translocation, based on their similarity to proteins in other import mechanisms such as from Paulinella chromatophora (UCYN-A analogue for photosynthesis). Potential chaperones analogous to heat-shock proteins were also included in the search. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. We then followed this by obtaining the tertiary structure of all candidate proteins using a structure prediction tool, and used docking tools to select candidate proteins likely to bind the previously identified transit motifs.

In addition to in silico experiments, we also aimed to investigate the transport mechanisms of UCYN-A in vivo. Instead of making use of plants as target organisms, we opted for using single-cell model eukaryote organisms, namely the yeast S. cerevisiae and the green alga C. reinhardtii. The initial in vivo characterization of the UCYN-A transport system involved examining the expression and localization of the UCYN-A transit peptides in these eukaryotic model organisms to test whether uTP would have any unexpected effect on cell viability and would not target any other organelle. To this end, we designed vectors, cloned them using Gibson-assembly, transformed bacteria, purified expression plasmids, and transformed those into S. cerevisiae and C. reinhardtii. We expressed uTP-tagged fluorescent proteins, together with controls targeting other organelles, and localization was assessed using fluorescence microscopy. Our preliminary results indicate that uTP did not target any other organelle and did not lead to alterations in cell morphology.

Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in S. cerevisiae using either E. coli or S. elongatus [12]. Another study successfully inserted Azotobacter strains into C. reinhardtii [13]. Building on this research, we initially aimed to develop a reliable protocol for transplanting a nitroplast into C. reinhardtii and S. cerevisiae as a proof-of-concept for transplantation into other eukaryotes, using polyethylene glycol (PEG) fusion protocols. However, due to time limitations, we started out with the model eukaryotic bacteria, E. coli, and refined a protocol for its fusion with S. cerevisiae.

We obtained B. bigelowii as a gift from Dr. Kyoko Hagino (Kochi University, Japan) and cultured it according to the protocol. For the transplantation of the nitroplast into a different model organism, we were also required to isolate this organelle from its host. Our project also implemented the protocol for nitroplast isolation from its host, according to the protocol and suggestions provided by Dr. Tyler Coale (University of California, San Diego, USA). Isolated UCYN-A could potentially be used for PEG fusion with other eukaryotes.

Finally, we have also wondered how our project would affect society. For this, our human practices team has actively worked to understand the possible consequences of our project. Also, wider acceptance of our idea goes hand in hand with educating the general population. Last but not least, we have also assessed the potential economic benefits of our idea and the omission of fertilizer use in agriculture. For this, we made an economic analysis and business plan.

Our project lays the foundation for the transplantation of nitroplast into eukaryotic hosts. The emergence of nitrogen-fixing plants could lead to a significant drop in the demand for fertilizers, and consequently in both carbon emissions and nitrogen pollution.

1. Nitrogen - WUR. url: https://www.wur.nl/en/research-results/dossiers/file/nitrogen.htm
2. The nitrogen strategy and the transformation of the rural areas — Nature and biodiversity — Government.nl. url: https://www.government.nl/topics/nature-and-biodiversity/the-nitrogen-strategy-and-the-transformation-of-the-rural-areas
3. National Oceanic US Department of Commerce and Atmospheric Administration. “What is eutrophication?”. https://www.noaa.gov/what-is-eutrophication
4. Jeff Elhai. “Engineering of crop plants to facilitate bottom-up innovation: A possible role for broad host-range nitroplasts and neoplasts”. In: (Apr. 2023). doi: 10.31219/OSF.IO/NY2RC. url: https://osf.io/ny2rc
5. Toename stikstofoverschot in landbouw door droge zomer 2022 — CBS. url: https://www.cbs.nl/nl-nl/nieuws/2024/05/toename-stikstofoverschot-in-landbouw-door-droge-zomer-2022
6. AR4 Climate Change 2007: Mitigation of Climate Change — IPCC. url: https://www.ipcc.ch/report/ar4/wg3/
7. Protesting farmers close roads and borders in nationwide campaign - DutchNews.nl. url: https://www.dutchnews.nl/2019/12/protesting-farmers-close-roads-and-borders-in-nationwide-campaign/
8. Statistics Netherlands. “Dutch agricultural exports worth nearly 124 billion euros in 2023”. In: (June 2024). url: https://www.cbs.nl/en-gb/news/2024/10/dutch-agricultural-exports-worth-nearly-124-billion-euros-in-2023
9. Team Wageningen 2021 homepage. url: https://2021.igem.org/Team:Wageningen_UR
10. Team — Stony-Brook - IGEM 2023. url: https://2023.igem.wiki/stony-brook/
11. Coale T.H. Loconte V. Turk-Kubo K.A. Vanslembrouck B. Mak W.K.E. Cheung S. Ekman A. Chen J.H. Hagino K. Takano Y. and T. Nishimura. “Nitrogen-fixing organelle in a marine alga”. In: Science 384 (2024), pp. 217–222.
12. Angad P. Mehta et al. “Engineering yeast endosymbionts as a step toward the evolution of mitochondria”. In: Proceedings of the National Academy of Sciences of the United States of America 115.46 (Nov. 2018), pp. 11796–11801. issn: 10916490. doi: 10.1073/PNAS.1813143115/SUPPL{\ _ }FILE/PNAS .1813143115 . SM02 . MP4. url: https://www.pnas.org/doi/abs/10.1073/pnas.1813143115
13. N.H. Nghia et al. “Uptake of Azotobacters by Somatic Fusion of Cell-wall Mutants of Chlamydomonas reinhardii”. In: Biochemie und Physiologie der Pflanzen 181.5 (Jan. 1986), pp. 347–357. issn: 0015-3796. doi: 10.1016/S0015-3796(86)80008-7.