NitroBLAST: Laying the foundation for nitrogen fixation.

Background

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: a solution for increasing global food supply while maintaining environmental standards.

Nitrogen Crisis in the Netherlands

Figure 1: Nitrogen manure production in kilograms/hectare in the Netherlands in 2010 [8].

The Nitrogen Action Programme, introduced by the Dutch government in 2015, aimed at reducing nitrogen deposition, was deemed insufficient in 2019 by the council of state. This declaration restricted the building of new residential areas, until the nitrogen emissions were compensated for, further augmenting the ongoing housing crisis of the Netherlands [1]. This emphasizes the urgency of addressing this crisis.

On the other hand, to combat global hunger, an increase in global food production is of the essence. This is addressed through the increase in crop yield, which is possible due to the Haber-Bosch process of fertilizer production, 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 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].

Altogether, there is a clear and urgent need for an alternative environmental-friendly solution to the nitrogen problem. This can not only make a huge impact on the Netherlands, but also globally, by enabling a sustainable and food secure future.

Figure 2: Farmer protests in the Netherlands [9].

Motivation

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 [10]. The Netherlands is also considered one of the front runners in terms of food and agriculture technology. The world's first lab-grown meat burger was a Dutch invention, introduced in 2013 [11]. Given our leadership in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 [12] and Stony-Brook 2023 [13] that have tackled similar challenges, alongside a recent publication in Nature in April [14].

Solution

The Nature publication by Coale et al. examines UCYN-A, a cyanobacterial species capable of converting N2 into organic nitrogen, and its relationship with the marine algae Bradurosphera 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 a common symbiont, but has instead evolved into a eukaryotic organelle for nitrogen fixation, termed the "nitroplast" [14].

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 millions of years of evolutionary optimization.

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 the 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.

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 not-so-distant 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.

Figure 3: Evolution of the nitroplast from the integration of the endosymbiotic UCYN-A bacteria into a eukaryotic B. bigelowii cell [14].

Our approach: NitroBLAST

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 need to better study the nitroplast and how it could be introduced into other cells.

Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in S. cerevisiae using E. coli and S. elongatus [15]. Another study successfully inserted Azotobacter strains into C. reinhardtii [16]. Building on this research, we aim 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. We will use polyethylene glycol (PEG) fusion protocols, starting out with analogous bacteria to UCYN-A (Azotobacter genus and Cyanothece ATCC51142, the closest free-living relative of UCYN-A).

It has been discovered that several essential UCYN-A proteins are expressed in the host, B. bigelowii, and imported into the symbiont, not unlike chloroplasts and mitochondria, though to a lesser extent [14]. Many of these proteins possess a C-terminal extension known as the “uTP” (UCYN-A Transit Peptide) [14]. We first aim to use bioinformatic analysis to identify the characteristic motifs required for a protein to be imported into UCYN-A. For this, we will make use of host and nitroplast's genome data as well as the proteomics data published by Coale et al..

To understand the functioning of the UCYN-A import mechanism, we will attempt to identify the proteins involved in translocating host-encoded proteins into UCYN-A. First, we will locate genes in the host genome which are potentially involved in the translocation based on their similarity to proteins in other import mechanisms such as the Paulinella chromatophora (UCYN-A analogue for photosynthesis) protein import or chaperones that seem analogous to heat-shock proteins. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. Following this, we will obtain the tertiary structure of all candidate proteins using a structure prediction tool, and use docking tools to select ones that are likely to bind the previously identified transit motifs.

The initial in vivo characterization of the UCYN-A transport system will involve examining the expression and localization of the UCYN-A transit peptides in the eukaryotic model organisms S. cerevisiae and C. reinhardtii to test for interference by cellular processes. To this end, uTP-tagged fluorescent proteins will be expressed in S. cerevisiae and C. reinhardtii, and the constructs will be confirmed using fluorescence microscopy.

Following up on this, we will test our candidate chaperone proteins by expressing them and recreating their interaction with uTP-tagged proteins in our model organisms, in order to demonstrate that the import mechanism can be reproduced outside of B. bigelowii. This will involve constructing plasmids to express fluorescently tagged chaperones and observing colocalization with transit peptide-tagged fluorescent proteins.

Finally, while the nitroplast could significantly reduce the need for nitrogen fertilizers, it would also consume energy from its host. Although photosynthesis should supply the plant with sufficient energy for nitrogen fixation, this energy expenditure might impact the growth rate or yield of crops. To assess the potential consequences of nitrogen-fixing staple crops, we will use metabolic models to predict effects on growth rate, and economic models to link crop yields to farmers’ budgets and profits.

Our project lays the foundation for the transplantation of nitroplast into their algal hosts, allowing for the creation of nitrogen-fixing eukaryote strains. The emergence of nitrogen-fixing plants could lead to a significant drop in fertilizer demand, and consequently in both carbon emissions and nitrogen pollution.

References

1. Nitrogen - WUR.
2. The nitrogen strategy and the transformation of the rural areas — Nature and biodiversity — Government.nl.
3. National Oceanic US Department of Commerce and Atmospheric Administration. 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. 4 2023.
5. Toename stikstofoverschot in landbouw door droge zomer 2022 — CBS.
6. AR4 Climate Change 2007: Mitigation of Climate Change — IPCC.
7. Protesting farmers close roads and borders in nationwide campaign - DutchNews.nl.
8. Potter, P., and N. Ramankutty, et al. (2010). Global Fertilizer Application and Manure Production.
9. The easy guide to the Dutch nitrogen crisis, farmers’ protests, and more - dutchreview.com.
10. Statistics Netherlands. Dutch agricultural exports worth nearly 124 billion euros in 2023. 6 2024. - cbs.nl.
11. The mission — Mosa meat - mosameat.com.
12. Team Wageningen 2021 - https://2021.igem.org/Team:Wageningen\_UR.
13. Team Stony-Brook 2023 - https://2023.igem.wiki/stony-brook/.
14. Loconte V. Turk-Kubo K.A. Vanslembrouck B. Mak W.K.E. Cheung S. Ekman A. Chen J.H. Hagino K. Takano Y. Coale, T.H. and T. Nishimura. Nitrogen-fixing organelle in a marine alga. Science, 384:217–222, 2024. 6
15. Angad P. Mehta, Lubica Supekova, Jian Hua Chen, Kersi Pestonjamasp, Paul Webster, Yeonjin Ko, Scott C. Henderson, Gerry McDermott, Frantisek Supek, and Peter G. Schultz. Engineering yeast endosymbionts as a step toward the evolution of mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 115(46):11796–11801, 11 2018.
16. N.H. Nghia, I. Gyurj ́an, P. Stefanovits, GY. Paless, and I. Turt ́oczky. Uptake of Azotobacters by Somatic Fusion of Cell-wall Mutants of Chlamydomonas reinhardii. Biochemie und Physiologie der Pflanzen, 181(5):347–357, 1 1986.