<p>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 <strong>insufficient</strong>, 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 <ahref="https://www.wur.nl/en/newsarticle/Nitrogen-crisis-in-the-Netherlands.htm"style="color: #C6EBE8;">[NitrogenWUR]</a>. 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.</p>
<p>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. <strong>Over-fertilization</strong> and its direct and indirect impact on the environment make agriculture the second leading contributor to short-term <strong>increases in global surface temperature</strong><ahref="https://www.nature.com/articles/s41598-023-34214-3"style="color: #C6EBE8;">[Elhai2023 Engineering Neoplasts]</a>.</p>
<p>We expected, as the results of the experiment, that the light emitted by the primitive lux operon was a very faint blue light (similar to a schematic diagram of PV), which is very technological, but far from being called "LAMPS". Our project first set out to make our enzymes emit brighter and more colorful light for practical applications. We use both <b>protein engineering</b> and <b>metabolic engineering</b> to achieve the design goal of <b>increasing light intensity</b>.</p>
<p>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 <strong>2% of global CO<sub>2</sub> emissions</strong><ahref="https://www.cbs.nl/en-gb/news/2023/increase-in-agriculture-s-nitrogen-emissions"style="color: #C6EBE8;">[ToenameCBS]</a>. In addition to <strong>water pollution</strong> by leakage of nitrate, <strong>air pollution</strong> due to the conversion of nitrates to N<sub>2</sub>O leads to a global greenhouse effect equivalent to 10% of that caused by the increase in atmospheric CO<sub>2</sub><ahref="https://www.ipcc.ch/report/ar4/wg1/"style="color: #C6EBE8;">[AR4IPCC]</a>. For staple crops like cereals and maize, <strong>up to 40% of a farm’s operating cost is spent purchasing fertilizer</strong><ahref="https://www.nature.com/articles/s41598-023-34214-3"style="color: #C6EBE8;">[Elhai2023 Engineering Neoplasts]</a>. 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 <ahref="https://dutchnews.nl/news/2023/farmers-protests-in-the-netherlands/"style="color: #C6EBE8;">[ProtestingDutchNews.nl]</a>.</p>
<p>Being a team from the Netherlands, we have actively followed the <strong>unfolding of the nitrogen crisis</strong> 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 <strong>agricultural exports</strong> being worth 124 billion euros in 2023 alone <ahref="https://www.government.nl/topics/agriculture-and-food/agricultural-exports"style="color: #C6EBE8;">[Netherlands2024 Dutch2023]</a>.</p>
<p>The Netherlands is also considered one of the <strong>front runners in terms of food and agriculture technology</strong>. 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 <ahref="https://2021.igem.org/Team:Wageningen"style="color: #C6EBE8;">[TeamHomepage]</a> and Stony-Brook 2023 <ahref="https://2023.igem.org/Team:Stony-Brook"style="color: #C6EBE8;">[Team2023]</a> that have tackled similar challenges, alongside a recent publication in Nature in April 2024 <ahref="https://www.nature.com/articles/nature2024"style="color: #C6EBE8;">[Coale2024 Nitrogen-fixing Alga]</a>.</p>
<p><strong>Symbiotic relationships</strong> 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 <strong>other crops do not have anything of the sort</strong>. Besides transgenic nitrogenase expression, the other main avenue currently being explored in nitrogen fixation is the <strong>engineering of external symbiosis</strong> 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.</p>
<p>Replicating endosymbiosis, while more ambitious than root-bacteria symbiosis, <strong>ensures by design that cell and organelle will work tightly together</strong>, preventing the difficulties associated with either root-dependence or nitrogenase expression. Our ideal <strong>long-term goal would be to introduce this organelle into crops</strong>. By doing this, it may be possible to <strong>reduce the reliance on synthetic fertilizers</strong>, 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.</p>
<p>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. <strong>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</strong>.</p>
<p>It has been discovered that, to ensure the endosymbiotic relationship, several proteins that are essential to UCYN-A are expressed in the host, <em>B. bigelowii</em>, and imported into the symbiont, similar to chloroplasts and mitochondria, though to a lesser extent <ahref="https://www.nature.com/articles/nature2024"style="color: #C6EBE8;">[Coale2024 Nitrogen-fixing Alga]</a>. 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 <ahref="https://www.nature.com/articles/nature2024"style="color: #C6EBE8;">[Coale2024 Nitrogen-fixing Alga]</a>. Our first aim was to employ bioinformatics analyses to identify the characteristic <strong>motifs required for a protein to be imported by UCYN-A</strong>. For this, we made use of host (<em>B. bigelowii</em>) and nitroplast (UCYN-A) genome data as well as the proteomics data published by Coale <em>et al.</em>. <strong>We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2</strong>.</p>
<p>To understand the functioning of the UCYN-A import mechanism, we attempted to <strong>identify the proteins involved in translocating</strong> 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 <em>Paulinella chromatophora</em> (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 <strong>obtaining the tertiary structure of all candidate proteins</strong> using a structure prediction tool, and used <strong>docking</strong> tools to select candidate proteins likely to bind the previously identified transit motifs.</p>
<p>In addition to <em>in silico</em> experiments, we also aimed to investigate the transport mechanisms of UCYN-A <em>in vivo</em>. Instead of making use of plants as target organisms, we opted for using single-cell model eukaryote organisms, namely the yeast <em>S. cerevisiae</em> and the green alga <em>C. reinhardtii</em>. The initial <em>in vivo</em> characterization of the UCYN-A transport system involved <strong>examining the expression and localization of the UCYN-A transit peptides in these eukaryotic model organisms</strong> 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 <em>S. cerevisiae</em> and <em>C. reinhardtii</em>. 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.</p>
<p>Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in <em>S. cerevisiae</em> using either <em>E. coli</em> or <em>S. elongatus</em><ahref="https://link.springer.com/article/10.1007/s00438-018-1448-1">[Mehta2018 EngineeringMitochondria]</a>. Another study successfully inserted <em>Azotobacter</em> strains into <em>C. reinhardtii</em><ahref="https://www.jstor.org/stable/24358669">[Nghia1986 UptakeReinhardii]</a>. Building on this research, we initially aimed to <strong>develop a reliable protocol for transplanting a nitroplast</strong> into <em>C. reinhardtii</em> and <em>S. cerevisiae</em> as a <strong>proof-of-concept</strong> for transplantation into other eukaryotes, using polyethylene glycol (PEG) fusion protocols. However, due to time limitations, we started out with the model eukaryotic bacteria, <em>E. coli</em>, and refined a protocol for its fusion with <em>S. cerevisiae</em>.</p>
<p>We obtained <em>B. bigelowii</em> 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.</p>
<p>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 <strong>economic analysis</strong> and business plan.</p>
<p>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 <strong>economic analysis</strong> and business plan.</p>
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<div class="h1">Reference</div>
<p style="text-align:left;">[1] Kaku T, Sugiura K, Entani T, Osabe K, Nagai T. Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer. Sci Rep. 2021;11(1):14994. Published 2021 Jul 22. doi:10.1038/s41598-021-94551-4</p>
