Abstract
The overuse of synthetic nitrogen-based fertilisers has caused significant damage to ecosystems across the globe. Our project aims to use synthetic biology to engineer E. coli to produce ammonia naturally, in order to decrease the fossil fuels and nitrogen pollution impact of the fertiliser industry. SOILutions aims to develop an oxygen-sensing feature that allows the bacteria to activate only under low oxygen conditions, increasing the longevity and usability of these engineered microorganisms. With the potential to increase ammonia production in soil, this synthetic biology solution may be the last piece of the puzzle for creating a consistent and far more sustainable supply of fertiliser for farms across the world. A renewable and sustainable way of producing nitrogen-based fertiliser, through our engineered bacteria, ensures the continuous production of modern crops, without causing ecological damage from nitrogen overuse.
Overview
Our project was to design microorganisms to improve soil quality. One of the nutrients that are limited in soil for plant growth is nitrogen. Nitrogen-fixing bacteria fix nitrogen from the atmosphere and make it available for plant growth. These bacteria have a complex enzyme called nitrogenase, which only works under low oxygen conditions. The main regulatory gene for making nitrogenase is nifA, and numerous people have found that just upregulating nitrogenase is not beneficial for the bacteria or the soil. So, we aimed to identify an oxygen-sensitive promotor that could control nifA and only make nitrogenase when the conditions were appropriate. Our project's main synthetic biology aspect is to make and test promotors that express genes under low oxygen conditions but not under high oxygen conditions. We think this is the first example of a demonstration of a successfully applied oxygen-sensitive promotor.
Project Inspiration
The inspiration for our project is guided by the increasing global concern for our planet as climate change becomes a prominent issue affecting us all, especially our generation. As such, a keen environmental concern guided our project, ideating on which aspect of nature needed the most help.
Our team settled on promoting soil health as we quickly discovered through our research that global fertilisers in our soils contribute greatly to excess nitrogen being emitted into the atmosphere. Not only this, but Australia has been striving to become a clean and greener country as a whole, however this is not the case. Unfortunately, our country has the largest Nitrogen footprint (47 kg of nitrogen per person, per year), primarily due to our large agricultural industry.
With all members of the team going to various different schools based in Sydney, Australia, the team are all acutely aware of the significant contribution of agriculture to our economy. With agriculture being Australia’s primary industry, SOILution’s product is aimed at providing a greener and more environmentally friendly source of fertiliser.
Current Problem
Current human population requires consistent use of fertiliser to sustain itself.
The consistent growth of the human population continues to place a continuous strain on global production chains across the globe. In order to ensure that products like vegetables, grains, clothing materials and feed for animals continue to sustain the population, the International Fertilizer Association estimates global fertiliser consumption at 150 million tonnes of nitrogen-based fertiliser per year.
In 2017, the Food and Agriculture Organisation of the United Nations estimated that the production of food by 2050 will have risen to 13.5 billion tonnes, in order to feed an expected 9.7 billion people. This unprecedented increase will almost certainly need an incredibly abundant supply of fertiliser, most of which will be sourced from nitrogen-based fertilisers like ammonia. In 2022, the Ammonia Energy Association (AEA) published a report that estimates that by 2050, the global demand for ammonia as a fertiliser will have risen from 150 million tonnes to 260 million tonnes.
The overuse of ammonia fertiliser results in an abundance of excess nitrogen not consumed by the crops, emitting more nitrogen into the atmosphere. This plays a significant role in climate change as this greenhouse gas is over 300 times more effective at trapping in heat, remaining in the atmosphere for 114 years (United Nations Environment Programme, 2023). Following from this, fertilisers utilised in farming account for around two thirds of global nitrogen emissions, increasing the rapid rate of global warming.
Current Solution
Overuse of synthetic fertiliser to maintain consistent production.
In order to address the constant demand for food and clothing material production, farmers must maintain consistent production. As a result, the global agricultural industry has become dependent on synthetic fertilisers. This demand is evident as the fertiliser industry in Australia has grown in revenue by 9.8% from 2018 to 2023, reaching $6.8 billion (IBISWORLD, 2023). Most of these products contain phosphorus, nitrogen and potassium, with urea and ammonium phosphate being the most commonly used in Australia. Urea and ammonium phosphate were the most commonly used in 2015-16, making up 47% and 38% of total nitrogen based fertilisers used in Australia (Australian Bureau of Statistics, 2017). In seeking to maintain productivity in agriculture, farmers who are applying synthetic fertilisers to their land regularly over-apply fertiliser. This often exceeds the nutritional requirements of the plants and soil, with estimates believing that a range of 24-32% of purchased nitrogen-based fertiliser is wasted (Trachtenberg & Ogg, 1994).
The nitrogen from this wasted fertiliser is not absorbed into the soil, and flows into nearby waterways causing nitrogen pollution. Nitrogen pollution, over an extended period of time, results in severe environmental and health issues, with excess nitrogen in the water increasing the chance and severity of large growths of algae called algal blooms. Some algal blooms can produce elevated toxins and bacteria growth, causing sickness in humans or animals who come into contact and consume it. In some of the most severe nitrogen pollution sites, this algae overgrowth prevents sunlight and oxygen reaching other organisms, making it impossible for any other aquatic life to survive.
Our Solution
Consistent fertiliser production without needing synthetic fertilisers.
SOILutions acknowledges the continuous need for farming for all our food and clothes, but the team has become aware of the urgent need for change in agricultural practices that utilise synthetic fertilisers due to their harmful impact on the environment. Current practices utilised by farmers meet the demand for materials, however the long lasting effects of using synthetic fertilisers far outweigh the short term benefits.
At SOILutions, our mission revolves around the development of an oxygen-sensitivity mechanism, enabling bacteria to activate exclusively in low-oxygen environments. This advancement significantly extends the lifespan and utility of these specially engineered microorganisms. With the potential to augment ammonia generation in soil, our synthetic biology innovation could represent the final piece of the puzzle in establishing a dependable and vastly more sustainable fertiliser supply for global agriculture. By offering a renewable and sustainable approach to nitrogen-based fertiliser production through our engineered bacteria, we ensure the continuous cultivation of modern crops without the ecological harm stemming from nitrogen overuse.
This is essential in allowing consistent fertiliser production without needing synthetic nitrogen-based fertilisers that cause significant damage to ecosystems across the globe. Instead, farmers can use our solution to continue to meet demand for necessities while helping the environment in the long term, preserving the earth’s environment and atmosphere. Our goal is to implement nitrogen fixation, as performed by diazotrophs, in e.coli.
Diazotrophs and Nitrogenase
Diazotrophs are a subset of bacteria species adapted to fix gaseous nitrogen (N2) from the atmosphere to bioavailable forms, typically ammonia (NH3). The most common mechanism by which diazotrophs fix nitrogen is via the Nitrogenase pathway, employing mainly two structural proteins, Reductase (coded by nifH) and Nitrogenase (coded by nifD and nifK). The former is a homodimer (2x alpha sub-units) which moves freely within the cell delivering electrons to the latter, a heterotetramer (2x alpha sub-units + 2x beta sub-units) imbedded in the cell wall (integral protein), which performs the bulk of N2 fixation. In addition to these proteins and their respective genes, the nitrogenase pathway also involves several other accessory proteins and regulatory genes necessary to initiate and complete the construction of the reductase and nitrogenase proteins. One such regulatory gene, nifA, is key in regulating the synthesis of nitrogenase proteins and thus was the target of much of our work.
Oxygen Sensor
In implementing nitrogenase within e.coli, a primary concern is that as nitrogenase is prone to reacting with Oxygen to yield reactive oxygen species (ROS) which can cause oxidative stress/damage to the cell, the nitrogenase mechanism should be regulated with respect to Oxygen abundance if it is to be efficient, where ≈ 2% O2 concentration is the documented upper limit (source). We thus chose to test fumarate and nitrate reductase (FNR) protein as an oxygen-responsive transcriptional regulator to manage the abundance of intracellular nitrogenase given it is part of a pre-existing, oxygen sensitive mechanism employed by e.coli to switch between aerobic and anaerobic metabolism. FNR functions as a transcriptional activator and upregulator in e.coli which can be used to promote the production of certain proteins by binding to DNA binding sites on promoters, while reacting via its Iron-Sulphur cluster with oxygen to be deactivated in the presence of high oxygen conditions.
Hence, by testing FNR as an oxygen-sensitive transcriptional upregulator, we aim to eventually implement it as a regulatory system for nitrogen fixation in e.coli.
Step 1: Gathering information, gene parts, reviewing papers about existing oxygen sensors
To effectively design a biological oxygen sensor, we conducted research on existing oxygen sensors commonly used in plants and microorganisms, settling on FNR as anaerobically purified samples have been shown to contain a [4Fe-4S]2+ cluster that is sufficiently unstable toward O_2 to make it a suitable oxygen sensor to rely on for appropriate upregulation of Nitrogenase production (Kiley, P. J., & Beinert, H. (1998)). As for optimising this FNR upregulation mechanism, increasing the yield of nitrogenase in low O2, our design and development utilised findings from “Control of nitrogen fixation in bacteria that associate with cereals,” (Ryu, M. H., et al., 2020) which tested different promoters for increase in transcriptional yield using fluorescent green protein (gfp) as an indicator. Following its findings, our test structure utilised gfp as a placeholder for nifA while varying the type of promoter interacting with FNR in hopes of optimising yield. In addition to differentiating via the intensity of fluorescence (indicating rate of transcription), different oxygen levels were also tested ensuring transcription is adequately oxygen-sensitive and is sufficiently reduced with >2% O_2 concentration.
Step 2: Designing and assembling the parts we found into a theoretical working oxygen sensor.
Our species:
Our designs tested 3 promoters, narGp, narKp and dmsAp each modified to possess an extra half binding site [TTGAT] (1.5 total binding sites) with the intention of increasing FNR sensitivity to maximise yield in low O2 conditions.
Our oxygen prototype consisted of the following 3 parts (parts 2 and 3 were put together in the registry):
- Part 1 constant (BBa_K4735012): lacIQ promoter which provides baseline rate of transcription for rbs-fnr followed by a TrrnBT1 terminator. This provides a constant supply of FNR protein
- Part 2 variable (BBa_K4735013, BBa_K4735014, BBa_K4735015 (all include part 3)): A T-Spacer and T7 binding site (for future positive control testing), followed by the promoter to which FNR will bind, each containing 1.5 binding sites, varies between a narGp, narKp and dmsAp promoter.
- Part 3 constant: Ribosome binding site (different to the rbs in part 1) rbs2 followed by superfolder fgfp and then a terminator TrrnBT1.
Design philosophy/explanation
When our plasmid is introduced into the e.coli, the lacQ promoter will synthesise the fnr gene constantly, allowing FNR to roam within the cytoplasm and hence either upregulate gfp transcription in low oxygen levels, or be deactivated in high oxygen and cease upregulation. When FNR is exposed to high levels of oxygen naturally, it will break down and not bind to the FNR binding sites, whereas when desired levels of oxygen are present, FNR will bind to its binding site and act as a medium for RNA polymerase to locate and start translating the following genetic code (gfp in this case). This will hence increase the unit of superfolder gfp synthesised which can be measured by monitoring the frequency and intensity of fluorescence in colonies under certain uv conditions.
Step 3: Prototypes
Prototype A consisted of:
- Part 1 - lacIQ-rbs-fnr-TrrnBT1
- Part 2 - T-Spacer-T7-3FNRbindingNarG
- Part 3 - RBS2-sfgfp + TrrnBT1
Prototype B consisted of:
- Part 1 - lacIQ-rbs-fnr-TrrnBT1
- Part 2 - T-Spacer-T7-3FNRbindingNarK
- Part 3 - RBS2-sfgfp + TrrnBT1
Prototype C consisted of:
- Part 1 - lacIQ-rbs-fnr-TrrnBT1
- Part 2 - T-Spacer-T7-3FNRbindingDmsA
- Part 3 - RBS2-sfgfp + TrrnBT1
Primers for PCR amplification and Golden Gate Assembly:
As for assembly, we utilised the NEBridge golden gate assembly tool to design primers for PCR using its software, yielding
- Prototype A = Part 1(ac) + Part 2a(a) + Part 3(ac)
- Prototype B = Part 1(b) + Part 2b(b) + Part 3(b)
- Prototype C = Part 1(ac) + Part 2b(c) + Part 3(ac)
In the form:Prototype n = Part 1(primer) + Part 2n(primer) + Part 3(primer)
Citations
Gielen, D. (2022, May 19). Renewable Ammonia's role in reducing dependence on Gas - Energy Post. Energy Post. Retrieved October 06, 2023 from https://energypost.eu/renewable-ammonias-role-in-reducing-dependence-on-gas/
Eric, Trachtenberg, & Clayton Ogg. (1994). POTENTIAL FOR REDUCING NITROGEN POLLUTION THROUGH IMPROVED AGRONOMIC PRACTICES [PDF]. Journal of the American Water Resources Association. https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1752-1688.1994.tb03356.x
Reasons why the world needs to limit nitrogen pollution (n.d.). UNEP. Retrieved October 06, 2023 from https://www.unep.org/news-and-stories/story/four-reasons-why-world-needs-limit-nitrogen-pollution
The Issue | US EPA. (2013, March 12). US EPA. Retrieved October 06, 2023 from https://www.epa.gov/nutrientpollution/issue
The Effects: Dead Zones and Harmful Algal Blooms | US EPA. (2013, March 12). US EPA. Retrieved October 04, 2023 from https://www.epa.gov/nutrientpollution/effects-dead-zones-and-harmful-algal-blooms
