@@ -18,7 +18,7 @@ Eventually, the co-polymer concept was discarded, and the team focused on trimmi
Finally, the team committed to a concept outline and began work on literature review and background research to prepare for entering the lab work phase of our project. *Vibrio natriegens* was selected as a chassis organism, with the combating eutrophication being selected from the several water treatment focuses possible.
# Purpose
The purpose of the wet lab design was driven by the team’s inspiration, tackling coastal eutrophication with a circular and sustainable approach. Natronaut’s aim is to restore ecosystem balance, mitigating the hypoxic effects of nitrate (NO3-) induced algal bloom and decomposition (Cosme and Hauschild, 2017; Ærtebjerg, 2001). Considering a fully circular approach, the team decided to further extend the project’s purpose to the prevention of eutrophication development. This would be done by recycling the cells into single-cell proteins, which would supplement animal feeds and lower fertiliser use in traditional animal feed production.
The purpose of the wet lab design was driven by the team’s inspiration, tackling coastal eutrophication with a circular and sustainable approach. Natronaut’s aim is to restore ecosystem balance, mitigating the hypoxic effects of nitrate (NO3-) induced algal bloom and decomposition (Cosme and Hauschild, 2017; Ærtebjerg, 2001). Considering a fully circular approach, the team decided to further extend the project’s purpose to the prevention of eutrophication development. This would be done by recycling the cells into single-cell proteins, which would supplement animal feeds and lower fertiliser use in traditional animal feed production. This is visualised in Figure 1.
**Figure 1.** Project purpose and goals outline. Created with Biorender.com
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@@ -45,7 +45,7 @@ In contrast, the assimilatory NO3- pathway leads to incorporation of nitrogen in
The assimilatory pathway in bacteria comprises several steps. First, NO3- is captured and internalised from the extracellular environment to the intracellular space. This step is mediated by a NO3–-transporter, which is most commonly an ATP-binding cassette (ABC)-type transporter located in the cytoplasmic membrane (Moreno-Vivián & Flores, 2007). The transporter consists of three subunits: a periplasmic protein that binds NO3- with high affinity (even at low extracellular concentration of NO3-), a transmembrane protein that facilitates the transport of NO3- across the membrane, and a cytoplasmic ATPase anchored to the membrane, which hydrolyses ATP to provide energy for the process (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007).
Once the NO3- is internalised, it is then reduced to NO2- by assimilatory nitrate reductase (Nas). This enzyme is NADH-dependent, and has two subunits: a large catalytic subunit, which contains the essential active site for the reduction of NO3- and a small NADH oxidoreductase subunit, which facilitates the transfer of electrons to the active site (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007). In the following step, NO2- is further reduced to NH4+ by the monomeric nitrite reductase Nir. Afterwards, the produced NH4+ is incorporated into amino acids, specifically glutamine and glutamate through the GS-GOGAT and GDH pathways (Moreno-Vivián & Flores, 2007; van Heeswijk et al., 2013). The GS-GOGAT pathway consists of two key steps. First, the enzyme glutamine synthetase (GS) catalyses an ATP-dependent reaction that converts glutamate to glutamine by incorporating an ammonium ion. Following this, glutamate synthase (GOGAT) transfers the amide group from glutamine to 2-oxoglutarate, resulting in the production of two glutamate molecules. In contrast, the GDH pathway employs a more direct approach. The enzyme glutamate dehydrogenase (GDH) catalyses the incorporation of an ammonium ion (NH₄⁺) directly into 2-oxoglutarate, forming glutamate in a single step. The resulting amino acids, glutamate and glutamine, undergo further transamidation and transamination, yielding various amino acids, which then serve as building blocks for the biosynthesis of proteins during translation (van Heeswijk et al., 2013).
Once the NO3- is internalised, it is then reduced to NO2- by assimilatory nitrate reductase (Nas). This enzyme is NADH-dependent, and has two subunits: a large catalytic subunit, which contains the essential active site for the reduction of NO3- and a small NADH oxidoreductase subunit, which facilitates the transfer of electrons to the active site (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007). In the following step, NO2- is further reduced to NH4+ by the monomeric nitrite reductase Nir. Afterwards, the produced NH4+ is incorporated into amino acids, specifically glutamine and glutamate through the GS-GOGAT and GDH pathways (Moreno-Vivián & Flores, 2007; van Heeswijk et al., 2013). As visible in Figure 2, The GS-GOGAT pathway consists of two key steps. First, the enzyme glutamine synthetase (GS) catalyses an ATP-dependent reaction that converts glutamate to glutamine by incorporating an ammonium ion. Following this, glutamate synthase (GOGAT) transfers the amide group from glutamine to 2-oxoglutarate, resulting in the production of two glutamate molecules. In contrast, the GDH pathway employs a more direct approach. The enzyme glutamate dehydrogenase (GDH) catalyses the incorporation of an ammonium ion (NH₄⁺) directly into 2-oxoglutarate, forming glutamate in a single step. The resulting amino acids, glutamate and glutamine, undergo further transamidation and transamination, yielding various amino acids, which then serve as building blocks for the biosynthesis of proteins during translation (van Heeswijk et al., 2013).
**Figure 2.** The pathways that result in the biosynthesis of glutamine and glutamate. The GDH pathway is shown in the left panel. The GS-GOGAT pathway is shown in the right panel. Created with BioRender.com
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@@ -53,14 +53,14 @@ Once the NO3- is internalised, it is then reduced to NO2- by assimilatory nitr
**Figure 3.** Genes in K. oxytoca
In K. oxytoca, the genes associated with the nitrate (NO₃⁻) assimilation pathway are arranged in a nasFEDCBA operon. Within this operon, the nasFED gene cluster encodes the transporter necessary for the uptake of extracellular NO₃⁻. Both the membrane-spanning subunit NasE and the ATP-binding subunit NasD play essential roles in the acquisition of nitrate. The genes nasA and nasC encode the large and small subunits of assimilatory nitrate reductase (Nas), respectively, while the nasB gene is linked to nitrite reductase (Nir). This genetic arrangement allows K. oxytoca to efficiently assimilate nitrate (Wu & Stewart, 1998).
In K. oxytoca, the genes associated with the nitrate (NO₃⁻) assimilation pathway are arranged in a nasFEDCBA operon (Figure 3). Within this operon, the nasFED gene cluster encodes the transporter necessary for the uptake of extracellular NO₃⁻. Both the membrane-spanning subunit NasE and the ATP-binding subunit NasD play essential roles in the acquisition of nitrate. The genes nasA and nasC encode the large and small subunits of assimilatory nitrate reductase (Nas), respectively, while the nasB gene is linked to nitrite reductase (Nir). This genetic arrangement allows K. oxytoca to efficiently assimilate nitrate (Wu & Stewart, 1998).
A biological system was developed for the uptake and reduction of NO3- into ammonium (NH4+) via a genetically introduced assimilatory reduction pathway (ANRA). The decision to introduce the ANRA pathway into V. natriegens is motivated by a key factor: the pathway is not naturally present in the organism, which restricts its ability to use NO3- as a source of nitrogen (He et al., 2021). By engineering the ANRA pathway, we aim to enhance its metabolic capabilities and enable it to thrive in nitrate-rich environments, such as coastal waters.
To further leverage this metabolic enhancement, the NH4+ produced from NO3- is then assimilated via the organism’s native GS-GOGAT and NADPH-dependent GDH pathways which enable glutamate (Glu) and glutamine (Gln) biosynthesis (Ohashi et al., 2011; van Heeswijk et al., 2013; Jiang & Jiao, 2016). This self-sustaining biological system supports accelerated growth, transforming V. natriegens into a powerhouse for the production of single-cell protein (SCPs). At the end of the organism’s life cycle, it would have accumulated a high protein content, making it a suitable alternative for livestock feed, reducing the demand for synthetically fertilised crop production.
## Selection of Genes and Genetic Parts
For the construction of this biological system, we selected three key components of the assimilatory nitrate reduction pathway: the nitrate transporter, nitrate reductase (Nas), and nitrite reductase (Nir). These proteins are encoded by a cluster of six genes derived from Klebsiella oxytoca (K. oxytoca) (strain: M5aI) (Wu and Stewart, 1998).
For the construction of this biological system, we selected three key components of the assimilatory nitrate reduction pathway: the nitrate transporter, nitrate reductase (Nas), and nitrite reductase (Nir). These proteins are encoded by a cluster of six genes derived from Klebsiella oxytoca (K. oxytoca) (strain: M5aI) as can be seen in Figure 4 (Wu and Stewart, 1998).
**Figure 4.** nasFEDCBA operon as seen in K. oxytoca. Sourced from Wu and Stewart, (1998).
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@@ -75,12 +75,12 @@ With these sequences obtained, codon optimisation was carried out in order to ad
In previous studies on the nasFEDCBA operon in *K. oxytoca*, RBS sites were found at the end of the coding sequence of each gene. However, these RBS were specific to *K. oxytoca*, which is why their efficacy in *V. natriegens* was uncertain. To ensure their proper functioning, RBS specific to V. natriegens were obtained from the Marburg Collection and inserted before the start codon of each gene.
Additionally, we sourced a native P1 promoter and a B0015 terminator from the Collection as well, which came together in the following sequence (Figure X.). The promoter and terminator were initially taken from Tschirhart et. al (2019) as they have previosly tested them, and have demonstrated high protein expression levels in *Vibrio natriegens.*
Additionally, we sourced a native P1 promoter and a B0015 terminator from the Collection as well, which came together in the following sequence (Figure 5.). The promoter and terminator were initially taken from Tschirhart et. al (2019) as they have previosly tested them, and have demonstrated high protein expression levels in *Vibrio natriegens.*
**Figure 5.** The sequence of genes, including the promoter, terminator, and RBSs selected from the Marburg Collection
To ensure the correct folding of the proteins encoded by the identified genes, their three-dimensional structures were simulated using AlphaFold2, an artificial intelligence tool with high accuracy of predicting protein structures based on primary sequences (Yang et al., 2023). Simulations were conducted using the AlphaFold2 Colab notebook. The amino acid sequences (represented in one-letter code) for each protein were entered into the AlphaFold2 pipeline as input. The predicted protein structures were then downloaded in PDB format and analysed using the molecular visualisation software ChimeraX-1.8. The resulting structures of the enzymes and the transporter are illustrated below.
To ensure the correct folding of the proteins encoded by the identified genes, their three-dimensional structures were simulated using AlphaFold2, an artificial intelligence tool with high accuracy of predicting protein structures based on primary sequences (Yang et al., 2023). Please refer to Figure 6. Simulations were conducted using the AlphaFold2 Colab notebook. The amino acid sequences (represented in one-letter code) for each protein were entered into the AlphaFold2 pipeline as input. The predicted protein structures were then downloaded in PDB format and analysed using the molecular visualisation software ChimeraX-1.8. The resulting structures of the enzymes and the transporter are illustrated below.
**Figure 6.** Proteins of interest. Simulated in AlphaFold2, visualised using ChimeraX-1.8.
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### Traditional Plasmid
For traditional plasmids with independent replication, multiple origins of replication (ORIs) were considered based on the research papers by Tschirhart et al. (2019), as well as Valenzuela-Ortega and French (2021), such as p15a, pBBR1, pJUMP26-1A, and pUC.
After thorough research on the subject, the team decided to opt for plasmid pSEVA261 with a p15a origin of replication due to its high maintenance, high stability, and low copy number in *Vibrio natriegens* (Tschirhart et al. 2019).
After thorough research on the subject, the team decided to opt for plasmid pSEVA261 with a p15a origin of replication due to its high maintenance, high stability, and low copy number in *Vibrio natriegens* (Tschirhart et al. 2019). The map for pSEVA261 can be seen in the figure below.
**Figure 7.** pSEVA261 plasmid. Image taken from SnapGene.
