Part:BBa_K5306006

ANRA Pathway

This composite part encodes for the whole assimilatory nitrate reduction to ammonium (ANRA) pathway.

Usage and Biology

Nitrate assimilation in bacteria is a process by which the organism uses NO₃^-, and NO₂^-, as nitrogen sources and convert it into ammonia (NH4+). Nitrates are transported in the intracellular space by a transporter, where they are converted through nitrites into ammonium (Lin et al., 1994). Our team has selected this pathway as it provides an effective means of nitrogen acquisition and assimilation, as well as avoiding the production of harmful byproducts that can harm the microorganism.

Vibrio natriegens is a fast-growing marine organism, that thrives in a high salinity environement, with a doubling rate of less than ten minutes, making it a potential chasiss for molecular and synthetic biology applications (Weinstock et al., 2016, Stukenberg et al., 2021). Natronaut has chosen to integrate the nitrate assimilation pathway in this organism in order to adress the problem of coastal eutrophication by capturing ran-off NO3-.

Composition

This composite part encodes for the three main enzymes involved in the ANRA Pathway:

1. Transporter (nasFED complex): This is an ABC (ATP-Binding Casette) enzyme located in the cytoplasmic membrane of the bacteria responsible for the uptake of nitrates (Wu & Stewart, 1998). It is composed of three subunits: a periplasmic binding protein that binds to extracellular NO₃^-, with high affinity, a transmembrane protein that allows the passage of nitrate through the lipid bilayer and a cytoplasmic ATP-binding protein (Wu & Stewart, 1998).
2. Nitrate Reductase (nasCA complex): The nitrate reductase is an NADH-depepndent enzyme that catalyzes the conversion of NO₃^-, once it has entered the cell to NO₂^-, (Lin et al., 1993, Lin et al., 1994). It has a large catalytic subunit that contains the active site for reduction and a small NADH oxidoreductase subunit, used for transfer of electrons through the biochemical reactions(Coelho & Romão, 2015). The active site is accompanied by a molybdenum-molybdoprotein responsible for catalyzing the reduction reaction(Coelho & Romão, 2015).
3. Nitrite Reductase (nasB): Nitrate reductase is the enzyme responsible for carrying out the last step of the pathway. Being a cytochrome enzyme, it facilitates the conversion of NO₂^-, into NH₄⁺, through different heme groups, where each one converts one molecule at a time (Einsle et al., 2002). The sequence encoding nasB contains a 6x His tag to be able to locate the protein and confirm the introduction of this pathway in the organism.

Overview of the assimilatory nitrate reductase pathway enzyme complexes (Wu & Stewart, 1998)

In our project, the converted NH₄⁺ is subsequently assimilated for the production of single-cell proteins (SCPs) through glutamate biosynthesis. This process occurs through two pathways: glutamine synthetase (GS)-glutamate synthase (GOGAT) and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutamate dehydrogenase (Ohashi et al., 2011; van Heeswijk et al., 2013; Jiang & Jiao, 2016).

The assimilatory nitrate to ammonium (ANRA) pathway and the Glutamate Biosynthesis for the production of single-cell proteins (SCPs) (Created with Biorender)

Operon Structure

According to Lin et al. (1994) the genes encoding the three enzyme involved in nitrate assimilation, function as an operon. They form one long molecule of mRNA that gets translated into multiple proteins. Therefore, we have chosen to integrate the whole operon sequence into the plasmid backbone under the control of only one promoter and a terminator. This allows for the coordinated expression of all three enzymes in a single messenger RNA, facilitating appropiate protein-protein interaction and communication within the pathway. By using only one promoter and terminator, we simplify the regulation of the gene expression. As it is an operon, we inserted an RBS sequence, native to V. natriegens, upstream of each gene to simulate native conditions.

Operon structure and size (Lin et. al, 1994)

The coding sequences of the three enzymes: transporter, nitrate/nitrite reductase have been obtained from the organism Klebsiella Oxytoca M5al by Wu & Stewart (1998) and has undergone codon optimization for Vibrio natriegens .

Assembly Design & Results

To ensure a succesful expression, our team has selected a strong promoter that has been found to be native to our chasiss organism Vibrio natriegens from iGEM Groningen 2019 team (BBa_K3171171). This promoter has been reported to induce consitutive transcription of our target gene and has been documented by Tschart (2019) to induce high gene expression levels. Moreover, Tschart (2019) has also tested the double terminator (B0015) confirming its use in V. natriegens .

We have also selected three RBS sequences synthesized by Marburg 2018 VibriGens that are similar in strength ( BBa_K2560008, BBa_K2560010, BBa_K2560016to acquire optimal translation efficiency for the expression of our target genes. These sequences were chosen to balance protein production rates and to avoid overwhelming the cell.

Due to the sheer size of the construct (10174 bp), a vector with a low-copy number was chosen to minimize the metabolic burden on the host cell. Therefore, the backbone that was used, pSEVA261, is a plasmid with a p15A origin of replication, that has been found by Tschart (2019) to have a high-transformation efficiency and high-maintanance.

For the purpose of this project, our team used Gibson Assembly as this method would be the most effective approach, since the fragments have a substantial length (2000 bp- 2900 bp). The construct was assembled into the pSEVA261 vector using the NEBuilder HiFi DNA Assembly by NEB and further cloned into the NEB® 10-beta Competent E. coli cells.

Characterization

To confirm the inital assembly, restriction enzymes digestion reactions were performed on the isolated plasmid. We have selected the NaeI and XhoI enzymes present in our construct and we simulated the gel electrophoresis expected results in SnapGene. After running the PCR we ran a 1% agarose gel and found that the results match the expectations confirming the succesful asembly of the construct.

Considerations

Due to time constraints, our team was unable to fully characterize the enzymatic activity of the pathway in Vibrio natriegens. However, we have developed a detailed plan for future analysis:

• Western-blot to localize the pathway: As we have added a 6x HIS tag on the nitrite reductase enzyme, and since it is a key component of the ANRA pathway, we will be able to determine not only its location but also where the other components are located.

Chemical tests:

• Griess Test: The purpose of this test is to test the enzymatic activity of the nitrate reductase protein.
• Indophenol Blue Test: This test is used to detect the presence of ammonia by exploiting the electrophilicity of the ammonium ion, subsequently confirming the enzymatic activity of the nitrite reductase.

From these experiments, the concentrations of the detected compounds can be measured by looking at the absorbance levels and using Beer Lambert’s Law.