16: Fixing Nitrogen Fixation

Nitrogen Fixation

We spent most of Chapter 22.1 discussing the biochemistry of nitrogenase which fixes the stable molecule N₂ to form NH₃/NH₄⁺.  It's a very complicated reaction conducted by symbiotic microbes (prokaryotes) that fix N₂ for plants.  The world uses the Haber-Bosch process to produce over 100 million metric tons of nitrogen fertilizer that supports half of the world's population's food supply. Only about half of the ammonium added to soil is taken up by plants.  The rest is released into waterways or used by microbes, which can produce from it the potent greenhouse gas nitrous oxide (N₂O).  It has a 300x greater effect than CO₂ based on weight. The oxidation number of N in NH₃  is -3 and in N₂O it is +1 showing that the NH₃ can be oxidatively metabolized for energy production by the microbes. Also, excess NH₄⁺ goes into waterways and leads to eutrophication, the overproduction of algae and plankton, which depletes O₂ from the waters and kills other organisms.  

N₂O emissions have increased dramatically since 1850, as shown in the interactive graph from Our World in Data in Figure \(\PageIndex{9}\) below.

Figure \(\PageIndex{9}\):  Our World in Data. https://ourworldindata.org/grapher/n...ions?tab=chart

So what can we do to "fix" nitrogen fixation to reduce our reliance on the Haber process and its collateral climate effects?  Perhaps we could use mutagenesis to make nitrogenase a better and more efficient enzyme.  That would prove very difficult given the complexity of both the enzyme and the mechanism of N₂ conversion to NH₃ that requires many metal ion cofactors.  A better alternative would be to express nitrogenase in plants so they could synthesize their own nitrogen fertilizer!

Genes of Nitrogen Fixation

The catalytic nitrogenase enzyme complex is encoded by three genes:

• nifH gene for the subunit Nitrogenase iron protein 1 (also called Nitrogenase component II, and Nitrogenase reductase).  It is a homodimer that binds one [4Fe-4S] cluster per dimer.
• nifD gene for the subunit Nitrogenase molybdenum-iron protein alpha chain (also called Dinitrogenase and Nitrogenase component I).  It catalyzes the key enzymatic reactions along with its partner nifK as part of a heterodimer. It binds one [8Fe-7S] cluster per heterodimer with nifK and binds 1 [7Fe-Mo-9S-C-homocitryl] cluster per subunit.
• nifK gene for the subunit Nitrogenase molybdenum-iron protein beta chain (also called Dinitrogenase and Nitrogenase component I).  With its partner nifD, it catalyzes the key enzymatic reactions as part of a heterodimer with nifD.  It binds one [8Fe-7S] cluster per heterodimer with nifD and binds 1 [7Fe-Mo-9S-C-homocitryl] cluster per subunit.

The nitrogenase enzyme complex has regulatory proteins as well:

• nifA - Nif-specific regulatory protein required for activation of most nif operons.  It senses N₂.  If there is insufficient quantities of N₂, the protein NtrC activates NifA expression which activates the rest of the genes.
• nifB - FeMo cofactor biosynthesis protein NifB (also called FeMo-cofactor maturase NifB, Nitrogenase cofactor maturase NifB, and radical SAM assemblase NifB).  
• nifL - Nitrogen fixation regulatory protein required for the inhibition of NifA activity (i.e. nitrogenase formation) in response to oxygen and low level of fixed nitrogen.
• nifE - Periplasmic [NiFe] hydrogenase small subunit.
• NifM - a possible peptidyl prolyl cis‐trans isomerase (i.e. a protein chaperone) which helps in the folding of NifH.

Figure \(\PageIndex{10}\) below shows a summary of these gene products.

Figure \(\PageIndex{10}\): Minimum set of nif genes essential for nitrogen fixation with molybdenum-iron nitrogenase.  EMILY M. BENNETT et al., BIODESIGN RESEARCH.  10 Jan 2023, Vol 5, https://spj.science.org/doi/10.34133/bdr.0005 .  Creative Commons Attribution License 4.0 (CC BY 4.0).https://doi.org/10.34133/bdr.0005

The stoichiometry depicted has not been adjusted. NifB contains one catalytic cluster (shown in white) and 2 substrate [4Fe-4S] clusters that react to produce the NifB cofactor. NifEN matures the NifB cofactor producing the FeMo cofactor. The molybdenum-iron (MoFe) nitrogenase (NifHDK) contains the FeMo cofactor at its active site. Electron donors transfer single electrons to the [4Fe-4S] cluster at the interface of the NifH homodimer. Electrons are moved from the [4Fe-4S] cluster into the active site of nitrogenase using energy produced by ATP hydrolysis by NifH. A minimum of 8 electrons are used to reduce each molecule of N₂.

It would be especially important to express nitrogenase in the main cereal food crops (rice, corn, and wheat) which get their nitrogen from soil microbes (in contrast to legumes, which contain nitrogen-fixing bacteria in nodules in their roots). It's a daunting task given the complexity of the protein complex, its metal cofactors, their inhibition by O₂, and the multiple genes required for nitrogenase regulation. Ideally, the relevant gene clusters could be moved into a chlorplast which is evolutionarily derived from bacteria so the gene regulation system might be more suitable. It also has low O₂ levels at night.  However, O₂ is produced in chloroplasts, which is problematic given the sensitivity of nitrogenase to O2.

Using synthetic biology, Saccharomycescerevisiae has been engineered to express the NifDK nitrogenase tetrameric protein in their mitochondria (after post-translational import).  Yeast is a model organism and tools have been developed for synthetic biology experiments using yeast, so much can be learned that could apply to other eukaryotic organisms like plants.  Mitochondria have high O₂ consumption (as opposed to production as in the chloroplast) and the ability to synthesize bacterial-type iron–sulfur clusters.  The Nif gene clusters were engineered into the XV chromosome as shown in Figure \(\PageIndex{11}\) below.  

Figure \(\PageIndex{11}\): nif gene assembly in yeast. Buren et al., ACS Synth. Biol. 2017, 6, 6, 1043–1055. https://doi.org/10.1021/acssynbio.6b00371.  CC-BY license

Panel (a) shows the assembly strategy for transcription units, subclusters, and full clusters inserted by homologous recombination in the genome of S. cerevisiae. Panel (d) shows a disgram of nif gene organization in DSN14, a strain of S. cerevisiae.

Nitrogenase activity has also been functionally expressed in transgenic rice containing the NifH with a [4Fe-4S] cluster from Hydrogenobacter thermophilus  and NifM (a peptidyl prolyl cis‐trans isomerase from Azotobacter vinelandii) which helps NifH fold.  They were correctly targeted to the mitochondria which again minimizes O₂ oxidative damage to the metal ion cofactors. The purified protein was able to transfer electrons to the MoFe protein (NfiDK dimer) but did so poorly.  It also assisted in the assembly of the FeMo cofactor.  However, the [4Fe-4S] cluster occupancy in the protein was poor. However purified protein was also able to reduce acetylene, HC=CH (an alternative substrate similar to N=N) after the addition of purified NifDK. 

Many steps have to be optimized to create a functional nitrogenase in plants like rice, corn, and wheat.  For example, mitochondrial-expressed NifD is readily cleaved by a mitochondria endoprotease. Some NifD subunits are more resistant to proteolysis and a single amino acid change (Y100Q) leads to enhanced stability on the protein.  AI will likely be extremely useful in maximizing the expression of nitrogen in crop plants.

Synthetic Biology to Express Nitrogenase in Bacteria

Bacteria can express nitrogenase that can fix atmospheric N₂ but they won't work with the critical cereal crops unless the bacteria can interact with roots in the "rhizosphere", the layers of dirt intimately in contact with roots. This life in this area is often called the holobiont, which consists of the plant host and all species interacting with it in a symbiotic relationship.  The metabolism in the holobiont is complex.  For example, plant carbohydrates are used by other organisms in the holobiont. It's similar in a way to the gut biome, which consists of an ecosystem of human and microbial cells.

Bacteria have now been engineered that express nitrogenase AND interact with corn roots to fix N₂.  The cells are derivatives of γ-proteobacterium (KV137), found on corns roots and which can fix N₂.  They have been engineered to turn nitrogenase genes on when N₂ fixation is needed.  The engineered bacteria is added to liquid fertilizer, reducing the need for chemical fertilizer by 25 lb/acre) and at the same time increasing yields.  This bacterial-based fertilizer does not wash into waterways with its concomitant negative environmental effects.  Likewise, no N₂O is produced on microbial metabolism of excess fertilizers, which decreases the release of this potent greenhouse gas. In 2021 it was used on 3 million acres of corn.

One problem with the use of biological N₂ microbes in agricultural settings is that high levels of chemical fertilizers can effectively inhibit microbial N₂ fixation, which is regulated (as mentioned above) to shut down if nitrogen is bioavailable.  Still, bacteria that fix N₂ can produce up to 10% of the nitrogen requirement.  Genetic engineering is needed to overcome this inhibition.  Such bacteria are diazotrophs as they can fix N₂ and grow without exogenous sources of N₂. Rhizobia are one example. which can fix N₂ in the nodules of legumes. The diazotroph isolated from corn roots and mentioned above, Kv137, was gene-edited to produce a modified strain (Kv137-1036) that fixes N₂ without inhibition by applied nitrogen fertilizers.

The Kv137 strain has nifA and nifL genes and proteins which, as described above, regulate the expression of nitrogenase based on the need for nitrogen. These two genes are on one operon under the control of a single promoter.  nifL was replaced with another promoter which removes the down-regulation of nifA since no nifL was present.  This allowed the expression and activity of nitrogen even in the presence of exogenous fertilizer. 

Figure \(\PageIndex{12}\) below shows that Kv137-1036 strain (red dots) does colonize corn roots.

Figure \(\PageIndex{12}\): Commercial efficacy of strain Kv137-1036 - Colonization of corn roots by microbes (red) after germination.  Wen et al., Enabling Biological Nitrogen Fixation for Cereal Crops in Fertilized Fields.  ACS Synth. Biol. 2021, 10, 12, 3264–3277. December 2021.  https://doi.org/10.1021/acssynbio.1c00049.  Attribution 4.0 International (CC BY 4.0)