Using N 2 as a final electron receptor in yeast to produce NH 3 in bioreactors

This research proposal aims to replacement of N molecule (instead of O ) as a final electron receptor in micro-organisms so that they can produce NH , which is important industrially, instead of H O. This idea is based on the hypothesis that replacement of the central atom in gas-carrying compounds in living organisms such as chlorophyll, heme and hemocyanin would replace the fixed gas. However, NH is an expensive product because it is produced using Haber-Bosch reaction technique which requires high temperature and pressure. Therefore, it is predictable here to produce NH so simply in bioreactors which is a great economic benefit.

Great scientific achievement: If our purpose succeeded, our new yeast strain, would be the first of its type ever; we would have an organism that "breath" N instead of O for the first time on Earth.

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Our new yeast strain would start new scientific horizons in terms of evolution, microbiology, adaptive mutations and life itself.

Introduction/Background
Photosynthesis and respiration are two vital processes that are the prerequisites for all life on Earth.The two pigments responsible for these processes are chlorophyll and heme respectively.The so-similarity between chlorophyll and heme has been a mysterious subject of research by some researches for a long (Vernon and Seely 1966); they are identical in structure except for the central atom: magnesium in chlorophyll and iron in heme (Bryson and Vogel 1965).Chlorophyll has been proven to have the ability to turn into hemoglobin (Hughes andLanter 1936, Kohler et al. 1939).
O is fixed by living organisms to be used as a final electron receptor, to be turned into H O. If we managed to replace N by O in yeast (because yeast contains heme) and green bacteria, we are going to get NH , and that is a real economic benefit.NH (ammonia or azane gas) is an important product; it has a number of vital uses such as fertilizers, cleaners, fermentation industry and antimicrobial agent in food products.NH is currently produced using Haber-Bosch reaction technique which requires high temperature (400-500°C) and pressure (150-250 bar) that is why it is so expensive, so the production of ammonia gas in our proposed way would be a great economic benefit.
Heme is the compound that forms, with some proteins, hemoglobin; the compound that exists in red blood cells in most animals.It has the ability to deliver O and CO .Its chemical structure is C H N Fe.Hemocyanin is the compound that delivers O and CO in mollusk and arthropod species the same way hemin does, but less effectively.Its chemical structure is C H N Cu.Chlorophyll is the compound that fixes O and CO in plants.It also absorbs sun light's energy necessary for photosynthesis.Its chemical structure is C H N Mg.Hemin has a semi-similarity with other compounds such as cobalamin (vitamin B ) which has Co atom as a central atom in its structure.

Research Approach and Methodology
Accurately, gases do not bind heme, as a whole, they bind only the central atom (iron) within it.This makes a sense that if we managed to change the central atom in heme, the fixed gas would be changed.When we have a breath in our lungs, only oxygen binds hemoglobin, not because oxygen is more suitable than other gases, but because of the central atom in heme, iron, which interacts with oxygen naturally.
Iron exists naturally in the form of oxides i.e.FeO, Fe O , Fe O .Copper, in hemocyanin, exists in nature in the form of oxides too i.e.Cu O CuO, CuO , Cu O .Magnesium, in chlorophyll interacts with oxygen and carbon dioxide easily too.Therefore, our hypothesis is "Changing the central atom in hemoglobin, hemocyanin and chlorophyll, changes the fixed gas by them".Although it is possible to replace the central atom in heme and chlorophyll, it looks greatly difficult to make specific genetic manipulations to reach the target of our proposal.Therefore, our goal can be possible if we follow an adaptive mutation technique.Adaptive mutation states that rather than mutations and evolution being random, they are responsive to certain stresses.In other words, the mutations which happen are more beneficial and specific to the given stress, neither random nor a response to anything in particular.The term stress refers to any alteration in the environment, such as temperature, nutrients, population size, etc. Tests with microorganisms have found that for adaptive mutation, more of the mutations remarked after a given stress were found to be more efficient at dealing with the stress than chance alone would suggest is possible (Foster 1993, Sniegowski andLenski 1995).Adaptive mutation is a very controversial topic so there have been many experiments to prove or discredit the theory.Three key examinations are the SOS response (McKenzie 2000), responses to starvation in Escherichia coli (Foster 2000) and testing for revertants of a tryptophan auxotroph of Saccharomyces cerevisiae (Foster 2000).

Proposed experiment
Model of research: • The micro-organism(s) that we are going to use in our experiment must have two properties: 1.
Obligate aerobic: Because if a facultative aerobic micro-organism was used, it is highly probable to simply turn into anaerobic pathway when aerobic conditions are not suitable to it.2.
• The advantages of using a yeast strain over using a bacterium strain as a model of research are: 1.
Ferrochelatase adds ferrous ion as a terminal step in heme biosynthesis, while magnesium chelatase adds magnesium ion as a first step in bacteriochlorophyll synthesis; therefore, if any modification was made in magnesium chelatase enzyme, it may be required to be followed by further modifications in the enzymes that synthesize bacteriochlorophyll which is more difficult.It is much easier to modify the top of a pyramid than modifying its base.

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The biosynthesis of heme looks simpler than the biosynthesis of bacteriochlorophyll.Besides, the structure of ferrochelatase is simple and its molecular weight is low relative to magnesium chelatase.
On the other hand, mutation rate in prokaryotes, in general, is much higher than mutation rate in eukaryotes which is a disadvantage of using a yeast strain over using a bacterium strain as a model of study in this proposal.Therefore, it is proposed here to use a yeast strain and a bacterium strain at the same time to avoid the need to use any mutants that may induce unpredictable, undesired mutations.Torulopsis psychrophile, T. austromarina, L. gelidum, and L. nivalis are examples for obligate aerobic yeasts (Anonymous 2023), so they are candidates to be models of study.
Fe , Cu and Co , are naturally selected by ferrochelatase, all lie in the fourth group of the periodic table.They are all bivalent, and their electronegativity ranges from 1.83 to 1.90.Besides, they are all reactive to O .Therefore, if we are to replace ferrous ions at the hemin of yeast, we should focus on an element that is: Have in-range or close electronegativity.
As previously mentioned, Zn is naturally selected by ferrochelatase (causing a disease in humans).Its electronegativity is 1.65, and it is reactive to N forming Zn N .Thus, Zn looks to be perfect to be our first candidate to be replaced by ferrous ions in hemin.The electronegativity of Ni is 1.91. it is reactive to N (forming NiN), so it likes to be a good fit to be our second candidate.At the third place, Mn comes.Its electronegativity equals 1.55.It is also reactive to N forming Mn N , Mn n and MnN .

Proposed experiment steps:
• In a lab fermenter, Torulopsis psychrophile, for instance, should be grown on a suitable medium with the replacement of ferrous with zinc under normal aerobic conditions.It is predictable here that Zn-heme will be synthesized in the yeast cells.• pH value of the medium may be needed to be modified several times because to G. A. Hunter (August 29, 2008), ferrochelatase selectivity is pH-dependent.• N gas should be replaced by the same volume of O gas gradually as a trial to induce an adaptive mutation.• N is less reactive than O (bond energy of oxygen equals 119 k.cal/mole, but equals 226 k.cal/mole for nitrogen), so temperature may be needed to be increased.As a result, the yeast strain to be chosen as a model of research should be thermophilic or superthermophilic.

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Test the aerobic environment of the bioreactor for ammonia gas periodically.If found, we have reached our goal.

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Mutants may be needed to be used.
While some researchers managed to replace the central atom in chlorophyll with copper and zinc in vitro(Petrovic et al. 2006), no attempts have been made to replace the central atom either in chlorophyll nor in heme in living organism for two reasons, to the author's point of view.They are: Firstly, hemin and chlorophyll are synthesized through a set of bioreactions, so if you need to do a specific modification in heme, for instance, you will need to make a number of "specific" genetic alterations or mutations in yeast, for instance, which sounds to be impossible.Secondly, if you replaced the central atom in heme with Ca in vitro, obtained Ca-Heme and managed to replace it with the heme of yeast (using microinjection technique), your new trait wouldn't be inherited to next generations because of ferrochelatase enzyme which is selective to Fe ions(Ogun et al. 2023).
Author-format, Bollivar 2006eviewed document posted on 01/04/2024.DOI: https://doi.org/10.3897/arphapreprints.e124141Ferrochelatase (or protoporphyrin ferrochelatase) is an enzyme which is encoded by the FECH gene in humans(Anonymous In Press).It catalyses the terminal (8 ) step in the biosynthesis of heme, turning protoporphyrin IX into heme B. The interesting fact (to this proposal) is that ferrochelatase can also insert other divalent metal ions into protoporphyrin.Some ions such as Zn , Ni and Co form other metalloporphyrins ( Medlock 2009).Zinc protoporphyrin (ZPP) is a compound found in red blood cells when heme production is inhibited by lead and/or by lack of iron(Labbe et al. 1999).Substrate inhibition happens in the order Cu > Zn > Co > Ni and can be alleviated by supplementation of metal complexing agents such as β-mercaptoethanol or imidazole to the reaction buffer(Hunter et al. 2008).Magnesium-chelatase is a three-component enzyme that catalyses the insertion of Mg into protoporphyrin IX.This is the first unique step in the synthesis of chlorophyll and bacteriochlorophyll(Willows 2003, Bollivar 2006).