First and foremost, as a set of genetic parts of biological methods, we believe that the most important application is to "provide tools" for other synthetic biology topics, that is, the end users should be mainly scholars of other synthetic biology and technological enterprises of synthetic biology. The set of gene tools that we have constructed can rapidly reduce the local oxygen concentration in the cell environment, which can better maintain the efficiency of reducing enzymes in cells, and may provide help or some feasible ideas for future research on synthetic biology.
There are a large number of reducing products in the current biological or chemical synthesis industry. Our intracellular hypoxia system may help existing biosynthetic routes to improve efficiency As a result, some products that can only rely on highly polluting chemical synthesis can achieve green and safe biosynthesis because they cannot complete industrial biosynthesis due to their high reducibility.
The biosynthesis of carotenoids, for example, consists of the biosynthesis of GGPP and the synthesis of carotenoids from GGPP to carotenoids, starting with the synthesis of octahydro-lycopene from GGPP catalyzed by PSY, on the basis of which other carotenoids are generated by further dehydrogenation and cyclization. The dehydrogenation process requires enzymes that are more efficient in a reducing environment. In particular, the biosynthesis of β-carotene often previously required the addition of antioxidants (e.g. Ethoxyquin) to the culture medium to ensure that the double bond structure of the compound is not damaged by oxygen during synthesis.
The introduction of our low-oxygen gene tool can save the cost of adding antioxidants and increase the production efficiency.
The current process of deep nitrogen removal in sewage treatment can be divided into physical and biological methods. The physical process is not commonly used in actual sewage treatment plants because of high operating costs and complex operation processes. The biological denitrification process, on the other hand, has been commonly applied in the deep denitrification of sewage treatment plants due to the advantages of high denitrification efficiency, low operation cost and simple operation process, among which nitrification-denitrification treatment using nitrifying bacteria and denitrifying bacteria is the most common in China. This method is divided into two stages of nitrification and denitrification, using nitrifying bacteria in the sewage to convert nitrogenous substances (including organic and inorganic nitrogen) into nitrate under aerobic conditions, and then using denitrifying bacteria in the sewage to reduce nitrate to gaseous nitrogen under anoxic conditions (dissolved oxygen <0.5 mg/L).
When both molecular oxygen and nitrate are present, denitrifying bacteria preferentially perform aerobic respiration. The key for microorganisms to change from aerobic to anaerobic respiration is the synthesis of enzymes for anaerobic respiration, and the presence of molecular oxygen inhibits the synthesis and activity of such enzymes. In order to ensure a smooth denitrification process, a strict anoxic state must be maintained. The metabolic water environment of denitrifying bacteria requires dissolved oxygen (0.2~0.5mg/L), in which the dissolved oxygen range, denitrifying bacteria are more active and metabolize faster. But the sewage field treatment process is generally anaerobic (dissolved oxygen below 0.2mg/L) - anoxic (0.2~0.5mg/L) - aerobic (2-4mg/L), denitrifying bacteria need to return from aerobic pool to anoxic pool, which will carry higher dissolved oxygen, which is not good for the metabolism of denitrifying bacteria.
If the low oxygen environment can be created in a specific denitrifying bacteria cell, then the denitrifying fine chrysanthemum will continue to function in the aerobic pond. So that the construction of the anoxic pond body will be eliminated, which will greatly reduce the investment cost of sewage plant construction and save floor space.
In addition, denitrifying bacteria are heterotrophic bacteria. In order to maintain their denitrogenation effect in the field of sewage treatment, a large amount of carbon sources (sodium acetate solution) need to be invested to ensure that they can degrade nitrate nitrogen (or nitrite nitrogen). If they can be genetically modified into autotrophic denitrogenation microorganisms, the input of carbon source agents in the sewage plant will be reduced, which is of great significance in the field of sewage treatment, and can reduce the operating cost of the sewage plant by 20-30%.
Nowadays, hydrogen is an important clean energy source and a widely used chemical feedstock, and how to produce hydrogen in an efficient and environmentally friendly way has become a key topic. However, these methods are generally costly, energy-intensive, and some of them also produce a lot of polluting by-products.
In 1939 and 1942, Gafforn and his co-workers first discovered that the alga Scenedesmus obliquus could both absorb hydrogen and fix CO2 under anaerobic conditions and emit hydrogen under light conditions, but only for a few seconds to a few minutes. Many subsequent studies have successively found that other green algae are also capable of hydrogen production, such as Chlamydomonas reinhardtii and Chlorellaf scal. Since these green algae can obtain hydrogen from the most abundant resources in nature (sunlight and water), and the hydrogenase activity in green algae is more than 100 times higher than that in photosynthetic bacteria and cyanobacteria. Therefore, in the 1998 International Energy Agency (IEA) assessment report, indirect photobiohydrolysis of hydrogen by reversible hydrogenases from green algae was described as one of the most promising methods for application.
The mechanism of hydrogen production by these organisms is the absorption of light energy in photosynthesis to produce electrons, which are later passed to hydrogenase (hydrogenase) to combine with hydrogen ions to produce hydrogen gas. However, at a certain concentration of oxygen, the hydrogenase activity is significantly inhibited, making sustained hydrogen production impossible. This "oxygen paradox" has been a pressing problem for hydrogen production.
Since our project is a genetic device (even a system may be formed in the future), we hope that this device can be applied to the research of relevant scientific principles or enterprise production requiring intracellular hypoxia environment. We have made contact with relevant experts, scholars and enterprises, and confirmed that "oxygen hunter" has great prospects in a wide range of fields such as biological carbon fixation, biological hydrogen production, biological carbon fixation, synthetic reducing drugs and biological control of water bodies. In the future, after the introduction of vitreous hemoglobin (which can increase the oxygen in cells), our project can evolve into an "oxygen controller" to control the oxygen concentration of cells to various levels.
We hope that after the introduction of hypoxia devices into cells, the oxygen concentration near the active sites of biochemical reaction enzymes related to hydrogen production can be reduced regionally and directionally, so as to alleviate the "oxygen paradox" and achieve efficient large-scale production of biological hydrogen production.
Biological carbon fixation is to convert inorganic carbon, that is fixing carbon dioxide in the atmosphere into organic carbon as carbohydrate, and fix it in plants or soil. There are two common biological carbon fixation methods: photosynthesis, such as various green plants and photosynthetic autotrophic microorganisms (such as cyanobacteria); Chemical energy synthesis, for example, nitrobacteria use ammonia oxide to synthesize organic substances.
The reductive tricarboxylic acid cycle is mainly found in photosynthetic green sulfur bacteria and anaerobic bacteria. One carboxylase called 2-Oxoglutarate synthase , is a strictly anaerobic enzyme whose working activity is influenced by oxygen concentration.
Since the reaction steps of biological carbon sequestration are many and the reaction efficiency is low, in order to improve the carbon sequestration efficiency, scientists want to design artificial carbon sequestration pathways with fewer steps. With the rapid development of synthetic biology, the newly designed biological carbon fixation enzymes can partially solve the defects of natural carbon fixation enzymes and make the artificial biological carbon fixation pathway significantly more efficient, which has a broad development potential and is the current hot spot of synthetic biology research.
However, the artificially designed carbon fixation process needs to be insensitive to oxygen. Many studies start with protein modification to make it insensitive, but if our low oxygen system can be introduced, we can consider using more natural carbon fixation pathways in organisms to improve.
Nitrogen plays an important role in agricultural production, contributing 50% to the final yield of crops. Biological nitrogen fixation accounts for 60% of the global total. Therefore, it is most promising to replace chemical nitrogen fertilizer as the main nitrogen source of food crops. It is the most potential direction in the field of sustainable agricultural development to achieve green and safe production.
Reference: Andrés R. Schwember, Joachim Schulze, Alejandro del Pozo and Ricardo A. Cabez. Regulation of Symbiotic Nitrogen Fixation in Legume Root Nodules.(2019)Plants.
In 2021, the amount of chemical fertilizer applied per hectare of crops in China will reach 506.11 kg/hectare, 2.05 times that of Britain and 3.69 times that of the United States, far higher than that of developed countries in the world.
The mechanism of biological nitrogen fixation is a unique physiological function of nitrogen fixing microorganisms under specific conditions (such as anaerobic or microaerobic, ammonium free or low ammonium, etc.), under the catalysis of nitrogenase, to directly use N2 molecules in the atmosphere. Leguminous plants and rhizobia are the most important biological nitrogen fixation system in nature. Their outstanding feature is that they can form nodules or nodule like structures, and their nitrogen fixation efficiency is very high. The reason is that they solve the "oxygen paradox" in the process of biological nitrogen fixation, and guarantee the efficiency of aerobic respiration while protecting nitrogenase through leghemoglobin.
If leghemoglobin is introduced into other biological nitrogen fixation systems or some artificial nitrogen fixation systems, nitrogen fixation efficiency can be greatly improved.
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