In order to solve the "oxygen paradox" in many biochemical reactions, we found leghemoglobin and laccase, and introduced their genes into the target strain through appropriate processing. It can enable Escherichia coli to maintain the cell environment with low oxygen concentration, and allow some oxygen to be transported to the position required for aerobic metabolism to provide energy for cells, while other physiological processes of high reducing biochemical reactions in cells that require low oxygen environment can not be affected. However, globins with different binding abilities or promoters with different strengths can achieve different levels of hypoxia concentration, and finally can complete a set of gene elements that meet the needs of different hypoxia concentrations.
In the brainstorming part of finding topics, we once had the idea of conducting research on biological hydrogen production, biological nitrogen fixation and other related research. However, due to the feasibility, public attitude, expert advice and other reasons, we did not choose these topics. (Click here to know more) However, when the brainstorming came here, different students' ideas collided with sparks.
In the process of symbiotic nitrogen fixation, the mechanism that rhizobia use leghemoglobin to complete nitrogen fixation is that leghemoglobin combines with oxygen to transport to specific areas of aerobic metabolism to generate energy. This is equivalent to that rhizobia uses a very efficient globin (leghemoglobin is also a globin), which not only ensures the hypoxia concentration in cells, but also does not weaken or even strengthen their respiratory metabolism[1].
In a document on biological hydrogen production, it was also mentioned that the introduction of leghemoglobin into Chlamydomonas reinhardtii detected a decrease in intracellular oxygen concentration and an increase in hydrogen production efficiency, which reflected our idea[2].
In addition to leghemoglobin, other globins that are highly efficient in combining with oxygen should also be able to achieve similar effects. The production of reductive products and reductive biochemical reactions in other organisms may also need to overcome the "oxygen paradox".
In the experiment, we used leghemoglobin and laccase to reduce the oxygen concentration in cells.
leghemoglobin
laccase
Leghemoglobin, which exists in soybean root nodules in nature, is a protein composed of 144 amino acids. We integrated the leghemoglobin gene fragment into the plasmid pET-28a (+) with lactose operon and introduced it into E. coli cells for recombinant expression.
Laccase is a kind of oxidoreductase which widely exists in microorganisms, animals and plants. Its basic principle is to catalyze the reaction of reducing substrate and oxygen, directly consume oxygen, and thus build a hypoxic environment in cells. We are going to choose CueO, a multi copper oxidase contained in Escherichia coli, as laccase.
First, we transferred the leghemoglobin plasmid and laccase plasmid with nirB detection plasmid into E. coli BL-21 to induce expression. Regularly detect the fluorescence intensity and OD value reflecting the strain concentration, draw a curve for analysis and modeling.
After that, laccase plasmid, leghemoglobin plasmid and nirB detection plasmid were simultaneously introduced to express in Escherichia coli BL-21, SDS-PAGE was performed, fluorescence intensity and OD value were detected regularly, and curves were drawn for analysis and modeling.
The final experimental results show that:
Detection module: The nirB promoter can be normally expressed in anaerobic medium and has no effect on cell growth, which proves that the detection module can play a role. The activity of Icd promoter was negatively correlated with cell oxygen consumption rate.
Function module: Compare the fluorescence intensity of the plasmid introduced into the function module with that of the plasmid introduced into the detection module. It can be seen that leghemoglobin and laccase play a role. The effect of leghemoglobin is more obvious. By observing the results of SDS-PAGE, the expression of leghemoglobin was more. Compared with the use of laccase or leghemoglobin alone, the fluorescence intensity shows that the effect is best when laccase and leghemoglobin are added at the same time.
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.
[1] Casey Van Stappen, Laure Decamps et al. The Spectroscopy of Nitrogenases. (2020,120) Chem. Rev. 5005-5081
[2] Wu, S., Yan, G., Xu, L., Wang, Q., & Liu, X. (2010). Improvement of hydrogen production with expression of lba gene in chloroplast of Chlamydomonas reinhardtii. International Journal of Hydrogen Energy, 35(24), 13419-13426.