Our project originates from the idea of biological hydrogen production and biological nitrogen fixation. We found that hydrogen producing enzymes and nitrogenase can be inactivated by combining with oxygen, but if the oxygen concentration is too low, the metabolism of microorganisms will be affected, which is the "oxygen paradox".
In response to this paradox, we propose to build a system that can build an intracellular hypoxic environment while maintaining normal cell metabolism, named Oxygen Hunter.
In order to achieve the two conditions of sufficient oxygen for cell supply and respiration and low oxygen concentration in the cytoplasm, we consulted the relevant literature and learned about various methods to reduce oxygen concentration, while also maintaining the normal growth of cells. The following design was carried out
We choose leghemoglobin to express near the cell respiration point. Leghemoglobin can combine with oxygen to enrich oxygen near the respiration point, promote respiration, and realize anaerobic environment in the cell.
In addition, we have also selected laccase (multi copper oxidase) to construct a hypoxic environment. It can catalyze the reaction of oxygen with a variety of reducing substrates, thereby converting it into water and reducing the oxygen concentration in the solution. The extracellular oxygen can contact with the cell membrane for respiratory purposes, so it will not affect cell growth.
The mechanisms of the two strategies are different, but they can complement each other. We decided to compare the effects of the two function modules, measure the amount of expression and optimize the results with mathematical models.
In order to detect the intracellular oxygen concentration, we selected the anaerobic promoter nirB, the element ArcA that is negatively related to the oxygen consumption rate, to connect the green fluorescent protein, which is used to characterize the strength of the promoter through the fluorescence intensity, thus obtaining the oxygen concentration and oxygen consumption rate in the system we built, and providing data for the model. Finally, we combine the formed detection module with the function module to get a complete system that can construct intracellular hypoxia and know the degree of hypoxia at the same time.
We hope to introduce application components for production. Our project can be applied to the production of reducing products, biological combined nitrogen fixation to promote production, biological hydrogen production, reduction of carbon dioxide, etc.
Our results show that our project can achieve anaerobic enzyme reaction production in cells at normal oxygen concentration while maintaining normal metabolic growth for continuous production in a stepwise manner. With this approach we expect to resolve the inhibition of reductive enzyme production by the oxygen paradox and construct systems for promoting production.