This year, our team Nanjing-China is aimed at nitrogen fixation. Nitrogen fixation is the process that coverts free nitrogen into compound form.
It is generally known that free nitrogen makes up 78%, a large proportion, of the air. But the inertia of nitrogen makes it difficult to react with other substances. Besides, only when this indispensable element exists in compound form, can it be utilized easily by most of the living beings. Fixed nitrogen plays a crucial part in food supply around the world. Fixation of nitrogen in vitro, Haber-Bosch process in other words, requires massive use of fuels, for it will only occur in the presence of enough energy. In urgent need of it, plants and crops have been too dependent on the use, which turns out to be abuse nowadays, of fertilizers. All these overuse has led to harsh problems not only constraining the ecological development but also affecting the environment of the whole world harmfully. However, biological conversion of gaseous nitrogen to ammonia as a natural and spontaneous reaction in vivo allows us a brand new angle to look into, a more feasible way of nitrogen fixation.
Various ways are discovered to fix nitrogen in numerous strains. Because pathways that generate high-energy electrons are evolved to be in their metabolism system. What’s more, plants cannot fix nitrogen as they do to carbon for lack of an efficient enzyme system, nitrogenase. But some microorganisms do. Together, the energy in these electrons can be utilized by nitrogenase to convert free nitrogen into ammonia.
Nitrogenase is a huge system varied by the essential metallic co-enzyme, it requires such as Mo-Fe, V-Fe and Fe nitrogenase. Wherein, Mo-Fe nitrogenase is better understood than other types. Therefore, in our project, we choose the best known to perform the needed reaction.
Meanwhile, the gene clusters that coded nitrogenase are different in distinct species. It is reported by previous studies that there is a small nitrogen fixation gene cluster consisting of nine relative genes from Paenibacillus. It is quite simple and has proved functional after being transferred into E. coli cells, besides its expression does not lead to obvious negative feedback regulation.
As illustrated in Fig.5 above, nitrogen is fixed by Mo-Fe nitrogenase. Most of the nitrogenase is Mo-dependent, which exists mainly in bacteria and archaea. Such nitrogenase is made up of two components, Mo-Fe protein and Fe protein. At room temperature and atmosphere pressure, it costs at least 16mol ATP to reduce 1 mol nitrogen to ammonia by the nitrogenase. The process marches as high-energy electrons passed by Fe protein to Mo-Fe protein. After the binding to such electrons, Mo-Fe protein is able to reduce free nitrogen. However, the cost of the reaction is not so economical. In 2016, our team established a system to produce hydrogen driven by light which is considerably cost-effective. Inspired by our previous project, this year, we choose to alter ATP with solar energy.
There has always been an interest in harvesting the most important renewable energy source, the solar energy, which meanwhile is the hardest to capture. The significant breakthrough reported by Katherine et al. showed that certain semiconductors, cadmium sulfide (CdS) nanocrystals, function to photosensitize the Mo-Fe protein, replacing ATP hydrolysis by light harvesting to obtain electrons for the reduction of N2 into NH3. The results contributed to the development of our system. Lead-specific binding protein is applied to biosynthesize such semiconductors.
Herein, we aim to establish a sound and ideal whole-cell photocatalytic nitrogen fixation system consisting of the following elements: (i) a biocompatible and highly efficient light-harvesting inorganic semiconductor; (ii) active engineered E. coli cells as biocatalysts. The engineered E. coli cells, which express nitrogenase as well as have the capability of in situ biosynthesis of CdS nanocrystals for the existence of the surface-displayed heavy lead-specific binding proteins, is developed. Such system is able to reduce N2 to NH3 driven by light instead of ATP-hydrolysis with considerably high efficiency. The whole-cell system will be more biocompatible and cost-effective than any other ones.
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