Team:USTC/Description
Background
Problems need to be solved
As science and technology develop so fast, manufacturing has became a mature business. Lots of different things can be synthesized by us human beings! Even something we can’t imagine 100 years ago.
However, nothing is perfect. We want things to become better and better, of course. And there are many troubles in the manufacturing! For example, there will be a lot of pollution if we synthesize something with a chemical method. In addition, chemical methods are lack of specificity, which means it will take a lot of troublesome work to purify the compound we want.
How to solve?
So people now begin to turn their attention to a new and hot research field——Microbial ElectroSynthesis(MES). MES entails the application of microorganism to the cathode of an electrochemical cell, in which those microbes can catalyze the process of fermentation or production of fuels and chemicals.
Figure 1. General structure of MES
Figure 2. A simplified scheme of a bio-cathode
MES can synthesize compounds, especially some biological products, in a more environmental-friendly way. This is what we gravely want as fossil fuel is so polluting and is about to run out. With MES, we can use a much cleaner energy--electricity, to synthesize carbohydrate even just from carbon dioxide. Besides, with higher specificity, MES can save companies a great amount of money. Last but not least, thanks to its property of self-replication, the reaction system can sustain a much longer time.
But as you can imagine, there are too many problems that limit the application field of bio-synthesis. Accordingly, there are three main challenges in bio-synthesis. The first one is that the contamination risks are too high for bio-synthesis. Although conditions have been better during the days, companies still don’t want to take this none-or-all risk. Secondly, it’s too hard to reach a high productivity, not to mention to maintain the productivity at a high stable level. Last but not least, there are too many varieties during the process of bio-synthesis, which can NOT be managed so easily. In a word, the benefit seems too pale compared with the risks, or the loss it may cause. So bio-synthesis still can not be scaled up and put into practice in normal cases.
To conquer these great challenges, making bio-synthesis a more practical technology that can be used by most pharmaceutical companies or manufacturing factories, we came up with our project——PELICAN!! The Photo-Electro-e.coLI-CAN provides you a brand new experience of bio-synthesis, one that is way more convenient and efficient!!
To build a universal platform, we conduct our project in a more common-used host--E.coli. But most researches about MES just focu on some microbes like Shewallena as they have the ability to transfer extracellular electrons into the cytoplasm originally. So what we are doing is kind of cutting-edge!
Regardless of all these obstacles, we make it! In the demonstrate section in our wiki, you can see that the three systems in our project can function as we expected, which strongly prove our concept!!
Reference:
1. Rabaey, K., & Rozendal, R. A. (2010). Microbial electrosynthesis—revisiting the electrical route for microbial production. Nature Reviews Microbiology, 8(10), 706-716. 2. Shin, H. J., Jung, K. A., Nam, C. W., & Park, J. M. (2017). A Genetic Approach for Microbial Electrosynthesis System as Biocommodities Production Platform. Bioresource Technology. 3. Kitching, M., Butler, R., & Marsili, E. (2017). Microbial bioelectrosynthesis of hydrogen: Current challenges and scale-up. Enzyme and microbial technology, 96, 1-13. 4. Sakimoto, K. K., Wong, A. B., & Yang, P. (2016). Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production. Science, 351(6268), 74-77.Design Summary
As we have mentioned in the background part, there are many obstacles in the field of bio-synthesis. One of them is that the productivity is too hard to reach a high level, not to mention to make it stay at a high level. So, to conquer this problem, making biosynthesis a more practical technology, we came up with our project. But our project still can’t cover all the reactions that might happen in different bio-synthesis pathway. The object we are dealing with is reduction reactions. All reduction reactions utilizing NADH can be sped up with our project !!!
So, to improve the efficiency of those reduction reactions that utilize NADH as coenzyme, the most simple solution is to improve the NADH’s concentration inside of the cytoplasm. But how?This is the main goal we need to achieve during the journey of our project!
As we all know, NADH plays the role as an electron carrier. In another word, it’s a form how electrons exist inside the cytoplasm. So how to increase the concentration of NADH inside the cytoplasm? The first thought that came into our minds was to pump as many electrons as possible into the cytoplasm! But where do those electrons come from? How to provide a great amount of electrons to our bacteria? A new and hot technology can help us remove this trouble!! The bio-cathode!!
What is a bio-cathode? As its name indicates, it stands for an electrode that can release electrons to the bio-element on it. In usual cases, there will be engineered bacteria on the electrode to catalyze some reactions. This technology has many different applications. It can, for example, generate electricity if coupled with an anode, treat organical pollutions in daily wastewater, and, more importantly, can be used in microbial-electro-synthesis! In our project, we mainly use this aspect to bring out the potential of bio-cathode.
Figure 3. Main principle of our project
So now, we have the electron source to provide electrons for our bacteria continuously! But usually, the bacteria people use for bio-cathode can transfer extracellular electrons into the cytoplasm originally. This set a boundary for the bio-cathode, or even bio-synthesis in some cases. So, to remove this boundary, we decide to use a common-used and well-studied bacteria for our bio-cathode! With this, we can also make our project more practical and convenient! The E.coli
If we want to realize our beautiful hope, we first need to enable the E.coli to transfer extracellular electrons into the cytoplasm, what E.coli can not perform originally. Here, we use the electron tunnel from another species--Shewanella. On the membrane of this kind of bacteria, there is a protein complex called MtrCAB, which can transfer electrons bidirectionally.
Besides, we need to have a harvest system in our engineered E.coli to demonstrate the improvement of our project’s efficiency. As we have mentioned, our project is a universal platform, so it does NOT matter what enzyme you use, as long as it is a NADH-dependent enzyme. So we use two different reductases, an alcohol dehydrogenases from Kluyveromyces marxianus which can reduce ethanal to ethanol and an reductase CmCR that can catalyze the reduction reaction from Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] to ethyl 4-chloro-3-oxobutanoate (COBE).
Moreover, the most shining point in our project is that we introduce a photocatalyst system to increase the efficiency further more! We use an aminotransferase that can generate S2- from cysteine to form CdS nanoparticles on the surface of our engineered bacteria! With these CdS nanoparticles, our bacteria can utilize the light energy to further improve the efficiency of our harvest system.
Figure 4. Whole pathway in our project
In a word, our project consists of these three systems——a conduction system to transfer extracellular electrons into the bacteria, a photocatalyst system to utilize light energy to better optimize our project, and, lastly, a harvest system to demonstrate the improvement!
Figure 5. Three systems in our project
Reference:
1. Rabaey, K., & Rozendal, R. A. (2010). Microbial electrosynthesis—revisiting the electrical route for microbial production. Nature Reviews Microbiology, 8(10), 706-716. 2. Shin, H. J., Jung, K. A., Nam, C. W., & Park, J. M. (2017). A Genetic Approach for Microbial Electrosynthesis System as Biocommodities Production Platform. Bioresource Technology. 3. Kitching, M., Butler, R., & Marsili, E. (2017). Microbial bioelectrosynthesis of hydrogen: Current challenges and scale-up. Enzyme and microbial technology, 96, 1-13. 4. Sakimoto, K. K., Wong, A. B., & Yang, P. (2016). Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production. Science, 351(6268), 74-77. 5. Jensen, H. M., Albers, A. E., Malley, K. R., Londer, Y. Y., Cohen, B. E., Helms, B. A., ... & Ajo-Franklin, C. M. (2010). Engineering of a synthetic electron conduit in living cells. Proceedings of the National Academy of Sciences, 107(45), 19213-19218. 6. Rosenbaum, M., Aulenta, F., Villano, M., & Angenent, L. T. (2011). Cathodes as electron donors for microbial metabolism: which extracellular electron transfer mechanisms are involved?. Bioresource Technology, 102(1), 324-333.