Team:NWPU/Design
Design
Former Method
Goal:
Design a system to achieve the re-use of carbon dioxide in the spacecraft. The system, in other words, will convert carbon dioxide into the sugar compounds offering heat supply, so as to maintain the carbon cycle of the spacecraft and ensure the astronauts' long-term space travel or space presence.
Former Method:
Since the photosynthesis efficiency of plant is only about 1%[1] , it is clearly not a practical way to use the green leaves to maintain the carbon cycle of the spacecraft where the space is narrow while the demand for energy is huge.
Last year, the iGEM team from Aachen University of Technology in Germany[2] designed a synthetic pathway for Glycogen with the methanol from E.coli and finally won the Best manufacture prize. They got the methanol,which is the raw material, through the electric conversion. And then, they combined Methanol Condensation Cycle with Glycogen Metabolism, and they succeeded to synthesize the glycogen. However, in this synthetic pathway, an obvious defect lies on the additional requirement of a five-carbon sugar used together with methanol and then to form glycogen. Since the five carbon compound must be obtained in other glycolytic pathways by E.coli, which will consume at least one molecule of six carbon sugar to synthesize a glycogen hexose sugar monomer.
Figue 1, Synthesis pathway diagram of AACHEN2015.
This route aforementioned cannot be used to synthesize Glycogen only with one carbon compound as the carbon source, so as to be deficient for the closed-loop system of spacecraft. But this idea about utilizing the Methanol Condensation Cycle and Glycogen Metabolism into the synthesis of glycogen has stimulated us greatly.
Direction:
- To synthesize Glycogen in the presence of one-carbon compound as the sole carbon source, we need to find or even design a new pathway for the synthesis of five carbon saccharide from formaldehyde.
- A simple and effective method for the complex reaction involved with so many enzymes should be found to enhance the interaction and cooperation within each enzyme and improve the efficiency of the overall reaction.
- The toxicity of the formaldehyde is high and unstable, especially for the cells. Thus, based on the ways to fix the carbon dioxide by electro-transformation, a one-carbon product with low toxicity and high electro-conversion efficiency should be found, and then we design the pathway of it to assimilate.
New Formaldehyde Assimilation
Background
In nature, few other species have this function, besides the C3 or C4 cycle of green plants can accumulate aone-carbon compound into multi-carbon compounds like saccharide. The team AACHEN 2015 used the Methanol Condensation Cycle[3]. It transforms methanol into formaldehyde that will be condensed into six-carbon sugar with five-carbon sugar produced in E. coli, and then goes to the subsequent glycogen polymerization.
How to synthesize five-carbon carbohydrates from formaldehyde?
New Formaldehyde Assimilation
we have designed a new formaldehyde assimilation pathway, which realized the efficient synthesis of D-xylulose by formaldehyde. This part is “Bearing before and after” part in Blue Leaf as well as the determined factor of whether the entire device can achieve the synthesis of starch by formaldehyde as the sole carbon source.
We found two enzymes with different condensation functions, BFD and TalB , the former could condense formaldehyde into two intermediate products: dihydroxyacetone and Glycolaldehyde. The latter could condense two intermediate products into five-carbon sugars compounds: D-xylulose. BFD derives from Pseudomonas putida and TalB derives from E. coli.
Figue 2, New Synthesis of D-xylulose by Formaldehyde Assimilation.
Though this new approach, we have achieved the synthesis of D-xylulose by formaldehyde as the sole carbon source. In the next route, we used Methanol Condensation Cycle. D-xylulose was phosphorylated to 5-phosphate-xylulose and then went into the methanol assimilation pathway. Finally we could synthesize glycogen though Glycogen Metabolism.
The synthetic pathway we designed was completely untested. So our experiments also focused on the validation of this pathway and the efficient expression and the efficient catalysis of both enzymes.
Self-assembly Protein Scaffold
In the case of Blue Leaf, a system in which multiple enzymes catalyze the reaction, the efficiency of the coordinated reaction between the enzymes determines the efficiency of the overall catalytic reaction.
We know that complex enzyme reaction often has a problem that the products of the last enzyme cannot be timely and competely transported to the next enzyme, making the overall reaction efficiency low due to the location of different enzymes in the space far apart.
In order to improve the efficiency of the multi-enzyme reaction system, Self-assembly Protein Scaffold was used. The Self-assembly Protein Scaffold has several adjacent binding sites. Microscopically, it could automatically assemble the fusion protein with the corresponding Dockerin, making different enzymes next to each other. From the macroscopic point of view, it can improve the local enzyme concentration and substrate concentration, thereby enhancing the multi-enzyme reaction system. [4]
We improved the scaffold protein on the basis of the IGEM CONCORDIA 2015 team[5] and used it in our designed pathway-from formaldehyde to D-xylulose. The scaffold protein consists of the following components:
Figue 3, Synthetic protein scaffolds improve the efficiency of multi - enzyme reaction.
The first part, Cohesins and Dockerins. Cohesins and Dockerins were the major components of protein scaffolds, each with a reactive group that recognizes each other and mosaics. Cohesins were expressed on the scaffold, while Dockerins co-expressed with the target enzyme to form a fusion protein. In this way, the target protein could be anchored firmly to the scaffold.
The second part, Linker. There is a linker between adjacent cohesin, and it is the linker’s length that determines the adjacent enzyme’s distance and influences the passing on of intermediates between co-located enzymes. Therefore, the linker’s length is a main influence of multi-enzyme system’s reaction efficiency.
The third part, CBM protein. CBM could specifically bind to the cellulose, so that the enzyme-scaffold complex could be sedimented by centrifugal force. With this function we could easily purified pure enzyme-scaffold complex.
Based on a registered cohesin(BBa_K1830004) submitted byiGEM15_Concordia’s, we isolated the cohesin module of this part from the linker, which makes following team could adjust linker’s length according to their requirement when they use this cohesin. Besides, we applied this part in our scaffold protein(BBa_K2155012) with other cohesins, and proved it was functional.
Electrocatalytic Reduction of Carbon Dioxide
Using electrocatalytic Reduction of Carbon Dioxide to synthesize formic acid, methanol and other one-carbon compounds is a highly efficient and a green carbon dioxide fixation method. In the reaction system, through the external power supply, the buffer can be ionized and then protonated with carbon dioxide, resulting in a series of reactants. By using different electrodes, buffer systems, and loading different voltages, different reaction products can be produced. Among them, formic acid and methanol are considered the most potential for the development of electrical conversion products.[6] The AACHEN 2015 team introduced one synthesis of methanol using electrocatalytic Reduction of Carbon Dioxide pathway as a sustainable carbon source. For the spacecraft, achieving he controllable recovery of carbon dioxide and making use of electricity are a safe and easy way.
Figue 4, CO2 Reduction Thermodynamics & The Strategy of Hydrogenation of CO2 to Formic Acid.
At present, some scientists have made carbon dioxide synthesis of formic acid conversion rate increased to 80% or more(up to 96%).[7]This is much more than the current conversion rate of about 0.2% of the actual conversion of methanol from carbon dioxide. [8] Moreover, the toxicity of formate is much lower than that of methanol. It is beneficial for the synthesis of carbohydrates to use formic acid as carbon source. It is clear that formic acid is more suitable as a sustainable carbon source than methanol.
So, how can we make E.coli assimilate formic acid? There are few pathways of formic acid assimilation in nature, and we have searched through the literature to find a path that has been validated. The use of two enzymes, ACS and ACDH can turn formic acid into formaldehyde.[9]
Figue 5, Investigator Peng Kang, from TIPC of CAS, designed a electrical device which can transform carbon dioxide into Formaldehyde. Amazingly, the conversion efficiency could be more than 80%.
The synthesis of glycogen was achieved by using the synthetic route of formaldehyde to D-xylulose, the partial methanol assimilation pathway and glycogen metabolic pathway.
Figue 6, Formic acid to formaldehyde conversion pathway.
Reference
[1] From: Treadway JA, Moss JA, Meyer TJ (1999) Inorg Chem38 (20): 4386-4387
[2] From the page: https://2015.igem.org/Team:Aachen
[3] From the page: https://2015.igem.org/Team:Aachen
[4] From: Kim, S., & Hahn, J. S. (2014). Synthetic scaffold based on a cohesin–dockerin interaction for improved production of 2, 3-butanediol in Saccharomyces cerevisiae. Journal of biotechnology, 192, 192-196
[5] From the page: https://2015.igem.org/Team:Concordia
[6] From: Kubiak et al. Chem. Soc. Rev., 2009, 38, 89
[7] From: Kang, Peng, Sheng Zhang, Thomas J. Meyer, and Maurice Brookhart. 2014. "Rapid Selective Electrocatalytic Reduction of Carbon Dioxide to Formate by an Iridium Pincer Catalyst Immobilized on Carbon Nanotube Electrodes." Angewandte Chemie - International Edition 53(33): 8709–13
[8] From: Energy Environ. Sci., 2012, 5, 7050–7059
[9] From: Siegel, Justin B et al. 2015. “Computational Protein Design Enables a Novel One-Carbon Assimilation Pathway.” Proceedings of the National Academy of Sciences of the United States of America 112(12): 3704–9.
