Over the last four months, we have designed and validated the new pathway of D-xylulose synthesis, adding the last but most important piece to the Blue Leaf system. In addition, for this approach, we used self-assembly protein scaffold technology, which has greatly improved the efficiency of the reaction.
Of course, our experimental results also need to be further confirmed and improved. Specifically speaking, D-xylulose can’t effectively accumulate owing to the metabolism of E. coli. In this way, we cannot accurately measure the state of our pathway in the body when only HPLC is used to detect the product.
Nevertheless, we have not integrated the pentose synthesis pathways we designed with the methanol assimilation pathway and the glycogen synthesis pathway. What if the new pathway brought into E. coli? Can each part be in a good convergence co-ordination? Whether does the efficiency of the overall reaction rise or decline? Honestly, we have no idea about these issues at present.
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.
In order to make the Blue Leaf come true, we plan to do the followings for the rest of the time:
- Detect the pathway of D-xylulose synthesis in vivo.
- Make BlueLeaf system-wide metabolic pathway modeling.
- Achieve the integration and debugging of the system-wide BlueLeaf.
Since only HPLC detecting the product, we must knock down the gene related to the metabolism of D-xylulose in E. coli so as to achieve the effective accumulation of D-xylulose, and then detect the working state of the D-xylulose synthesis in vivo. From our previous model results, xylulokinase (EC 18.104.22.168, BRENDA) will phosphorylate D-xylulose into D-xylulose 5-phosphate, which is then metabolized by the pentose phosphate pathway. Therefore, we need to knock out the xylulokinase gene (GO: 0004856, BRENDA). We can choose the Cre / loxp technology which is commonly used or the CRISPR / Cas9 technology which becomes popular nowadays.
Before integrating the pathways of D-xylulose synthesis, methanol assimilation and glycogen synthesis into E. coli, we need to model the whole system to simulate the influence of this exogenous pathway on E. coli and recognize its own working condition. Using the data from the last step and the other two pathways, we can model the metabolic pathways of BlueLeaf system and provide the theoretical guidance for the next experiment.
We need to construct the complex system into an expression vector, and then transform it into E. coli, achieving the system of BlueLeaf as a whole. Certainly, lots of unexpected problems are bound to arise at that time. Thus, we are supposed to make stable movements and use various synthetic biology methods to solve problems in the later exploration about the relevant theories and practices.
BlueLeaf is the essential device for human to explore the vast universe and improve the earth ecology in the future. There are also many scientists involving in the great exploration. The work we have done this year is only a small step towards this great goal. We firmly believe that as long as we are still curious about the vastness of the universe and still hope for the future of human, BlueLeaf will provide beneficial services for human and the Earth one day.
Stay true to the mission and do not forget our initial heart, and move on!