Team:NWPU/Proof
Proof of Concept
Introduction
In the nature, in addition to the green plants and algae, almost none of the species can transform one-carbon compounds to multi-carbon compounds. Even the AACHEN 2015 project could not synthesize Glycogen using one-carbon compounds as sole the carbon source. So it is not suitable for the closed-loop system in the spacecraft.
Using one-carbon compounds to synthesize a pentose becomes the last piece but the most important piece of the puzzle in the whole system.
This year, we designed a new and unproven pathway to synthesize pentose. This way is capable of condensing formaldehyde into D-xylulose. We used two enzymes with different condensation functions, BFD enzyme and TalB enzyme. The former can condense formaldehyde into two intermediate products: dihydroxyacetone and hydroxyacetaldehyde. And the latter can condense the two intermediate products into five-carbon saccharide compounds, D-xylulose. For such a Multiple enzyme - catalyzed reactions, we used multi-enzyme self-assembly protein scaffold technology to improve the efficiency of this reaction.
Therefore, this year our goal are:
- To verify the new way of synthesizing D-xylulose.
- To test whether the multi-enzyme self-assembly protein scaffold technology can improve the efficiency of synthesizing D-xylulose.
Our biological experiments was also carried out around the two goals.
Experiments
Our experiments include the following sections:
- in Vitro:
- Scaffold:
- in vivo
The fusion protein expression technology with His-tag was purified to obtain BFD enzyme and TalB enzyme respectively, and the extracellular enzyme reaction experiment was carried out to verify the function of these two enzymes, thus to verify the pathway of synthesizing D-xylulose.
Scaffold expression vector was constructed and the extracellular enzyme reaction was performed to test whether the multi-enzyme self-assembly protein scaffold technology could improve the efficiency of synthesizing D-xylulose.
The alive cells expressing BFD enzyme and TalB enzyme reacted with the substrate directly for intracellular reaction experiments. In addition, a multicistronic expression vector with BFD and TalB sequence was constructed, BTT. The intracellular reaction experiment was carried out to further validate the new pathway synthesizing D-xylulose .
Proof of Concept
We used High Performance Liquid Chromatography (HPLC) to test the intermediates and D-xylulose and used SDS-PAGE to check whether the enzyme was normally expressed. The experimental results and analysis are as follows:
- in Vitro:
- Benzoylformate Decarboxylase (BFD)
- Transaldolase B (TalB)
- Conclusion
- Scaffold:
- Result
- Conclusion
- in vivo
- BFD+TalB
- formaldehyde into two intermediate products.
- the accumulation of D-xylulose was little.
- BTT(BFD-TalB-TalB)
- The accumulation of two intermediate products obviously increased.
- the accumulation of D-xylulose was little.
- Analysis
- Conclusion
The purified protein was analyzed by SDS-PAGE. The result showed that E. coli expressed BFD enzyme normally.
Figue 1, BFD enzyme SDS-PAGE analys result. Lane M, protein marker; Lane 1, supernatant of E.coli's lysis containing BFD; Lane 2, purified BFD enzyme.
We made a reaction system with formaldehyde and BFD pure enzyme, and detected it by HPLC after the reaction ceased. The results showed that BFD enzyme could condense formaldehyde into dihydroxyacetone and hydroxyacetaldehyde.
Figue 2, HPLC analysis of samples from in vitro experiment of BFD.
The purified protein was analyzed by SDS-PAGE, and the results showed that E. coli could express TalB enzyme normally.
Figue 3, TalB enzyme SDS-PAGE analys result. Lane M, protein marker; Lane 1, supernatant of E.coli's lysis containing TalB; Lane 2, purified TalB enzyme.
We purified the reaction mixture containing BFD pure enzyme and intermediate products and TalB pure enzyme into the reaction system， and detected it by HPLC . The results showed that TalB enzyme can transform the two intermediate products, dihydroxy acetone and hydroxy acetaldehyde into D-xylulose by condensation reaction.
Figue 4, HPLC analysis of samples from in vitro experiment of TalB.
From the above experimental results we can confirm that the new pathway we designed to synthesize D-xylulose is effective. Using the BFD enzyme and TalB enzyme did transform formaldehyde into D - xylulose by condensation reaction. Our concept has been proven!
In addition, the reaction parameters were optimized by summing up the results of previous experiments. That made the conversion efficiency from formaldehyde to D-xylulose improved to 4.78%.
The purified protein was analyzed by SDS-PAGE, and the results showed that E.coli could express the scaffold protein normally.
Figue 5, SDS-PAGE analys result. Lane M, protein marker; Lane 1, purified Scafold enzyme; Lane 2, purified BFD enzyme; Lane 3, purified TalB enzyme.
The results of HPLC showed that BFD enzyme and TalB enzyme could synthesize D-xylulose by formaldehyde with the help of protein scaffold, and the highest conversion rate was 14.02% ,which is three times of conversion rate when the scaffold protein was not used!
Figue 6, Synthetic protein scaffolds improve D-xylulose synthesis efficiency.
From the experimental results we can confirm that we used the multi-enzyme self-assembly protein scaffold could greatly improve the efficiency of the D-xylulose synthesis reaction. Our concept has been proven!
Previous experimental results have confirmed BFD enzyme and TalB enzyme could express normally.
The intracellular reaction experiments of the live bacteria which expressed the BFD enzyme and TalB enzyme was carried out.
The live bacteria expressing BFD enzyme could condense
Figue 7, HPLC analysis of samples from in vivo experiment of BFD.
Figue 8, HPLC analysis of samples from in vivo experiment of TalB.
We detected BTT bacteria by SDS-PAGE. The results of SDS-PAGE test showed that E.coli could express both enzymes at the same time.
Figue 9, SDS-PAGE analys result. Lane M, protein marker; Lane 1, supernatant of E.coli's lysis containing BFD and TalB.
The intracellular reaction experiments of the live bacteria which expressed the BFD enzyme and TalB enzyme was carried out.The results of HPLC showed that:
Figure 10, HPLC analysis of samples from in vivo experiment of BTT(BFD-TalB-TalB).
In fact, D-xylulose is a pentose that provides heat. It can be catabolized by E.coli through the pentose phosphate. We constructed the mathematical model for the metabolic pathways in vivo, and the results showed that the synthesized D-xylulose was quickly phosphorylated and broken down by the pathway of pentose phosphate.
According to the results of our in vitro pure enzyme experiments in vitro, it was confirmed that BFD and TalB enzymes could synthesize D-xylulose using formaldehyde. we could also detect the normal expression of both enzymes, as well as the interpretation derived from modeling results. We were able to conclude that the use of viable bacteria can also be used to synthesize D-xylulose by formaldehyde.
Summary
We used High Performance Liquid Chromatography (HPLC) to test the intermediates and D-xylulose and used SDS-PAGE to check whether the enzyme was normally expressed. Based on the above experimental results, we could determine that we achieved our desired goals.
- The new D-xylulose synthesis pathway we designed is effective. The use of BFD enzyme and TalB enzyme could transform the formaldehyde into the D-xylulose by condensation reaction.
- The multi-enzyme self-assembled protein scaffolds used in this project could greatly improve the efficiency of synthesizing D-xylulose
Our concept has been proven!
