Team:BUCT-China/Design
In this project, our idea is to combine PE biodegradation and PHFA biosynthesis process together, converting waste PE into brand new PHFA, such that reducing costs of PHFA production and meanwhile the solving PE disposal problem.
Specifically, we will establish both enzyme reaction system for PE degradation and fermentation system for PHFA synthesis.
Specifically, we will establish both enzyme reaction system for PE degradation and fermentation system for PHFA synthesis.
| PE degradation system |
Previously, we have obtained a bacterium with a striking capability of biodegradation of polymer like polyethylene. Base on this foundation, we plan to harness it for the central part to drive the enzyme reaction system. To observe the enzyme directly and to realize immobilization of enzyme, we also incorporated spore surface display technique in our enzyme expression system. As a result, we developed an engineered Bacillus Subtilis spore with the novel oxidase anchored at the outside spore. PE can be degraded within an enzyme reactor with the help of the spore.
The spore surface display system is a technique that can fix enzymes on the outer surface of the spore. By doing this, enzyme is immobilized. Besides, we can easily see them by electron microscope scanning or simply detect them by naked eye, if a fluorescent protein is also anchored on the surface along with the target protein.
Spore surface display system requires three components: the target protein, the anchoring protein, and a link. The target protein is the one that are displayed and serves a particular function. In this case, our target protein is the novel lacasse. Anchoring proteins are all one kind of outer coat protein of the spore of B. subtilis, which naturally fix on the outer surface of B. subtilis. Among those proteins, cotG is perceived as the most commonly used in the field of Industrial biocatalysis. Hence, we use cotG as our anchoring protein. The link is a short peptide that connect target protean and anchoring protein together. Previous research shows that a flexible link (such as GGGGS) is instrumental in prompting catalyzing efficiency, compared to systems that do not incorporate a link. Aside from that, our previous research indicates that an unpliable link (such as EAAAK) works fine in redox systems. Consequently, we decided to experiment on both links (GGGGS and EAAAK). We expressed a fusion protein that have three domains (cotG-link-oxidase) discussed above. CotG will carry the oxidase to the outer surface and fix it on the surface.
The spore surface display system is a technique that can fix enzymes on the outer surface of the spore. By doing this, enzyme is immobilized. Besides, we can easily see them by electron microscope scanning or simply detect them by naked eye, if a fluorescent protein is also anchored on the surface along with the target protein.
Spore surface display system requires three components: the target protein, the anchoring protein, and a link. The target protein is the one that are displayed and serves a particular function. In this case, our target protein is the novel lacasse. Anchoring proteins are all one kind of outer coat protein of the spore of B. subtilis, which naturally fix on the outer surface of B. subtilis. Among those proteins, cotG is perceived as the most commonly used in the field of Industrial biocatalysis. Hence, we use cotG as our anchoring protein. The link is a short peptide that connect target protean and anchoring protein together. Previous research shows that a flexible link (such as GGGGS) is instrumental in prompting catalyzing efficiency, compared to systems that do not incorporate a link. Aside from that, our previous research indicates that an unpliable link (such as EAAAK) works fine in redox systems. Consequently, we decided to experiment on both links (GGGGS and EAAAK). We expressed a fusion protein that have three domains (cotG-link-oxidase) discussed above. CotG will carry the oxidase to the outer surface and fix it on the surface.
Furthermore, to avoid losses of gene, we integrate the gene of the fusion protein along with its promoter to genome of B. subtilis through a double cross-over reaction.
Finally, there is still one issue remained---how to test whether our enzyme is successfully expressed and anchored to the spore? The most straightforward solution is to fuse a GFP to the fusion protein above, so that we can simply detect whether prokaryotic expression system is functioning well. However, this method cannot demonstrate the location of our target---the fusion protein might either be on the outer surface or hide inside of spores. Facing this dilemma, we develop a system that do not contain anchoring protein. In this system, the fluorescent protean must be inside of spores or do not exist on spores at all. By comparing the fluorescent intensity, we can determine whether protein is successfully fixed to surface, since a successful system must have a stronger fluorescent intensity than that do not. In the view that GFP will influence activity of oxidase, we develop systems that are used particularly for biodegradations, which do not contain a GFP.
In conclusion, we developed four system, expression 4 kinds of fusion protein shown in the figure below.
| PE degradation system |
We chose E.coli as our chassis bacteria in consideration of safety and operability of chassis organisms. Having degraded PE as raw materials, the bio-engineered E.coli synthesis PHFA within a fermentation system. We constructed the metabolic pathway for bio-synthesis by packing all the genes of required enzymes within a double plasmid system, which are further transferred to E.coli MG1655.
We devised the pathway shown below. E.coli will absorb the PE degradation product---alkane by passive transport and then transform into PHFA through several steps of oxidation and polymerization. We chose enzymes that shows a pleasant result in previous study regarding to expression in E. coli. Two P450 enzymes are involved in this pathway, and cofactor of them is fused to P450 enzymes to create a more compact expression system, as well as enhancing the efficiency of them. There are 8 enzymes in total, that are inserted, along which a RBS for each of them, into pET28a(+) and pDEUT as shown in the map below.
We devised the pathway shown below. E.coli will absorb the PE degradation product---alkane by passive transport and then transform into PHFA through several steps of oxidation and polymerization. We chose enzymes that shows a pleasant result in previous study regarding to expression in E. coli. Two P450 enzymes are involved in this pathway, and cofactor of them is fused to P450 enzymes to create a more compact expression system, as well as enhancing the efficiency of them. There are 8 enzymes in total, that are inserted, along which a RBS for each of them, into pET28a(+) and pDEUT as shown in the map below.
| Reference |
2. Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications
3. Potent Impact of Plastic Nanomaterials and Micromaterials on the Food Chain and Human Health
4. Bacterial Surface Display of GFPUV on Bacillus subtilis Spores