Abstract
Our project aims to construct a recyclable cellulosome nanomachine that breaks down cellulose and PET in textiles efficiently. To achieve this goal, we break down our design into three parts: the assembly of the cellulosome complex, the production of enzymes, and the construction of an intracellular ferritin magnetic recycling system. After rounds of experiments, we carried out proof of concept experiments to show the validity of our design, which verified that our cellulosome-like complex system is capable of effective degradation of celluloses and PET, and magnetic recycling. Our proof of concept demonstrated that our cellulosome-like complex holds the potential to be further adjusted to revolutionize the industries for recycling waste clothing materials by degrading and converting textile waste into valuable materials through an environmentally friendly approach.
Protein scaffold construction
We constructed the protein scaffolds on the cell surface of the E. coli host in order to exploit the increase in efficiency brought by enzyme-microbe synergy and to use magnetic characteristics of intracellular ferritin molecules for magnetic recycling. To verify the successful construction of the protein scaffolds, we carried out experiments with eforRed-fused scaffold subunits, testing the functionality of our mini-scaffold, respectively.
We made the host E.coli express Neae-Nb fusion protein that anchors on the surface of the host bacterium, which has a nanobody domain exposed towards the extracellular side. An eforRed-fused antigen domain was produced and adhered to the nanobody domain, and the adhesion was suggested by the fluorescent pellet collected after centrifugation (Fig.2C).
We expressed fusion protein OlpB containing type II cohesin domain and antigen domain. The proteins were mixed with nanobody presenting host E. coli and fusion proteins of a type I dockerin domain ligated with an eforRed domain. The red fluorescence in the pellet of the centrifuged mixture suggested successful cohesion of type I dockerin to type I cohesin (Fig.2D).
Similar to the previous testing type II cohesin dockerin interactions, we constructed the whole scaffold system by further mixing eforRed-fused type I dockerin domain with nanobody presenting cell, secondary scaffold and primary scaffold. The type II cohesin-dockerin interaction was confirmed by the red fluorescence displayed by the pellet after centrifugation (Fig.2E).
By proving the binding of our cellulosome subunits, we ensured the effectiveness of cohesin-dockerin interaction, which set the foundation for incorporating cellulases and other enzymes into the cellulosome complex.
Enzyme Assay
Using various enzyme essays, we proved the functionality of PETase, cellulases, and cellulase boosters.
We mixed the PETase obtained with PET samples, and the activity of the enzyme was verified as samples of terephthalic acid and ethylene glycol were obtained, as suggested by the result of acidity analysis(Fig.3B).
The fungal cellulases we used were produced as fusion proteins ligated with type I dockerin. The enzymes produced were allowed to operate after cellulose was added at 40 degree Celsius for 24h. The results of Benedict’s test verified the enzyme activity of cellulases in degrading cellulose and the ability of cellulase boosters to boost the efficiency of the degradation process.
Assembling cellulosome-like complexes displayed on the surface of E. coli and their superior ability to degrade cellulose.
We assembled the cellulose-like complex (Celly complex) on the surface of E.coli and added cellulose. After 24h, the mixture was filtered and tested for glucose by Benedict's test. From the result, we determined that the cellulosome-like complexes were able to degrade cellulose at a higher efficiency than the cell-free cellulases mixture (Fig.4A), which is proven by the apparent red color of Benedict's test that indicates higher concentration of reducing sugar in cellulosome sample. This conclusion was also supported by the measure of light absorbance at 635nm in each sample (Fig.4B).
The capability of the magnetic characteristics of ferritin molecules for the magnetic recycling of E. coli hosts
The pictures of the Petri dishes with magnetic strips placed along the diameter were compared at the start of the attraction test and after 24h. The high concentration of red fluorescence around the magnetic strip (Fig.5A) suggested the ability of ferritin molecules to act as the medium for magnetic recycling.
The results successfully proved that our design made cellulose degradation more efficient. In the future, further experiments can be conducted to measure the degrading capability of larger cellulosome complexes with increased number of cellulase subunits fused, therefore bringing our design of a highly-powered cellulose degrading nanomachine into realization. The magnetic recycling ability of ferritin enables repeated usage of cellulosome complexes, making our approach more sustainable.