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Engineering Success

Abstract

The rapid expansion of the fast fashion market has led to overproduction and overconsumption of cheap, disposable clothes. However, the majority of them end up as textile waste at incineration plants or landfill sites, exacerbating the global climate problem. To minimize the environmental pollution of the fashion industry, GreatBay_SCIE proposed a sustainable and eco-friendly method to upcycle unwanted clothes using the cellulosome-like complex nanomachines. These protein complexes are composed of scaffold proteins OlpB and CipA, with cellulolytic enzymes and PET-degrading enzymes fused on them to enable the effective biodegradation of cellulose fiber and PET fiber in clothes, converting them into reusable end products including bacterial cellulose (BC) and PET beads. With Fabrevivo, we aim to revolutionize textile recycling, making fast fashion more environmentally-friendly.

Fig-1
Fig-1
Fig. 1 The schematic representation of Fabrevivo design. Two cellulosome complexes were constructed, with cellulase and PETase subunits binding to primary scaffold CipA1B2C via type I cohesin-dockerin interaction; Attachment of CipA scaffold to OlpB-Ag3 was made possible by type II cohesin-dockerin interaction, Neae-Nb3-Ag3 association displays the whole complex on E. coli surface. Ferritin expression within E. coli host enables magnetic recycling for cellulosome complexes to be reused.

Surface display system

In order to display the cellulosome complex on the surface of E. coli, we decided to use nanobody (Nb)-antigen (Ag) interaction between Neae-Nb3 and Ag3.

In the construction of Neae-Nb3 vector we fused ribozyme RiboJ, which is a genetic insulator that increases the protein expression of downstream sequence. The effect of RiboJ fusion was first verified using the construction of prha-riboJ-RFP plasmid (Fig.2A). Comparison of fluorescence intensity between prha-riboJ-RFP and control group absence of riboJ suggests significant increase in level of RFP expression (Fig.2C and Fig.2D).

The prha-riboJ-Neae-Nb3 plasmid were then constructed (Fig.2A) and transformed into E. coli BL21 strain for rhamnose-inducible expression. SDS-page was performed and Neae-Nb3 was presented in sediment, indicating the successful expression of Nb3 on bacterial cell surface (Fig.2B).

Fig-2
Fig-2
Fig. 2 E. coli surface display system. (A) Construction of prha-riboJ-Neae-Nb3 and prha-riboJ-mRFP1 vectors. (B) SDS-page analysis for Neae-Nb3 expression. (C) Comparison of RFP fluorescence intensity between prha-riboJ-mRFP1 construct and prha-mRFP1 set as control. (D) Stronger positive correlation was shown between RFP fluorescence intensity and concentration of rhamnose in prha-riboJ-mRFP1 construct.

Mini-scaffold construction of cellulosome We constructed E. coli expression vectors for the mini-scaffold protein subunits. The scaffoldin components of the wild-type cellulosome subunits are large protein scaffolds that impose massive burden on the bacterial host secreting them. Therefore, we modified the coding sequences for the wild-type cellulosome protein scaffold, as shown in (Fig.3A and Fig.3B). The mini-scaffolds were successfully expressed by our host, verified by the SDS-PAGE analysis shown in (Fig.3C and Fig.3D).

Fig-3
Fig-3
Fig 3. Mini-scaffold expression in E. coli BL21. (A) Construction of primary scaffold CipA1B2C (i.e., 1 CBM3 and 2 type I cohesin) (B) Construction of anchorage scaffold OlpB-Ag3 (i.e., 3 type II cohesin). (C) SDS-page analysis for CipA1B2C. (D) SDS-page analysis for OlpB-Ag3.

In order to visualize the three levels of protein-protein interaction involved in the assembly and display of our cellulosome complex, reporter protein eforRED was fused to antigen and dockerins. Ag3-eforRED, DocI-eforRED, and DocII-eforRED vectors were constructed and cultured for IPTG-inducible expression (Fig. 4D). SDS-page analysis was performed with lysed cells and all three targeted proteins were identified in both whole cell and supernatant (Fig. 5A and 5B).

The functionality of these proteins was then tested step by step.

The nanobody-antigen interaction was verified by mixing intact E. coli cells displaying Neae-Nb3 with the supernatant of Ag3-eforRED (Fig. 4A). Red fluorescent characteristics were observed in the pellets after resuspension of the centrifuged mixture, which is absent in the control group that only contains Neae-Nb3 (Fig. 5C).

After that, the type II cohesin-dockerin interaction was tested using the mixture of Neae-Nb3, OlpB-Ag3, and the type II dokerin fused with eforRED (Fig. 4B). A negative control lacking OlpB-Ag3 was set up for result comparison. Centrifugation was used to remove supernatant and the red fluorescence was only identified in pellets of the sample group, confirming the type II cohesin-dockerin interaction (Fig. 5D).

Finally, the association between type I cohesin and type I dockerin was validated using the mixture of Neae-Nb3, OlpB-Ag3, CipA1B2C, and DocI-eforRED (Fig. 4C), red fluorescence was detected in the pellets of the resuspended mixture while it was not observed in the control group lacking the primary scaffold CipA1B2C (Fig. 5E), verifying the type I cohesin-dockerin interaction.

Fig-4
Fig-4
Fig.4 Cellulosomal scaffold system construct (A) Antigen-nanobody interaction between Nb3 and Ag3 domain reported by the ligated eforRed fluorescent domain (B) Type II cohesin -dockerin interaction between a fixed secondary scaffoldin component and a type II dockerin domain reported by the ligated eforRed fluorescent domain (C) Type I cohesin-dockerin interaction between a fixed primary scaffoldin component and a type I dockerin domain reported by a ligated eforRed fluorescent domain (D) Genetic circuit designed for the expression of ligated form of Ag3, type I dockerin and type II dockerin domains fused with an efoRed fluorescent domain at N terminus for reporting the adhesive functions of those domains.

Fig-5
Fig-5
Fig.5 Production and assay of the scaffold proteins (A) SDS-PAGE analysis for the presence of type I dockerin (B) SDS-PAGE analysis of Ag3 and type II dockerin containing secondary mini-scaffold protein (C) The fluorescence indication for antigen-nanobody interaction for E. coli surface display with an Ag3 control group and a sample group (D) The fluorescence indication for type II cohesin-dockerin interactions with a type II dockerin control group and a sample group (E ) The fluorescence indication for type I cohesin-dockerin interactions with a type I dockerin control group and a sample group

Magnetic recycling

A novel design feature of our engineered E. coli was the inclusion of intracellular ferritin expression. The Fe2+ ions in ferritin allows the E. coli cells displaying cellulosome complex to be attracted by strong magnets, therefore enabling the magnetic recycling of cellulosomes to be reused.

We've constructed three ferritin plasmids: the ferritin wild type (Pfuferritin), the existing part (BBa_K1189065), and the wild type ferritin fused with Nb3 (Pfuferritin-Nb3) (Fig.6A). All three vectors were transformed and cultured for IPTG-inducible expression. The target proteins of all three ferritins were detected in the whole cell and supernatant samples of SDS-page (Fig.6B).

To further prove that the host E. coli cells carrying ferritin is magnetically attractive, we transformed prha-mRFP1 plasmids into competent cells containing Pfuferritin and BBa_K1189065 respectively. The co-expression of ferritin and RFP allowed magnetic recycling results to be better visualized. In both ferritin wild type and ferritin part, aggregation of red fluorescence were found near the strong magnets, proving the feasibility of magnetic recycling system (Fig.6C).

Fig-6
Fig-6
Fig. 6 Ferritin expression and magnetic recycling. (A) Genetic circuit construction for three types of ferritin: ferritin wild type (PfuFerritin), IGEM existing ferritin part (BBa_K1189025), and ferritin-Nb3 (PfuFerritin-Nb3) adapted for surface display system. (B) SDS-page analysis of PfuFerritin-Nb3, BBa_K1189025, Ferritin wild type (WT) respectively. (C) Magnetic recycling was conducted with Ferritin control group, RFP control group, BBa_K1189025-RFP, and Ferritin WT-RFP. Apparent aggregation of RFP fluorescence shown in cells co-expressing ferritin and RFP verified the ability for ferritin to be attracted by strong magnets.

PET degradation

The degradation of PET polymers using an enzymatic approach requires the synergetic functions of PETase and MHETase, producing terephthalic acids and ethylene glycol by hydrolytic cleavages (Fig.7A). We constructed E. coli expression vectors for the production of PETase and MHETase that can be induced by IPTG (Fig.7B). The production of the proteins was verified by SDS-PAGE analysis (Fig.7C).

The degrading efficiency of PETase and fusion protein PETase-t were tested by adding PET string and PET membrane to PETase 5 and PETase 5-t respectively. The PH difference after 48h of four sample groups alongside two control groups without PET substrate were tested separately. Larger decrease in PH level were measured in the sample groups, suggesting the production of acidic compounds during PET degradation (Fig.7D).

Fig-7
Fig-7
Fig. 7 FAST-PETase expression (A) Metabolic pathway of PET degradation, PETase catalyzes the cleavage of PET into MHET (mono-2-hydroxyethyl terephthalate) and EG (Ethylene glycol). (B) Genetic circuit constructions of FAST-PETase and FAST-PETase-t with type I dockerin fused to anchor the enzyme subunit onto the cellulosome complex. (C) SDS-page analysis for PETase 5 and PETase 5-t (D) The PH values of different samples of PET degraded by PETases either fused or not fused with type I dockerin domain.

Cellulases and cellulase boosters expression

The enzymatic digestion of the polysaccharide chains of cellulose was completed by exoglucanase, endoglucanase and 1-4 betaglucosidase, and this series of reactions are catalysed by LPMO and CDH. We constructed expression vectors for yeast Kluyveromyces marxianus with the unique origin of replication and antibiotic selection marker for the culturing of Kluyveromyces marxianus . Expression vectors were made distinct by the insertion of different sequences coding for the ligated form of the cellulase enzymes, LPMO and CDH. The enzymes were ligated with an alpha-mating factor secretion signal for Kluyveromyces marxianus at the N-terminus and a type I dockerin domain at the C-terminus (Fig.8A).The successful production and secretion of the protein NpaBGS, MtCDH and TrEGIII are examined by SDS-PAGE and western blot analysis (Fig.8D).

Fig-8
Fig-8
Fig.8 Construction of expression vectors for fusion proteins production in yeast Kluyveromyces marxianus and the analysis of the secreted enzymes (A) The design of our expression vector for production of cellulases and cellulase boosters in Kluyveromyces marxianus; the coding sequences for the cellulases and cellulase boosters were ligated with an alpha-mating factor secretion signal for Kluyveromyces marxianus at the N terminus and a type I dockerin domain at the C terminus linked by a flexible linker (B) The growth curve of recombinant yeasts transformed with expression plasmids coding for different enzymes (C) The agarose gel electrophoresis image of coding sequences for different enzymes, respectively NpaBGS, TaLPMO, CBHII, MtCDH and TrEGIII (D) Western blot result for TrEGIII and MtCDH

Cellulosome construction

We assembled the cellulose-like complex on the surface of E. coli by adding primary scaffold proteins, cellulases and cellulase boosters onto E. coli expressing secondary scaffold proteins. The mixture was centrifuged and resuspended in tris-HCl. The mixture underwent centrifugation and resuspension using tris-HCl, and cellulose was added to the mixture. After 24h, the mixture was filtered and tested for glucose by Benedict's test. From the result, we determined that the cellulosome-like complexes are able to degrade cellulose at a higher efficiency than cell-free cellulases mixture (Fig.9A and 9B). The overall success in engineering our project was verified by the successful construction of cellulosome complex and degrading cellulose to reducing sugars.

Fig-9
Fig-9
Fig.9 The Benedict’s quantitative and qualitative tests for reducing sugar produced by the enzymatic or cellulosomal degradation of cellulose (A) Benedict’s qualitative test result for reducing sugar production through 24h of cellulose degradation by cellulosome, cellulosome without boosters, nanobody presenting cell+free cellulases+cellulase boosters, nanobody presenting cell+cellulases and nanobody presenting cell control from left to right (B) Benedict’s quantitative test for absorbance of the samples obtained from the Benedict’s qualitative test at 635 nm wavelength.

Conclusion and discussion

In conclusion, we successfully constructed cellulosome complexes containing cellulases and PET-degrading enzymes respectively. Type I cohesin-dockerin interaction fixes enzyme subunits on CipA primary scaffold; Type II cohesin-dockerin interaction is involved in the association between primary scaffold and secondary scaffold OlpB. The whole complex is then displayed on E. Coli cell surface using Nb3-Ag3 interaction, with intracellular expression of ferritin to allow magnetic recycling. Overall, the complex was proven more effective in degrading cellulose and PET fibers in textiles than free enzymes.

As a highly-diversified protein complex with unlimited potential, we had plans to build on what we have accomplished with the construction of cellulosome so far. In the future, larger scaffold proteins such as CipA2B9C and OlpB with 7 type II cohesin domains could be expressed to maximize the number of enzyme subunits that can bind to the cellulosome complex. Additionally, we would try to increase the expression of all five types of cellulases and cellulase boosters, aiming to further enhance cellulose degradation efficiency of the complex. Similarly, more effective PET breakdown could also be anticipated with increased protein expression of PETase 5-t and MHETase-t.

In the closed-loop upcycling of unwanted textile we conceived, our end products from cellulose and PET fiber degradation were expected to re-enter the fashion industry. With the sugar we obtained, we decided to culture bacterial cellulose (BC) that can be made into clothes and bags. The TPA and EG monomers collected from PET degradation could be polymerized into PET beads again, which may be used in clothes manufacturing again.