The severity of the environmental problem
Fast fashion is the production of “low-priced but stylish clothing that moves quicky from design to retail stores to meet trends” [1]. Fast fashion, with a rapid increase in textile consumption and production, marks the world's economic growth and societal development.
However, while fast fashion provides affordable prices and increased profits for firms, most people are unaware of its downsides: pollution, massive textile waste, and the exploitation of natural resources. Thus, it is a highly unsustainable industry.
First, the textile waste created by fast fashion is immense: Globally, 92 million tonnes of textile waste is produced globally every year [2]. Looking closely at our own country, China, the textile industry is the largest in the world in overall production and exports; around 26 million tons of clothes are thrown away annually[3].
However, the recycling rate of textiles is low. Globally, only 12% of waste textile ends up being recycled. Domestically, less than 20% of the 26 million tons of textile waste was recycled in China in 2022[3]. The overwhelming majority of textile waste ends up in landfills and incinerators, creating massive environmental issues. In 2018, 17 million tonnes of textile waste ended up in landfills[4], which can take up to 200 years to decompose. Today, 84% of clothing is still in landfills or incinerators[5].
According to the Quartermaster Equipment Research Institute, every kilogram of recycled textile waste will reduce carbon dioxide emissions by 3.6 kg and save 6,000 liters of water [3]. Therefore, GreatBay_SCIE recognized the importance of recycling and felt urgent to increase the efficient recycling rate of waste textiles by developing an innovative synthetic biological method.
“The price of fashion is increased global warming” [5]. If more textiles were recycled, we could decrease the 93 billion cubic meters of water used by industry annually; we could reduce the 1.2 billion tonnes of greenhouse gases that the fashion industry emits.
Current recycling methods
What are the common destinations of the small percentage of waste textile that is not incinerated or burned? First, donation. However, this only accounts for 10% of total textile waste. While many people donate their worn clothing to charities, according to the EPA, only 16 percent of donated clothes are used, and 84 percent still end up in landfills and incinerators [6].
Second, some unwanted clothing is exported overseas, an unsustainable solution that is not encouraged. By “dumping” the extra waste into other countries, people purposely avoid confronting the problem's severity. The sheer volume of exported clothing would ultimately face the doomed destiny of landfills and incineration, simultaneously suppressing local clothing industries.
Third, recycling. While it is true that there are efforts made by textile recycling companies, downcycling still takes up a significant proportion of recycled products. Specifically, clothing is sent to textile recycling centers where it will be cut into rags and processed into furniture and building insulation fillings. Down-cycling turns textile waste into lower-valued products, which eventually will still end up in landfills.
Our project
Therefore, our project is proposed to tackle the above problems faced by the fast fashion industry and the textile recycling industry. We, Greatbay_SCIE, designed a project named “FabRevivo,” designated to revive the fabric, namely textile waste.
According to The Secondary Materials and Recycled Textiles Association (SMART), “95 percent of all clothing and household textiles can be recycled or repurposed”[7].
We want to help achieve the number.
Cellulosome – the finest cellulolytic nanomachine
With the goal of accomplishing complete and efficient biodegradation of unwanted textiles in mind, we did extensive literature research and identified an interesting protein complex in nature known as the “cellulosome”. The cellulosome complex was first discovered in an anaerobic cellulolytic bacterium Clostridium thermocellum . It is a supramolecular multienzyme complex comprised of primary scaffold protein, an anchoring scaffold protein, and a wide variety of cellulose-degrading enzymes [8].
In order to maximize the degrading efficiency of cellulose fiber, we referred to literature that successfully constructed the largest cellulosome complex on the surface of yeast species K. marxianus, which enables up to 63 cellulases to be anchored on the complex [9]. Our cellulosome design is a modified version of the original literature and consists of three essential components: the anchoring scaffold protein, outer layer scaffolding protein B (OlpB) (Fig7.), the primary scaffold protein: Cellulosome integrating protein A (CipA1B2C) (Fig7.), as well as five enzyme subunits that we selected, including NpaBGS, CBHII, TrEGIII, TaLPMO, and MtCDH (Fig7.).
The structure of the cellulosome complex is stabilized via vital cohesin-dockerin interaction, which is one of the strongest non-covalent interactions found in nature. Two types of cohesin-dockerin interaction contribute to the cellulosome structure. Type 1 cohesin-dockerin interaction between type I cohesin on enzyme subunits and type I dockerin on CipA1B2C; Type 2 cohesin-dockerin interaction between type II cohesin on CipA1B2C and OlpB scaffolding.
In the assembly of cellulosome complex, OlpB fused with 3 type II cohesin interact with the type II dockerin on CipA1B2C. The primary scaffolding CipA1B2C possesses 2 type I cohesin and 1 cellulose-binding module (CBM) that fixes the cellulose molecule. The 3 cellulases and 2 cellulase boosters are all fused with type I dockerin (with the suffix “-t” added) to convert them from free fungal cellulase into cellulosomal mode that can be assembled into the complex.
Bacterial cell surface display system
We designed a E. coli(BL21) surface display system based on the combining of nanobodies and antigens, in order to anchor our cellulosome onto the bacterias and achieve synergetic action of the cellulose and PET degradation enzymes.
Nanobodies are the variable domains of camelid heavy-chain antibodies. Nb consists of three antigenic complementary determining regions (CDR) and four frame regions (FR). Among them, three CDR are the binding regions of Nb to the antigen, while the traditional antibody needs six CDR to maintain the binding to the antigen. Compared with monoclonal antibodies, Nb showed considerable or even stronger antigen-binding ability. [10]
Nanobodies can be expressed on bacterial surfaces due to their small size and stability under various conditions. The combination between the single-domain structure and the intimin N terminus (Neae, which includes a short N-terminal signal peptide to direct its trafficking to the periplasm, a LysM domain for peptido-glycan binding, and a b-barrel for transmembrane insertion) allows the entirety of a highly specific, cell surface-bound adhesin to be encoded as a single fusion protein. Thus, we expressed Neae-Nb, Nb3 fused to the C terminus of Neae with E. coli(BL21) to act as a 'pedestal' for the cellulosome.
Neae-Nb will adhere to a corresponding antigen(Ag) via the Nb-Ag interaction. The interaction was proved to be both orthogonal (i.e., that adhesins only interact with designed partners) and composable (i.e., that arbitrary combinations of multiple adhesins function simultaneously within one cell). Ag3 was constructed at one end of outer layer scaffolding protein B, which connects the rest parts of the scaffoldin system of the cellulosome.
![Fig.8 the largest cellulosome complex on the cell surface expressed by the yeast [9]](https://static.igem.wiki/teams/4275/wiki/description/fig-8.png)
Cellulolytic enzyme system
To hydrolyse cellulose into monosacchoride beta glucose, a group of enzymes has been identified, among which endoglucanase (TrEgIII), exoglucanase (CBHII), β-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH)[10]are selected as a efficient combination according to our research.
Cellulose has a highly crystalline structure due to the compact hydrogen bonds, and thereby hardly broken down by pure cel cellulase. Broadly accepted, CDH with an elusive mechanism[11] acts as an electron donor to initiate reduction process of TaLPMO, which enables it to catalyze the oxidative cleavage of glycosidic chains found in complex, recalcitrant environments, including the crystalline lattices formed by cellulose. Consequently, new ends are created for processive hydrolases and promote the loosening of the cellulose structure. [12] Once the polymer of cellulose is degraded into celluoligomer, TrEgIII internally cleaves β-1,4-glycosidic bonds in the amorphous regions of cellulose thereby releasing reducing and non-reducing chain ends. Meanwhile, CBHII removes the disaccharide — cellobiose—— from the end of cellulose chain and the cellobiose can be cleaved by the NpaBGS into monosaccharide, glucose.[13]
With scaffolding proteins from cellulosome, we constructed a cellulolytic enzyme system by fusing the type 1 dockerin with three cellulases and two cellulase boosters, which enables those enzymes to bind type 1 cohesin on the primary scaffolding protein — cellulosome integrating protein A (CipA); CipA can then bind to the secondary scaffolding protein— outer layer protein B (OlpB) by type 2 dockerin-cohesin interaction. Due to the enzyme-substrate–microbe complex synergy, the hydrolysis activities of those enzymes are boosted to maximize the cellulose degradation efficiency. Thus, our cellulosome-like nanomachie is able to degrade natural fiber in unwanted clothes into simple sugar, β-glucose, as our primary product.
PET degradation enzyme system
Apart from natural fiber, current fashion products contain a large proportion of synthetic fiber. According to the previous study, about 61.3% of fiber produced in 2011 was synthetic fiber.[14]Hence, we have designed another cellulosome-like protein complex by fusing PETase and MHETase with dockerins, enabling these two enzymes to bind on the scaffolding proteins.
PETase super 5 ( PETase 5, BBa_K3715005) with 163 times greater enzyme activity than wildtype from BJEA_China 2021 has been chosen to hydrolyze the ester bonds in polyethylene terephthalate (PET) into monomeric mono-2-hydroxyethyl terephthalate (MHET). The MHET is cleaved into terephthalic acid (TPA)and ethylene glycol (EG) by MHETase reported in the literature, where a highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers. Similarly, we expect improvement in degrading efficiency of PETase and MHETase after assembling them into cellulosome-like protein complex.[15]
Overall design of Fabrevivo
The main objective of Fabrevivo is to construct an engineered E. Coli with a cell surface display system to present the cellulosome complex on bacterial surface. Two types of cellulosome complexes were designed: one with cellulolytic enzymes and one with PET-degrading enzymes to accomplish effective degradation of both natural fiber and synthetic fiber in waste clothes. Yeast species Kluyveromyces marxianus was selected for the expression of five cellulases and boosters, with the fusion of K. Marx MFa to enable effective secretion of targeted enzymes. E. Coli BL21 was selected to express PETase and MHETase. The species was also used in the expression of the surface display system, including Neae-Nb3, Ag3, alongside the miniscaffold OlpB-Ag3 and CipA1B2C. Three levels of protein-protein interaction contributes to the structure of cellulosome complex. The Interaction between Neae-Nb3 and Ag3 displays OlpB scaffold on E. Coli cell surface, type II cohesin-dockerin interaction anchors CipA1B2C to OlpB, and type I cohesin-dockerin interaction fixes enzyme subunits on primary scaffold, completing the assembly of the whole complex. Intracellular ferritin expression characterized the sustainability of our degradation approach, which involves magnetic recycling to reuse cellulosome complexes and reduce unnecessary cost. In conclusion, Fabrevivo offers an innovative method to upcycle waste textiles, converting excessive waste into valuable products that makes fashion recyclable.
References
[1]Hayes, Adam. “Fast Fashion.” Investopedia, 29 Apr. 2021, www.investopedia.com/terms/f/fast-fashion.asp.
[2]Beall, Abigail. “Why Clothes Are so Hard to Recycle.” Www.bbc.com, BBC, 13 July 2020, www.bbc.com/future/article/20200710-why-clothes-are-so-hard-to-recycle.
[3]Liqiang, Hou. “China to up Its Textile Recycling Capability.” THE STATE COUNCIL the PEOPLE'S REPUBLIC of CHINA, 20 Apr. 2022, english.www.gov.cn/statecouncil/ministries/202204/20/content_WS625f649fc6d02e5335329a8f.html. Accessed 11 Oct. 2022.
[4]US EPA. “Textiles: Material-Specific Data.” US EPA, 30 July 2018, www.epa.gov/facts-and-figures-about-materials-waste-and-recycling/textiles-material-specific-data.
[5]Smith, Delilah. “Fast Fashion's Environmental Impact: The True Price of Trendiness.” Good on You, 14 Feb. 2021, goodonyou.eco/fast-fashions-environmental-impact/.
[6]De Souza, Anna. “This Is What Really Happens to Your Used Clothing Donations.” Reader's Digest, 26 Feb. 2020, www.rd.com/article/what-happens-used-clothing-donations/. Accessed 12 Oct. 2022.
[7]“For Communities.” SMARTASN, www.smartasn.org/resources/for-communities/. Accessed 12 Oct. 2022.
[8] Hirano, Katsuaki, et al. “Enzymatic Diversity of the Clostridium Thermocellum Cellulosome Is Crucial for the Degradation of Crystalline Cellulose and Plant Biomass.” Scientific Reports, vol. 6, no. 1, 19 Oct. 2016, 10.1038/srep35709. Accessed 19 Apr. 2019.
[9] Anandharaj, Marimuthu, et al. “Constructing a Yeast to Express the Largest Cellulosome Complex on the Cell Surface.” Proceedings of the National Academy of Sciences, vol. 117, no. 5, 17 Jan. 2020, pp. 2385–2394, 10.1073/pnas.1916529117.
[10]Uchański, Tomasz, et al. “An Improved Yeast Surface Display Platform for the Screening of Nanobody Immune Libraries.” Scientific Reports, vol. 9, no. 1, 23 Jan. 2019, p. 382, www.nature.com/articles/s41598-018-37212-3), 10.1038/s41598-018-37212-3. Accessed 5 May 2021.
[11]Glass, David S., and Ingmar H. Riedel-Kruse. “A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns.” Cell, vol. 174, no. 3, July 2018, pp. 649-658.e16, 10.1016/j.cell.2018.06.041. Accessed 7 Feb. 2022.
[12]. Bissaro, Bastien, et al. “Molecular Mechanism of the Chitinolytic Peroxygenase Reaction.” Proceedings of the National Academy of Sciences, vol. 117, no. 3, 6 Jan. 2020, pp. 1504–1513, 10.1073/pnas.1904889117. Accessed 12 Oct. 2022.
[13]. Andlar, Martina, et al. “Lignocellulose Degradation: An Overview of Fungi and Fungal Enzymes Involved in Lignocellulose Degradation.” Engineering in Life Sciences, vol. 18, no. 11, 27 June 2018, pp. 768–778, 10.1002/elsc.201800039.
[14]. Senthilkannan Muthu, Subramanian. “Assessing the Environmental Impact of Textiles and the Clothing Supply Chain - 2nd Edition.” Www.elsevier.com, 20 Mar. 2020, www.elsevier.com/books/assessing-the-environmental-impact-of-textiles-and-the-clothing-supply-chain/muthu/978-0-12-819783-7. Accessed 12 Oct. 2022.
[15]. Knott, Brandon C., et al. “Characterization and Engineering of a Two-Enzyme System for Plastics Depolymerization.” Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 41, 13 Oct. 2020, pp. 25476–25485, pubmed.ncbi.nlm.nih.gov/32989159/, 10.1073/pnas.2006753117.