Abstract

The accelerated expansion of the textile industry has given rise to a global environmental crisis¹. Only 25% of textile waste is recycled or repurposed, while the remaining 75% is deposited in landfills. Inefficient and toxic textile disposal practices, such as incineration and harsh chemical degradation, contribute to over 80% of total waste accumulation worldwide. Moreover, the nature of the polymers used in the textile industry represents an environmental threat, as many secrete harmful leachate which intoxicates soil and groundwater. Thus, there is significant interest in developing novel sustainable technologies and practices concerning textile waste management and recycling. Spider silk, a biocompatible natural polymer with desirable physical properties, is an ideal replacement for synthetic textile materials such as polyester and nylon. Spider silk has been studied for its use in biomedical and tissue engineering as well as a biofabric, which expands the scope of our project beyond the textile industry. However, farming spider silk is both unethical and inefficient, requiring territorial breeds that cannibalize each other and create small quantities of silk. Our project will repurpose keratin-based waste that would otherwise be dumped into landfills as a sustainable feedstock for spider silk production^1,2.

We intend to Contribution Abstract
genetically engineer a dual-bacterial recycling system which incorporates fluorescent reporters to monitor the rate of keratin degradation and spider silk synthesis. Using E. coli, one culture will secrete enzymes that depolymerize keratin-based textiles into amino acids, and the second culture will take up the degradation products to assemble spider silk fibers. A biosensor will generate fluorescent emissions to report whether the keratin waste was successfully degraded or if silk is successfully synthesized. With this system, we will be able to screen large amounts of native and modified enzymes to enable further economical waste degradation. In addition, the biosensor assay can be adjusted to monitor the efficiency of various degrading enzymes. This will allow us to identify different keratin degrading enzymes and continue our zero-waste approach with tools at our disposal. Our solution creates a closed loop system for recycling keratin-based biofabrics, expands on current biotextiles research and provides an ethical alternative to spider silk farming. This research serves as a proof-of-concept which can be scaled up and applied to other significant waste products such as low-quality wool unfit for textile use, feather waste from poultry slaughterhouses, and much more.

Motivation

The renowned Climate Change Microbiology lab, led by Dr. Lisa Stein at the University of Alberta, hosted the 2023 UAlberta iGEM team. Their project, like ours, aligns with the motives of the Climate Change Microbiology lab; that microorganisms are capable of influencing climate change mitigation.

Our host lab’s previous work informed our initial project ideas. These included researching new ways to produce PHB, a biologically produced and easily degradable plastic polymer, using only methane as feedstock. However, as we reviewed the literature, we discovered this topic is already extensively described. Instead, our search yielded information about other useful, value-added materials that genetically engineered bacteria are capable of producing. Specifically, the rising interest in biologically produced textiles grabbed our attention. Previous research which successfully engineered E. coli to produce spider silk—a material with incredible physical properties that is difficult to sustainably and ethically farm from spiders—inspired the final direction of our project. We wanted to find a new way to produce this highly valuable textile through sustainable means, ideally by creating a circular economy within the textile industry.

Through our research into the textile industry, we procured knowledge about how fast fashion trends contribute enormously to waste accumulation worldwide. Through fervent literature review and interviews with experts in the field, we learned more about the textile industry, waste management, and the common culprits being dumped in landfills. We discovered that keratin is very valuable in clothing and, being a protein-based material, is an ideal material to biodegrade and repurpose via genetic engineering. Unfortunately, synthetic fibers have been on a sharp rise in past decades, leading to more wool textiles in landfills, and less in use. Articles detailing the successful breakdown of keratinous fibers into amino acids by engineered E. coli provided the groundwork for the missing half of our circular economy.

Thus, ReneWool was born. We have designed a dual-culture system for the breakdown of keratinous waste into amino acids, which will then be provided as feedstock for spider silk-producing E. coli. With a valuable end product in mind our circular economy will recycle the unfortunate byproducts of fast fashion trends into new, useful materials.

Background

The textile industry is one of the oldest and most valuable industries, worth approximately USD 1.3 trillion market value, and with hundreds of millions of employment opportunities (Kasavan et al. 2021). For centuries, wool textiles were at the forefront of the global industry but, beginning in the 20th century synthetic materials became a major competitor, leading to sharp declines in wool textiles on the market (Rajabinejad et al. 2018). However, sheep must still be sheared, and with the low demand for sheep wool, a significant portion is then treated as waste (Pattinson et al. 2015; Rajabinejad et al. 2018).

Keratin based materials such as wool, felt, and leather are highly valuable for having good thermal insulation, resilience, softness, durability, and elasticity (Surjit 2024). They are compatible with other synthetic and natural materials, making them valuable additions to textile manufacturing processes, and there are many different ways that producers have included wool into their products to confer valuable properties (Surjit 2024). According to interviews our Human Practices team conducted with experts in the textile and waste management industries, a major barrier to recycling within textile waste management is the prevalence of mixed blend fabrics, which often include polyester, cellulose, and keratin based materials.

Keratin is one of the most abundant proteins in mammals, birds, and reptiles, and is an area of concern for waste management as a whole. Feathers are a major waste product from poultry slaughterhouses and contribute to accumulation of waste (Sharma and Gupta 2016). The sheer abundance of keratin being produced from livestock to be thrown away is alarming, and the keratin being used in textiles are likely woven with synthetic fibers, and still fill our landfills.

Current Problem

The valorization of recycled keratin waste is an expanding area of research receiving greater and greater attention in recent years. New recycling methodologies are being developed to move away from incineration, or the harsh conditions required for its slow natural degradation (Perța-Crișan et al. 2021). With countries like the USA, Brazil and China producing over 40 million tons of keratin waste per year in the food and wool industries and with harmful fast fashion trends increasing worldwide, new strategies are necessary to handle the growing influx of textile waste (Sharma and Gupta 2016; Hussain et al. 2020; Niinimäki et al. 2020). Keratin products produced naturally by animals are highly stable, insoluble in most organic solvents, and remain undigested by pepsin and trypsin (Perța-Crișan et al. 2021). However, to say that all associated animal products (such as hair, skin, fur, horns, scales, or feathers) are purely keratin is incorrect, as various keratin-associated proteins are present (Perța-Crișan et al. 2021). Still, according to Perța-Crișan and team, keratin biomass consists largely of keratin protein—for example, chicken feathers contain 90% crude protein, while wool is composed of up to 95% keratin by weight. Therefore, focussing on keratin degradation in the management of these waste products is a viable solution. A major obstacle to the valorization of keratin proteins is the large number of disulphide bridges that require highly reducing conditions to disrupt, making it difficult to transform processed keratin fibers into useful products, according to several interviews our Human Practices team conducted with textile and waste management experts. These covalent bonds are a significant player in keratin’s natural hardness and stability. Various fields including cosmetics, biodegradable composites, compostable packing, biomedical technologies and more are looking at applications of keratinous proteins (Parbhu et al. 1999; Eslahi et al. 2013). Most sources of keratin waste are not, however, being put towards new uses; rather, they are being incinerated or put into landfills. The slow degradation of the ever-accumulating keratin waste in landfills is a health hazard, and the high sulfur concentration becomes apparent during burning, as pollution, landscape damage, and soil/groundwater contamination affects our environments (Hussain et al. 2020; Perța-Crișan et al. 2021).

Our Solution

We introduce "ReneWool," a circular economy based product that focuses on a genetically engineered dual-bacteria system designed to recycle wool, which is primarily composed of keratin, and convert it into spider silk—a valuable biotextile material. ReneWool not only breaks down keratin into free amino acids but also utilizes these amino acids to produce spider silk, a high-strength, biodegradable material.

Biological Mechanism

Keratin Degradation

Engineered Bacteria and FRET-Based Biosensor: ReneWool utilizes E. coli engineered to express both a keratinase enzyme (kerDZ, 1845 bp) and a Förster Resonance Energy Transfer (FRET) based biosensor.

Keratinase Enzyme: The keratinase enzyme we employed in our system is KERDZ derived from the gene kerDZ. KERDZ is a 1,845 bp chymotrypsin-type serine keratinolytic protease isolated from Actinomadura viridilutea strain DZ50. KERDZ is synthesized as a pre-protein with an encoded signal sequence which is later cleaved in the maturation process. It has a conserved catalytic triad at H35, D61, and S143 and has a broad cleve specificity with preference towards aromatic and hydrophobic amino acids (Elhoul et. al., 2016). Like its homologous, KERDZ is able to cleave disulfide bonds and peptide bonds allowing for the complete degradation of keratin into amino acids which can then be used by our secondary E. coli system (Elhoul et.al., 2021).

Biosensor: Our biosensor allows the real-time monitoring of the keratin degradation process. The biosensor complex includes:

1. mScarlet3: A fluorescent donor protein
2. Pseudo Keratin:Pseudo Keratin: A low-affinity keratin mimic
3. Nanobody:Nanobody: Binds to pseudo-keratin with low affinity, while binding to true keratin with high affinity.
4. RShadow:RShadow: A quencher recipient protein

The biosensor system will be constructed in the order of mScarlet • pseudo-keratin • nanobody (specific to keratin) • RShadow, inspired by the Fungalescene system incorporated into the Fungal Early Detection Drone System (FEDDS) designed by iGEM UAlberta 2023 . Each component within the FRET system is separated by medium flexibility linker peptides, allowing free movement of the fluorescent molecules.

In the absence of introduced keratin, the biosensor initially shows no fluorescence due to the close proximity of mScarlet and RShadow, which causes fluorescence quenching. Upon the introduction of wool waste, primarily composed of keratin, the nanobody • RShadow pair detaches from the pseudo-keratin • mScarlet pair, resulting in fluorescence. As keratin is degraded by keratinases, the reduction in keratin concentration causes the pseudo-keratin to rebind to the nanobody, bringing mScarlet3 and RShadow closer together again, resulting in decreased fluorescence.

Spider Silk Synthesis

Practical Application and Benefits

Biological Recycling Process: ReneWool offers a sustainable alternative to the energy-intensive chemical methods currently used for wool recycling. By employing a biological approach, ReneWool significantly reduces the environmental impact and energy costs associated with keratin waste management.

Closed-Loop Circular Economy: The system promotes a closed-loop circular economy by transforming keratin waste into valuable spider silk. This biotextile material can be used in various applications, contributing to sustainable practices in the textile industry.

Real-Time Monitoring for High Precision: The integration of the FRET-based biosensor ensures precise control and monitoring of the recycling process. This real-time tracking capability is crucial for maintaining high-quality standards required by textile manufacturers.

Key Components and Customization

Gibson Assembly Method: The genetic engineering of ReneWool is performed using the Gibson Assembly method, known for its precision and ease. This method allows for the flexible customization of the system's components, enabling the recycling of various biotextile materials beyond wool, as long as compatible degrading enzymes are available.

Scalability and Adaptability: ReneWool's modular design makes it adaptable to different industrial needs. While our prototype focuses on wool recycling to produce spider silk, the system can be tailored to recycle other textiles, providing a versatile solution for various biotextile recycling challenges by tackling other forms of keratin waste.

Eco-Friendly Textile Companies: ReneWool is particularly beneficial for clothing companies that prioritize eco-friendly practices. By offering a cost-effective and environmentally sustainable recycling method, ReneWool helps these companies comply with regulatory standards and reduce their ecological footprint. In addition to current efforts to slow down waste accumulation, ReneWool aims to reduce the current waste filling our landfills.

Economic and Environmental Benefits: The adoption of ReneWool not only supports eco-friendly practices but also offers significant economic advantages. The reduction in energy consumption and waste management costs, combined with the production of high-value spider silk, provides a compelling case for industrial implementation.

Future Directions

We chose to begin our research with common sheep’s wool as waste material. This allows us to target textile and fashion industries and learn more about the associated waste challenges. Furthermore, by converting sheep’s wool to spider silk we are practicing a circular economy within the same industries. However, ReneWool does not have to be limited to these products. Future directions include optimization of both degradation and production processes, using our Biosensor measurements to aid in optimization. As this process develops, greater attention can be directed to applying this technology to other forms of keratin waste, such as feathers, leather, etc. Furthermore, other protein products can be produced and optimized, and there may be other products that are even more efficiently produced from the degradation of keratin waste.