Team:BNDS China/Description
Leather industry has significant economic impacts on our daily life such as the manufacture of luxury bags, shoes, clothes, and so on1. According to the statistics from Food and Agriculture Organization (FAO) in 2001, the estimated annual leather production was about 1.67×109 m2. Moreover, International Trade Center (ITC) in 1999 also suggested that global trades in leather sector was estimated as seventy billion dollars. Therefore, leather industry occupies a large part in our economy.
However, the manufacture process of leather is harmful to animal resource conservation2. To be more specific, wild animal’s leather is the raw material for leather industry. Additionally, some luxury products like crocodile bag requires leather from rare species. Hence, this profitable business drives poachers to illegally hunt and hurt those wild animals. According to World Wide Fund for Nature (WWF), over the past 40 years, the number of wildlife has dropped 67% due to the raging poaching. The demands of leather put the wild animals in a dangerous place and the harms caused by poaching for species conservation is unthinkable. Furthermore, to manufacture leather, glutaraldehyde is adopted as a tanning agent in order to crosslink proteins (especially collagens) together3. However, glutaraldehyde performs great biotoxicity, which means that the wastewater disposed from leather factories would contaminate natural organisms and even human4. Therefore, from the perspective of animal and environment conservation, leather production should be regulated in order to save animal resource. Nevertheless, the huge economic benefit brought by leather industry must be considered, so simply reducing leather production is not a long-term solution. As a result, there is a fierce conflict between the conservation of animal and the development of leather industry. So, at this point, the need for leather alternatives is crucial.
Current solutions of leather alternatives are mostly artificial leathers made of PU, PVC, and other plastic materials, in which the plasticizer and wastewater disposed during the manufacturing process still have inevitable damage to the environment5. Moreover, the non-biodegradable plastics used in artificial leathers have a tremendous impact on the sustainable future: synthetic leathers buried underground will keep on polluting the soil for hundreds of years6. To be more specific, those plastic leathers would break down into small pieces or nanoparticles. There is a high possibility that they might be mistakenly ingested by earthworms and other soil organisms, shuttling through the food chain, and hence creating inevitable damage to the entire eco-system7. The situation for marine animals is even worse: pieces of plastics has been found in 86% of all sea turtle species, 44% of all seabird species, and 43% of all marine mammal species8. Research have shown that nano-plastic inside an organism can lead to brain damage and behavioral disorders9. Thus, current artificial leather cannot effectively solve the conflict between animal conservation and leather industry.
Considering the importance to solve this fierce conflict, our project aimed to design and manufacture a brand-new kind of artificial leather through bacterial cellulose backbone and protein crosslinking.
First of all, bacterial cellulose is the polymer of glucose (Figure 1), which is one of the most important organic polymers as a kind of polysaccharide. Particularly, the intramolecular covalent bond and intermolecular hydrogen bond between glucose monomers make bacterial cellulose a great structural strength. Therefore, we choose it as the backbone of our artificial leather to ensure enough tenacity.
Figure 1. Molecular structure of bacterial cellulose.
Bacterial cellulose is naturally produced by several Acetobacteraceae, especially G. xylinus11 (Figure 2). Investigations about the production of bacterial cellulose via G. xylinus originated from Schramm, M. and S. Hestrin11. Particularly, the methods to culture G. xylinus like the culture media and incubation temperature were well-documented in their research article and is still widely adopted today in several researches12-15.Therefore, we chose to G. xylinus as the chassis organism to produce bacterial cellulose Moreover, it’s noteworthy that we also adopted the dynamic culture strategy in order to ferment G. xylinus and produce bacterial cellulose in large scale16. See more details in our hardware page.
HARDWARE
Figure 2. Biosynthesis process of bacterial cellulose by G. xylinus (Created with BioRender.com).
Besides a backbone with strong structural strength, leather typically also contains another part — protein network17. To be more specific, the tanning step in the manufacture of natural leather is just to denaturalize structural proteins like collagen, adding tanning agent to make them interconnect with each other, which not only further enhance the overall structure strength of leather, but it also integrates the function of structural proteins into leather as a whole17. Considering the potential negative effects caused by tanning agent to the environment, we designed an alternative solution regarding protein crosslinking strategy — SpyCatcher and SpyTag.
SpyCatcher and SpyTag derive from a protein adhesive domain named CnaB2 in Streptococcus pyogenes, in which CnaB2 is stabilized by the spontaneous reaction between Lys and Asp side chains to form an isopeptide bond18,19 (Figure 3). Researchers studying protein crosslinking split CnaB2 into two polypeptides named SpyCatcher and SpyTag20. So far, several improvements have been made on the original verion of SpyCatcher and SpyTag, generating SpyCatcher003 and SpyTag00321.
Considering that isopeptide bond is a strong covalent bond, we adopted this technology for our protein crosslinking with structural protein modification.
Figure 3. Amidation between SpyCatcher and SpyTag.
An Overview Of Design
In order to combine the bacterial cellulose backbone and protein crosslinking network altogether, we recombined SpyCatcher003 with cellulose binding domain (CBM) to make SpyCatcher003 bind with bacterial cellulose pellicle. On the other hand, we recombined SpyTag003 with structural proteins — spider fibroin and collagen-like protein — as the tanning-like agent. To be more specific, we would purify these two recombinant proteins. Then, SpyCatcher003 with CBM would be firstly put with bacterial cellulose so that they would bind together. After that, SpyTag003 with structural proteins would be put with cellulose-SpyCatcher003 complex so that SpyTag003 and SpyCatcher003 would further bind altogether to form an interconnected protein network, which could achieve the purpose of tanning (protein crosslinking), while avoiding the use of toxic tanning agent. See more details in our design page.
DESIGN
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2. Williams, E. E. Why animals matter: the case for animal protection. (Prometheus Books, 2010).
3. Fein, M. L. & Filachione, E. M. (Google Patents, 1960).
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10. Festucci-Buselli, R. A., Otoni, W. C. & Joshi, C. P. Structure, organization, and functions of cellulose synthase complexes in higher plants. Brazilian Journal of Plant Physiology 19, 1-13 (2007).
11. Schramm, M. & Hestrin, S. Synthesis of cellulose by Acetobacter xylinum. 1. Micromethod for the determination of celluloses∗. Biochemical Journal 56, 163-166, doi:10.1042/bj0560163 (1954).
12. Brown, R. M., Willison, J. H. & Richardson, C. L. Cellulose biosynthesis in Acetobacter xylinum: visualization of the site of synthesis and direct measurement of the in vivo process. Proceedings of the National Academy of Sciences 73, 4565, doi:10.1073/pnas.73.12.4565 (1976).
13. Castro, C. et al. Structural characterization of bacterial cellulose produced by Gluconacetobacter swingsii sp. from Colombian agroindustrial wastes. Carbohydrate Polymers 84, 96-102, doi:https://doi.org/10.1016/j.carbpol.2010.10.072 (2011).
14. Florea, M. et al. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proceedings of the National Academy of Sciences 113, E3431, doi:10.1073/pnas.1522985113 (2016).
15. Ishihara, M., Matsunaga, M., Hayashi, N. & Tišler, V. Utilization of d-xylose as carbon source for production of bacterial cellulose. Enzyme and Microbial Technology 31, 986-991, doi:https://doi.org/10.1016/S0141-0229(02)00215-6 (2002).
16. Pae, N. Rotary discs reactor for enhanced production of microbial cellulose. Tp Chemical Technology (2009).
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18. Oke, M. et al. The Scottish Structural Proteomics Facility: targets, methods and outputs. Journal of Structural and Functional Genomics 11, 167-180, doi:10.1007/s10969-010-9090-y (2010).
19. Hagan, R. M. et al. NMR spectroscopic and theoretical analysis of a spontaneously formed Lys-Asp isopeptide bond. Angew Chem Int Ed Engl 49, 8421-8425, doi:10.1002/anie.201004340 (2010).
20. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697, doi:10.1073/pnas.1115485109 (2012).
21. Keeble, A. H. et al. Approaching infinite affinity through engineering of peptide-protein interaction. Proc Natl Acad Sci U S A, doi:10.1073/pnas.1909653116 (2019).
