How will our project address the problem?

The fashion industry is an industry closely related to human society, but it also poses a significant pollution problem. Our team is extremely concerned about the environmental problems facing the fashion industry and has creatively proposed the use of microbial cultivation technology to produce new types of colored fibers to replace traditional materials, aiming to try to solve the pollution and ethical problems. Komagataeibacter xylinus ATCC 700178 in the microbial world has a natural ability to express cellulose, and relying on the fact that our supervisor's laboratory project has bacteria that can express indigoidine dye, our team will build a microbial system that can directly express colored fibers. Microbial fibers have excellent physical properties and natural advantages, such as biodegradability, with high practicality.

1. Background and significance of our project

The fashion industry is a vital sector in human society, playing an irreplaceable role in livelihood, employment, and import/export trade. China is the world's largest producer and consumer of textiles and clothing, with an annual total fiber consumption of about 30 million tons and a per capita fiber consumption of 22.4 kilograms. In 2021, global clothing and shoe sales hit a staggering 1.7 million dollars, with China alone accounting for 25%^[1].

Figure 1. Global shoe and apparel consumption scale and main market share in 2021

However, the fashion industry also faces severe environmental issues. Various types of pollution, such as water pollution, air pollution, heavy metal pollution, and chemical pollution, pose significant risks to both human health and the environment. According to surveys and statistics, the textile and clothing industry has become the second-largest water consuming industry globally, and the wastewater it generates accounts for roughly 20% of global wastewater output each year [6]. In addition, the amount of greenhouse gas emissions generated by the textile and clothing industry has surpassed the total emissions of international flights and shipping. The acquisition of fashion materials such as leather also poses certain ethical concerns. Environmental pressures on the industry place €110 billion of value at risk [5]. Globally, pollution from fashion industry has become the second-largest polluting industry, second only to the petroleum industry.

Therefore, our team hopes to use biosynthesis technology to directly produce colored fibers by microorganisms as new materials, to achieve our goal of sustainable development goal of fashion industry — low harm, low energy consumption, low pollution and sustainable.

2. How will our project address the problem?

2.1 Bacterial cellulose (BC)

Bacterial cellulose (BC) is a natural biopolymer, with biological activity, biological adaptability, and unique physical, chemical and mechanical properties, such as high crystallinity, high water holding, hyperfine nanofiber network, high tensile strength and elastic modulus, etc., so it has become a research hotspot of new biomedical materials in the world in recent years.

At present, there are two types of bacteria commonly used to synthesize bacterial cellulose. One is the genetically modified Escherichia coli . By recombinant expression of both the BC synthetase operon (bcsABCD) and the upstream operon (cmcax, ccpAx) , researchers change the metabolic pathway of E.coli ; The other type is the Komagataeibacter strains, which have nature enzymes to produce cellulose.

Given the need to introduce dye expressing plasmids into the bacteria later, E. coli may not be able to afford multiple plasmids for co-expression. In addition, through searching the data, we found that some Komagataeibacter strains exhibited higher cellulose production than E. coli , which was more in line with the original intention of our project and the purpose of sustainable development.

Synthetic route of BC in Komagataeibacter xylinus ：The first is the synthesis of the cellulose precursor uridine diphoglucose (UDP-Glu), and then the oligo-Cs complex, also known as terminal complex (TC), continuously transfers pyranose glucose residues from UDP-Glu to the newly formed polysaccharide chain. Dextran chains are formed, secreted through the outer membrane, and finally assembled, crystallized and combined by several dextran chains to form supramolecular texture.

Figure 2a. The metabolic pathways of K. xylinus

Figure 2b. BC membrane

Time permitting, we plan to further increase cellulose production by genetically modifying K. xylinus on the basis of cellulose expression. For example, strong promoters (J23104, J23100, J23119 etc.) and strong terminators (ECK120033736, ECK120033736, ECK120010799, etc.) were replaced in the expressed genes to improve gene expression efficiency. Moreover, some extra enzymes are planned to be added to metabolically engineer K. xylinus for enhancing BC production.

Additional features

Ultraviolet discoloration

In order to make the cellulose product more in line with the aesthetic needs of the public and meet more color choices, we try to modify the surface of BC nanofibers with spiropyran photochromes. When they are subjected to UV irradiation, their color changed from colorless to pink, and returned again to colorless by visible light.
Antibacterial

Judging from the feedback from the questionnaire, many people are worried about whether cellulose produced by bacteria will cause bacterial contamination if it is used in real life. In fact, cellulose-producing bacteria are strict with living conditions and die quickly without suitable conditions. Moreover, bacterial cellulose can be modified to have antibacterial properties. Ag nanoparticles have been studied for their antimicrobial activity, apart from their extremely developed specific surface. In this case, BC network serves as a template for the precipitation of Ag nanoparticles through different methods. We can create Ag-BC material by soaking purified BC in a solution of silver ammonia. Modified cellulose would not cause bacterial contamination, but inhibits bacterial growth and is used as a biological material to help wound healing.

2.2 The dye we use: indigoidine

The synthesis of indigoidine requires glutamate as a raw material, which would be catalyzed by indigoidine synthase to synthesize the end product. We would genetically modified Corynebacterium glutamicum to heterologously express bpsA (encoding indigoidine synthase). Because K. xylinus does not have the native PPTase that is necessary for activating bpsA, we need to transfect the target gene both bpsA and pcpS (encoding PPTase) for indigoidine synthesis into K. xylinus using pSB1A2 as a plasmid vector, and synthesize indigoidine fibers using K. xylinus which is capable of producing cellulose in high yield. Based on indigoidine, a series of chemical modifications could produce more colored fibers, such as blue 5,5'-dichloroindigoidine, orange 6,6'-dichloroindigoidine, and purple 6,6'-dibromoindigoidine.

Figure 3a. Metabolic pathway of the dye.

Figure 3b. Blue pigment indigoidine secreted by IPTG-induced/pTac-ind Corynebacterium glutamicum was induced with 1 mM IPTG at 30 °C for 12 h. A small amount of indigoidine was secreted into the culture medium, with the majority still remaining inside Corynebacterium glutamicum.

2.3 Our solution: bulid a cell factory producing colorful bacterial cellulose

Currently, dyestuffs are mainly derived from plant extracts and chemical synthesis, among which plant extracts are costly and inconvenient, while chemical synthesis has become the mainstream dye synthesis route with low cost and high efficiency. However, this method not only consumes a large amount of water in the synthesis stage, but also brings huge water pollution in the dyeing stage. Therefore, we want to combine synthesis and dyeing of the cellulose in one step by genetic modification of K. xylinus , omitting the intermediate steps of solvent extraction and dyeing with organic solvents to further reduce pollution and produce environmentally friendly colored cellulose.

We believe that the combination of two critical stages in textile industry----fiber production and color decoration would help combat problems mentioned above. K. xylinus produces bacterial cellulose from carbon sources as a protection coat from ultraviolet radiation and harsh chemical environments. Heterologous expression of bpsA in C. glutamicum converts glutamate, a common, cheap, self-produced and envorionmentally sound compound into natural dyes such as indigoidine and its derivatives.

As we want to bulid a cell factory producing colorful bacterial cellulose in a large-scale, we decide two different strategies:

symbiotic co-culturing of K. xylinus and C. glutamicum
expressing bpsA and pcpS involved in the synthesis of indigoidine from nutrients

The two different strategies are exemplified in Figure 4.

Figure 4. The two different strategies.

The second approach allows the strain K. xylinus to efficiently synthesize dyes and BC (bacterial cellulose). Inspired by Yadav et al.(2010), we expect the BC or UDP- glucose molecules intermix with indigoidine molecules and end up being polymerized by the cell’s cellulose biosynthesis machinery. Since no iGEM team or journal article has ever reported success in combinations of dyes and BC in vivo, little to nothing is known about the machnisms or the possible results. For example, it is unknown whether the dyes would bind tightly to the BC or do we need to add the cellulose binding domain (CBD) as the link between our colorful dyes and the cellulose. However, we are certain that the co-polymer produced by this method can be naturally degraded and reshape our knowledge of fashion and biomanufacturing revolution.

2.4 Future working directions

Currently, the extraction of lipophilic dyes often relies on organic solvents such as DMSO, which are not environmentally friendly. We are making preliminary attempts at microbial synthesis of bacterial cellulose dyed with indigoidine. Once the system is successfully established, we will use it to produce cellulose combined with other lipophilic dyes, such as violacein, Tyrian purple, melanin and indigo. We have conducted research on the synthesis pathways of these dyes and found that the process requires relatively few enzymes, making the construction of a metabolic system feasible.

Figure 5. Overview of the metabolic pathways for the production of colorful bacterial cellulose ^[16].

Furthermore, most of the pigments are prone to light-induced degradation ^[15]. To prolong the pigment's retention time, we will consider glycosylation modifications in the future.

References

Background

[1] McKinsey & Company. 2022 China Fashion Industry White Paper: From quantity to quality: Start a new journey for China's fashion industry[J], 2022.

[2] GB20814—2014 Limitation and Determination of Heavy Metal Elements in Dye Products [S].

[3] LEATHER STANDARD by OEKO-TEX[S].

[4] SHEN Yun, HUANG Xinxia, CHEN Yan, CHEN Meng, YU Mengting. Source and pollution control of heavy metals in leather chemicals [J]. China Leather, 2023,48(Z1):39-43.

[5] Sustainability and circularity in the textile value chain - Global stocktaking[R]. United Nations Environment Programme. 2020.

[6] Sustainability and Circularity in the Textile Value Chain - A Global Roadmap[R]. United Nations Environment Programme. 2022.

[7] UN Comtrade Database.

[8] Liang Long Action on "Clothing" and Zero Abandonment of Old Clothing - White Paper on Environmental Benefit Evaluation of Old Clothing Recycling and Resource Utilization [J]. China Textile, 2021 (Z2): 112.

[9] Be alert to the health hazards brought by clothing [J]. Tianjin Political Consultative Conference, 2010 (05): 62-63.

[10] 2022 Chinese Fur-bearing Animal Skinning Quantity Statistical Report[R]. China Leather Industry Association. 2022.

Project

[11] Yadav, V.; Paniliatis, B.J.; Shi, H.; Lee, K.; Cebe, P.; Kaplan, D.L.(2010). Novel in vivo-degradable cellulose-chitin copolymer from metabolically engineered Gluconacetobacter xylinus. Appl. Environ. Microbiol, 76, 6257–6265.

[12] Singh, A., Walker, K. T., Ledesma-Amaro, R., & Ellis, T. (2020). Engineering Bacterial Cellulose by Synthetic Biology. International Journal of Molecular Sciences, 21(23), 9185.

[13] Min Yan Teh, Kean Hean Ooi, Shun Xiang Danny Teo, Mohammad Ehsan Bin Mansoor, Wen Zheng Shaun Lim, and Meng How Tan. (2019). An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae. ACS Synthetic Biology,8 (4), 708-723.

[14] Sehrish Manan, Muhammad Wajid Ullah, Mazhar Ul-Islam, Zhijun Shi, Mario Gauthier, Guang Yang. (2022). Bacterial cellulose: Molecular regulation of biosynthesis, supramolecular assembly, and tailored structural and functional properties. Progress in Materials Science, Volume 129,100972,ISSN 0079-6425.

[15] M. R. Ghiffary, C. P. S. Prabowo, K. Sharma, Y. Yan, S. Y. Lee and H. U. Kim. (2021). High-Level Production of the Natural Blue Pigment indigoidineidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes. ACS Sustainable Chemistry & Engineering Vol. 9 Issue 19 Pages 6613-6622.

[16] Groeneveld, Iris & Kanelli, Maria & Ariese, Freek & Bommel, Maarten R.. (2022). Parameters that affect the photodegradation of dyes and pigments in solution and on substrate – An overview. Dyes and Pigments. 210. 110999. 10.1016/j.dyepig.2022.110999.

[17] Yang, D., Park, S. Y., Lee, S. Y., Production of Rainbow Colorants by Metabolically Engineered Escherichia coli. Adv. Sci. 2021, 8, 2100743. https://doi.org/10.1002/advs.202100743