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Team:Berkeley/Project
From 2013.igem.org
| The world consumes over 40 million kilograms of indigo annually, primarily for dyeing denim. Indigo is currently derived from petroleum using a high energy process, and commercial dyeing involves the use of reducing agents to solubilize the dye. The development of biosynthetic and bioprocessing methodologies for indigo dyeing could have environmental and economic advantages. By combining the biosynthesis of indigo and the use of the natural indigo precursor indican, we propose a more sustainable dyeing method as an alternative to chemically-reduced indigo in the large scale production of indigo textiles. We achieved in vivo indigo production in high titers, and efficient cleavage of indican using a non-native glucosidase. Inspired by natural systems, we isolated and characterized several plant and bacterial glucosyl transferases hypothesized to produce indican. Lastly, we compare the cost and environmental impact of our alternative with the present chemical process. |
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The world needs to dye 3 billion pairs of jeans with indigo annually. Currently, industrial dyeing uses an extensive process, first producing indigo from the petroleum product, benzene, and then solubilizing indigo to allow it to adhere to cloth. The chemicals used in the process include strong acids, strong bases, and the reducing agent sodium dithionite. The table shown below shows the NFPA diamonds for each chemical highlighting their reactivity and hazards. Given previous iGEM interest in indigo by teams Cornell, KAIST , and Chalmers, as well as the potential for a greener, biosynthetic alternative to denim dyeing, we started our project – Project Blue Genes. 
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Upon beginning our search for biosynthetic routes for dyeing jeans, we learned that certain species of plants produce indigo under stressed conditions. Below is polygonum tinctorium, an indigo producing plant. In the healthy, green part of the leaf, this plant naturally sequesters a toxic compound produced in plants called indole into a glucosylated form called indican. Indican is soluble and harmlessly stored in the vacuoles of plant cells. However, the plant's behavior changes when it is under stress. The leaf that is turning blue has been sprayed with ethanol, and parts of the plant may have begun to degrade. Though the plant is still producing indican in the vacuoles, a glucosidase enzyme previously isolated away from the indican gains access due to the failure of compartmentalization in the cell, and cleaves the glucose ring off of indican. This produces an unstable intermediate called indoxyl which quickly oxidizes and dimerizes to indigo, turning the leaf blue. 
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Drawing on our knowledge of the indigo chemistry in plants, we believed we could take advantage of the indican production pathway for jeans dyeing. Because indican is soluble, it adheres to jeans and can be mass produced as a dyeing agent. After jeans are coated in the soluble indican, they can then be exposed to a glucosidase similar to the mechanism in stressed plants, producing indigo dyed jeans. This process would circumvent many of the hazardous chemicals used industrially to produce blue jeans. 
We aim to establish a pathway in E. coli to use soluble indican to dye jeans. Our pathway begins with indole, a toxic intermediate generated in E. coli as well as plants, and uses a heterologously expressed FMO enzyme to produce indoxyl which would then be taken to indican using a GT or glucosyltransferase enzyme. After using indican to coat cloth as desired, a B-glucosidase enzyme, or GLU, would cleave the sugar from indican and generating indoxyl which oxidizes to blue indigo. |
