SBIR Phase I: Development of a novel indigo dye process using biosynthesized molecules

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project focus on improving the indigo dyeing process. Indigo, a dye integral to denim production, is produced on an industrial scale of 65,000+ tons annually by chemical synthesis, and the molecule must be solubilized with a reducing agent before dyeing. Both processes involve hazardous chemicals like formaldehyde and hydrogen cyanide that are damaging to dye workers, production facilities, and the surrounding environment. Aniline, the petroleum byproduct required to create indigo, is toxic when ingested, inhaled, or in contact with skin and may be carcinogenic. Reducing agents are deadly for aquatic life when dumped as wastewater in the dyeing process or costly to clean up as part of wastewater treatment dyeing. Today the indigo market is approximately $500 M, with indigo selling just under $10/kg. The proposed project will develop a novel biological method to synthesize the reduced form of indigo directly. This Small Business Innovation Research (SBIR) Phase I project will create a method to use Escherichia coli to biosynthesize an indigo precursor called indican, a stable, water-soluble molecule. With the introduction of a b-glucosidase, the indican is hydrolyzed and is converted to the water-soluble reduced indigo form, thus circumventing both the indigo chemical synthesis as well as the harmful reducing agent step. This indican method has been shown to work as an effective indigo dye on cotton fabric and yarns. To improve the process, the indican titer must be improved from the host strain. Two parallel strategies are proposed in this Phase I project: the indican pathway enzymes will be improved by directed evolution, and the fermentation process will be elongated by feeding key nutrients that the cell depletes, thus prolonging the productive lifespan of the fermentation. Currently the cells are fed tryptophan as a pathway precursor; this Phase I project also proposes engineering the cell to produce its own tryptophan. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.