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

这项小企业创新研究（SBIR）第一阶段项目的更广泛影响/商业潜力侧重于改进靛蓝染色工艺。靛蓝是牛仔布生产中不可或缺的染料，每年化学合成的工业规模为6.5万吨以上，染色前必须用还原剂将分子溶解。这两种工艺都涉及甲醛和氰化氢等有害化学物质，会对染料工人、生产设施和周围环境造成损害。苯胺是制造靛蓝所需的石油副产品，当摄入、吸入或与皮肤接触时是有毒的，可能致癌。还原剂在印染过程中作为废水倾倒或作为印染废水处理的一部分清理时对水生生物是致命的。今天，靛蓝市场约为5亿美元，靛蓝每公斤售价不到10美元。该项目将开发一种新的生物方法来直接合成靛蓝的还原形式。这个小企业创新研究（SBIR）第一阶段项目将创造一种方法，利用大肠杆菌生物合成一种叫做indican的靛蓝前体，这是一种稳定的水溶性分子。随着b-葡萄糖苷酶的引入，靛蓝被水解并转化为水溶性的靛蓝还原形式，从而避免了靛蓝的化学合成和有害的还原剂步骤。这种印度方法已被证明是一种有效的靛蓝染料，适用于棉织物和纱线。为了改进这一过程，必须从宿主菌株中提高印度人的滴度。在这个一期项目中提出了两个平行的策略：通过定向进化来改善印度途径酶，通过喂养细胞耗尽的关键营养物质来延长发酵过程，从而延长发酵的生产寿命。目前，细胞被喂食色氨酸作为途径前体；这个第一阶段的项目还建议改造细胞，使其产生自己的色氨酸。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.