(130b) Auto-Inducible Synthetic Metabolic Pathway Enhanced De Novo Production of Indigo from Glucoose in E. coli.

Indigo is widely used in textile industries to dye jeans and denim garments and is mainly produced by eco-unfriendly chemical synthesis. Despite the feasibility of indigo production using recombinant E. coli, existing methods are economically incompetent due to low yields and the need for an inducer. Here we first characterized several synthetic promoters and demonstrated the feasibility of inducer-free indigo production from tryptophan using the auto-inducible promoter. We next coupled the tryptophan-to-indigo and glucose-to-tryptophan pathways to generate a de novo glucose-to-indigo pathway. By rational design and combinatorial screening, we identified the optimal promoter-gene combinations and created a new E. coli strain that exploited an alternative indole pathway to enhance the indigo titer to 123 mg/L. We further assessed a panel of heterologous tryptophan synthesase homologs and identified a plant indole lyase (TaIGL) that improved the indigo titer to 235 mg/L while reducing the tryptophan byproduct accumulation. The optimal strain expressed mFMO, ppsA, tktA, trpD, trpC, TaIGL and feedback-resistant aroG and trpE. Fed-batch fermentation in a 3-L bioreactor with glucose feeding further increased the indigo titer (≈965 mg/L) and quantity (≈2183 mg) at 72 h. This new synthetic glucose-to-indigo pathway enables high-titer indigo production without the need of inducer and holds promise for bio-indigo production.