A Two-Phase Enzymatic Cascade for Indigo Synthesis From Anthranilate

We here describe an enzymatic in vitro synthesis route to indigo, the dye that gives denim its renowned color. The proposed 5-step enzymatic pathway is derived from the natural biosynthesis route that starts from anthranilate. In the context of the bio-based economy, this method provides an alternative to the current method of indigo synthesis, which relies on petroleum-based building blocks. First, the enzymes were assessed by stepwise addition to detect intermediate compounds, to ensure cascade function. A two-phase system was designed in response to the difference in melting temperatures of the enzymes. A titer of 183 mg/L indigo was reached through stepwise parameter optimization for each phase, followed by process engineering to enhance the indigo titer. Continuous supplementation of the co-substrate phosphoribosyl pyrophosphate was necessary to maintain indole-3-glycerol phosphate (IGP) formation in the first phase. During the second phase, complete uptake of IGP by ZmBX1 remained challenging, and formation of a significant amount of byproducts was observed. Despite attempts to resolve these issues, the underlying mechanism remained unclear. Although the yield must be significantly improved for an economically viable process, an enzymatic cascade based on bio-based materials remains a potential alternative to facilitate sustainable denim dye synthesis.