A Two‐Phase Enzymatic Cascade for Indigo Synthesis from Anthranilate

Herein, an enzymatic in vitro synthesis route to indigo, the dye that gives denim its renowned color, is described. The proposed five‐step enzymatic pathway is derived from the natural biosynthesis route 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 are assessed by stepwise addition to detect intermediate compounds, to ensure cascade function. A two‐phase system is designed in response to differences in melting temperatures of the enzymes. A titer of 183 mg L −1 indigo is reached through stepwise parameter optimization for each phase, followed by process engineering to enhance the indigo titer. Continuous supplementation of the cosubstrate phosphoribosyl pyrophosphate is necessary to maintain indole‐3‐glycerol phosphate (IGP) formation in the first phase. During the second phase, complete uptake of IGP by Zm BX1 remains challenging, and formation of significant byproducts is observed. Despite attempts to resolve these issues, the underlying mechanism remains 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 for sustainable denim dye synthesis by circumventing host toxicity observed in whole‐cell approaches.