Biocatalytic Indigo Synthesis From L‐Tryptophan Using a Three‐Step Cascade Without Cofactor Regeneration

Indigo is currently produced from petrochemical sources, which poses significant environmental challenges. Sustainable biotechnological alternatives are therefore highly desirable. Enzymatic synthesis of indigo from L-tryptophan via indole has been demonstrated, but conventional pathways based on flavin-containing monooxygenases require costly coenzymes such as NAD(P)H, limiting their practical applicability. In this study, we present a novel, self-sufficient, NAD(P)H-independent enzyme cascade for indigo biosynthesis from the renewable feedstock L-tryptophan. The cascade starts with conversion of L-tryptophan into indole and pyruvate by a tryptophanase. As next steps, the system couples an engineered bacterial tyrosine hydroxylase, which converts indole into indoxyl using hydrogen peroxide, with a pyruvate oxidase that generates the required peroxide in situ. The cascade thereby transforms a reaction byproduct into the oxidizing equivalent needed for the subsequent step, establishing a closed catalytic cycle with minimal auxiliary inputs. After optimizing cascade parameters, the system produced 0.25 mM indigo from 5 mM L-tryptophan. Although the overall yield remains moderate, this proof-of-principle demonstrates a sustainable and cost-effective enzymatic route for indigo production from biobased starting materials, providing an environmentally friendly alternative to petrochemical synthesis.