Production of indigo through the use of a dual-function substrate and a bifunctional fusion enzyme.

The current chemical process for industrial indigo production puts a heavy burden on the environment. An attractive option would be to develop an alternative biotechnological process which does not rely on a petrochemical. This study describes a new biotransformation approach in which l-tryptophan is used as starting material. Its conversion to indigo can be achieved through recombinant overexpression of a bifunctional fusion enzyme, flavin-containing monooxygenase (FMO) fused to tryptophanase (TRP). First, TRP converts l-tryptophan into pyruvate, ammonia and indole. The formed indole serves as substrate for FMO, resulting in indigo formation, while pyruvate fuels the cells for regenerating the required NADPH. To optimize this bioconversion, different fusion constructs were tested. Fusing TRP to FMO at either the N-terminus (TRP-FMO) or the C-terminus (FMO-TRP) resulted in similar high expression levels of bifunctional fusion enzymes. Using whole cells and l-tryptophan as a precursor, high production levels of indigo could be obtained, significantly higher when compared with cells containing only overexpressed FMO. The TRP-FMO containing cells gave the highest yield of indigo resulting in full conversion of 2.0 g l-tryptophan into 1.7 g indigo per liter of culture. The process developed in this study provides an alternative biotransformation approach for the production of indigo starting from biobased starting material.