[A dual-enzyme cascade for production of indigo from L-tryptophan]

Indigo, as a water-soluble non-azo colorant, is widely used in textile, food, pharmaceutical and other industrial fields. Currently, indigo is primarily synthesized by chemical methods, which causes environmental pollution, potential safety hazards, and other issues. Therefore, there is an urgent need to find a safer and greener synthetic method. In this study, a dual-enzyme cascade pathway was constructed with the tryptophan synthase (tryptophanase, EcTnaA) from Escherichia coli and flavin-dependent monooxygenase (flavin-dependent monooxygenase, MaFMO) from Methylophaga aminisulfidivorans to synthesize indigo with L-tryptophan as substrate. A recombinant strain EM-IND01 was obtained. The beneficial mutant MaFMO^D197E was obtained by protein engineering of the rate-limiting enzyme MaFMO. MaFMO^D197E showed the specific activity and k_cat/K_m value 2.36 times and 1.34 times higher than that of the wild type, respectively. Furthermore, MaFMO^D197E was introduced into the strain EM-IND01 to construct the strain EM-IND02. After the fermentation conditions were optimized, the strain achieved the indigo titer of (1 288.59±7.50) mg/L, the yield of 0.86 mg/mg L-tryptophan, and the productivity of 26.85 mg/(L·h) in a 5 L fermenter. Protein engineering was used to obtain mutants with increased MaFMO activity in this study, which laid a foundation for industrial production of indigo.