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, TnaA) from and flavin-dependent monooxygenase (flavin-dependent monooxygenase, FMO) from to synthesize indigo with L-tryptophan as substrate. A recombinant strain -IND01 was obtained. The beneficial mutant FMO was obtained by protein engineering of the rate-limiting enzyme FMO. FMO showed the specific activity and / value 2.36 times and 1.34 times higher than that of the wild type, respectively. Furthermore, FMO was introduced into the strain -IND01 to construct the strain -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 FMO activity in this study, which laid a foundation for industrial production of indigo.
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