Construction of Escherichia coli Cell Factory for Efficient Synthesis of Indigo

Graphical Here, we have constructed a dual-enzyme ( Ec TnaA, Ma FMO) cascade pathway for the biosynthesis of indigo from L-tryptophan and enhanced the enzymatic activity of Ma FMO through mechanism-guided rational design. Subsequently, by optimizing promoters and increasing copy numbers to regulate the expression levels of Ec TnaA and Ma FMO D197E , under optimal conditions, E. coli IND-13 achieved the highest yield to date using glucose as the substrate. Indigo is widely used in dyes, medicines and semiconductors materials due to its excellent dyeing efficiency, antibacterial, antiviral, anticancer, anti-corrosion, and thermostability properties. Here, a biosynthetic pathway for indigo was designed, integrating two enzymes ( Ec TnaA, Ma FMO) into a higher L-tryptophan-producing the strain Escherichia coli TRP. However, the lower catalytic activity of Ma FMO was a bottleneck for increasing indigo titers. To overcome this limitation, the enzyme activity of Ma FMO was enhanced through mechanism-guided rational design. The optimal mutant obtained in this study, Ma FMO D197E , whose k cat / K m was 1.34 times that of the wild type, and its specific activity was 2.36 times that of the wild type. In addition, the expression levels of Ec TnaA and Ma FMO D197E were regulated by optimizing the promoters and increasing the copy number to generate the strain E. coli IND-13. Finally, in the optimal fermentation conditions (220 rpm, 0.05 % Tween-80), the strain E. coli IND-13 achieved the indigo titer of 568.52 mg/L in a 5-L bioreactor, with the yield and productivity of 2.62 mg/g and 12.96 mg/L/h (the highest to date), respectively. The results presented here can lay a foundation for further construction of cell factories for indigo and its derivatives with industrial application potential.