Enhancing indirubin biosynthesis in Escherichia coli by engineering Methylophaga aminisulfidivorans favin-containing monooxygenase.

Indirubin, a naturally occurring bisindole alkaloid, has demonstrated clinical efficacy in the management of chronic myeloid leukemia while exhibiting multiple pharmacological activities including antitumor, anti-inflammatory, and broad-spectrum antibacterial effects. To enhance indirubin biosynthesis in the engineered Escherichia coli BL21 (DE3) host, an integrated metabolic engineering strategy was systematically executed in a sequential manner. Firstly, the Methylophaga aminisulfidivorans flavin-containing monooxygenase (MaFMO) was heterologously overexpressed under the regulation of the strong Ptac promoter. Then, fermentation conditions were systematically optimized, including adjusting the pH to 9.5 and supplementing with 15 mM cysteine to facilitate the conversion of indole to indirubin. Under the conditions of 15 mM cysteine and pH 9.5, the indirubin yield reached 162.16 mg/L. Guided by hotspot prediction and literature precedents, rational mutagenesis was performed on MaFMO. Through mutation of key residues, engineered strain A125C achieved a yield of 187.36 mg/L. Subsequent structural analysis revealed that substitution with cysteine at residue 125 introduced enhanced side chain polarity and steric volume. Additionally, a NADPH regeneration plasmid was introduced to facilitate the recycling of cofactors. Finally, exogenous tryptophan (4 g/L) was added as the precursor, engineered strain A125C-mdh leaded to 203.64 mg/L indirubin.