摘要
Utilization of substrate promiscuity in oxygenases to engineer indole hydroxylation activity for indigoid production has been actively pursued. Indole alkaloids such as indigo and indirubin are plants derived natural products with textile dyeing and pharmaceutical applications. While the precision of protein design has been enhanced by structural prediction tools, random mutagenesis can offer surprising insight into key residues involved in binding and function that are difficult to identify through rational models. Using mutagenesis coupled to screening of blue-hue forming colonies, here we identified two important residues M185 and V402 of the flavin-containing monooxygenase from Corynebacterium glutamicum (CgFMO) and generated a spectrum of variants with enhanced indole hydroxylation activity. The best performing mutant M185L/V402A exhibited nearly 10 fold higher catalytic efficiency toward indole compared to wild type CgFMO, with greater improvement in the kcat level. Mutation M185L sits on the FMO identifying motif while V402A locates near the presumed substrate binding cavity. Reversion checks revealed that while V402A plays a more influential role, presence of both mutations is required for the observed improvement. Final accumulation of indigo and indirubin using the engineered CgFMO reached 0.8 g/L and 0.4 g/L respectively, demonstrating by far the highest indirubin titer and ratio without supplementation of cysteine using FMO. This study provides insight into the CgFMO active site and helps future design of FMO biocatalysts.
•Identified key residues of the monooxygenase not easily picked up by rational models.•Indole hydroxylation activity greatly enhanced by random and saturated mutagenesis.•Best mutant exhibited 10 fold higher catalytic efficiency toward indole.•Engineered FMO mutant demonstrated by far the highest indirubin to indigo production ratio.