From discovery to engineering: revealing and optimizing the indigoids biosynthesis by FMO from Indigofera tinctoria

Indigo-producing plants have been used for millennia as natural dyes and traditional medicines. Despite evolutionary divergence, these species converge on a conserved indigo biosynthesis pathway, yet the molecular basis for differences in catalytic efficiency remains largely unknown.

This study aimed to identify and characterize the key genes involved in indigoid biosynthesisfrom Indigofera tinctoria, and to elucidate the structural determinants underlying their catalytic properties.

We performed transcriptome mining and functional screening of candidate genes fromI. tinctoria, followed by enzyme kinetic characterization, substrate specificity profiling, molecular dynamics (MD) simulations using the catalytically competent state, alanine scanning mutagenesis, and metabolic engineering through co-expression and co-cultivation strategies in Escherichia coli.

Three genes, Flavin-containing monooxygenase (ItFMO),Tryptophan synthase α-subunit (ItTSA), and Indole synthase(ItINS), were identified and functionally validated. ItFMO exhibited higher catalytic efficiency (k_cat/K_m = 0.22 ± 0.05 s⁻¹*mM⁻¹) toward indole than other plant FMOs tested, and converted 16 substituted indoles into variously colored indigoids. When 2-oxindole was supplied, ItFMO co-produced indirubin (∼70% of total pigments) via condensation of enzymatically generated indoxyl with 2-oxindole. Co-expression of ItFMO with E. coli TnaA (Tryptophanase) yielded the highest indigo titer (41.27 ± 0.84 mg/L), followed by ItINS (30.54 ± 0.33 mg/L) and ItFMO alone (27.58 ± 0.19 mg/L), whereas ItTSA severely reduced production (5.68 ± 0.20 mg/L). Co-cultivation of the two strains achieved a 2.7-fold increase over the monoculture, with a titer of 60.03 ± 0.82 mg/L. MD simulations and alanine scanning identified Q51, Y160, K212, S213, F373, and N427 as critical residues governing ItFMO catalysis, and which were authenticated by site-directed mutagenesis.

Our findings establish a mechanistic basis for understanding FMO-mediated indigo formation in plants and provide a structural insight for engineering plant-derived enzymes in microbial systems.