Antibiotic- and inducer-free biosynthesis of indican from crude glycerol using engineered probiotic Escherichia coli Nissle 1917

Chemical synthesis of indigo relies on aniline and formaldehyde feedstocks, inevitably generating carcinogenic byproducts and imposing severe environmental burdens, driving the urgent demand for sustainable bio-manufacturing alternatives. As a water-soluble glycoside precursor of indigo, indican circumvents the cytotoxicity and product accumulation issues caused by hydrophobic indigo precipitation, emerging as an ideal intermediate for green indigo production. Herein, we developed an antibiotic- and inducer-free biosynthesis platform using the probiotic Escherichia coli Nissle 1917 (EcN). By harnessing its native high-copy plasmids, constitutive expression of the flavin-containing monooxygenase FMO K223R and glycosyltransferase PtUGT1 was achieved to enable the de novo synthesis of indican. To redirect metabolic flux toward indican accumulation, competing pathways were blocked via inactivation of pheA , tyrA , pykA and ppc , while disruption of the trpR gene alleviated feedback inhibition to the tryptophan biosynthetic pathway. Combined overexpression of rate-limiting genes tnaA and the feedback-resistant trpE S40F synergistically enhanced indican accumulation. Further semi-rational mutagenesis of PtUGT1(S112A), coupled with fusion expression optimization, substantially elevated indican titers to 449.3 mg/L in shake-flask cultivation, and crude glycerol-fed-batch fermentation reached a peak titer of 1923.2 mg/L at 32 h. This study demonstrates that the EcN endogenous plasmid engineering eliminates dependence on antibiotics and inducers, establishing a robust and eco-friendly alternative to traditional chemical synthesis.