Experimental study demonstrates antibiotic- and inducer-free indican biosynthesis in engineered Escherichia coli Nissle 1917, highlighting a green biomanufacturing route for indigo dye.
Key Points
To construct an antibiotic- and inducer-free biomanufacturing platform in probiotic Escherichia coli Nissle 1917 for the sustainable synthesis of the indigo precursor indican from crude glycerol.
Engineered Escherichia coli Nissle 1917 using its native high-copy plasmids to constitutively express flavin-containing monooxygenase (FMO K223R) and glycosyltransferase PtUGT1.
Redirected metabolic flux toward indican by deleting competing pathway genes (pheA, tyrA, pykA, ppc) and trpR, alongside overexpressing tnaA and feedback-resistant trpE S40F.
Optimized PtUGT1 via semi-rational mutagenesis (S112A) and fusion expression, assessing performance in shake flasks and crude glycerol-fed-batch bioreactor fermentation.
Engineered strain with mutagenized PtUGT1(S112A) and optimized fusion expression achieved an indican titer of 449.3 mg/L in shake-flask cultivation.
Crude glycerol-fed-batch fermentation attained a peak indican titer of 1923.2 mg/L at 32 hours without requiring antibiotics or chemical inducers.