Modifying the Probiotic Escherichia coli Nissle 1917 for the Biosynthesis of Indirubin via Metabolic Engineering

doi:10.13560/j.cnki.biotech.bull.1985.2025-0718

Abstract

Abstract:

Objective Indirubin, a bi-indolic alkaloid derivative isolated from the traditional Chinese herbal medicine Indigo Naturalis, represents a potent therapeutic compound having broad-spectrum antibacterial, anti-inflammatory, and anti-tumor properties. Currently, it is clinically utilized primarily in combination therapy regimens for the treatment of chronic myeloid leukemia (CML), psoriasis, and related pathologies. This study investigates the potential of the safe Escherichia coli Nissle 1917 (EcN) as a chassis for constructing a green supply system for the active components of the traditional Chinese medicine indirubin. Method Based on the engineering modification of the natural endogenous plasmid of EcN, the product output efficiency of the indirubin biosynthesis pathway constructed using the flavin monooxygenase fmo and its mutants (fmo^K223R and fmo^K223R/D317S )) from Methylophaga sp. SK1 was systematically evaluated. Combined with metabolic engineering, key genes in the competitive branch of the tryptophan biosynthesis pathway were knocked out, including pheA, a key gene for phenylalanine biosynthesis, and tyrA, a key gene for tyrosine biosynthesis. Further optimization was achieved by inactivating the upstream genes of the tricarboxylic acid cycle, including pykA (pyruvate kinase) and ppc (phosphoenolpyruvate carboxylase). Finally, the product synthesis level was further improved by deleting the repressor protein gene trpR in the tryptophan pathway and strengthening the anti-feedback inhibition gene trpE^S40F in the tryptophan pathway. Result After 48 h of shaking culture in a 250 mL flask (50 mL culture), the indirubin titer reached (176.9 ± 4.5) mg/L, representing a 3.5-fold increase compared to the parent strain harboring only the wild-type fmo gene. The production in the 5 L bioreactor was (379.3 ± 12.3) mg/L. Conclusion Employing the endogenous cryptic plasmid of the probiotic EcN to express the heterologous flavin monooxygenase mutant gene fmo^K223R, and integrating this with systematic metabolic engineering of endogenous tryptophan biosynthesis pathways, the production of indirubin reached over 300 mg/L, demonstrating the excellent potential of EcN as a cell factory for biosynthesis of active ingredients in traditional Chinese medicines.

Key words: Escherichia coli Nissle 1917, flavin monooxygenase bFMO, traditional Chinese medicine Indigo Naturalis, indirubin, biosynthesis

MAO Li-jing, JIN Xiao-xuan, SHI Wan-ting, HU Fei-yang, ZHANG Yuan-rong, XIONG Liang-bin, REN Lu. Modifying the Probiotic Escherichia coli Nissle 1917 for the Biosynthesis of Indirubin via Metabolic Engineering[J]. Biotechnology Bulletin, 2025, 41(12): 74-81.

Figures/Tables 5

Fig. 1 Construction of the biosynthesis pathway of indirubin in Escherichia coliG6P: glucose-6-phosphate; G3P: glyceraldehyde-3-phosphate; PEP: phosphoenolpyruvate; E4P: erythrose-4-phosphate; DAHP: 3-deoxy-D-arabino-heptulosonate-7-phosphate; CHA: chorismate; Prep: prephenate; Phe: phenylalanine; Tyr: tyrosine; PYR: pyruvate; OAA: oxaloacetate; 2-HID: 2-hydroxyindole; 3-HID: 3-hydroxyindole

Fig. 2 Effect of introducing the fmo gene and its mutants into E. coli Nissle 1917 on the production of indirubinA: Agarose gel electrophoresis analysis of colony PCR for recombinant strains EIR01-03. Lanes show verification results for colonies of constructs: pI2e::fmo, pI2e::fmoK223R and pI2e::fmoK223R/D317S. B: Indirubin production by engineered strains EIR01-03 overexpressing fmoand its mutants. Significant differences were determined using student’s t-test (***P<0.001)

Fig. 3 Effects of knocking-out of gene related to the precursor synthesis on indirubin productionSignificant differences were determined using student’s t-test (**P<0.01; ***P<0.001; ns: not significant)

Fig. 4 Effects of deleting genes in the competitive pathway of chorismate biosynthesis on the production of indirubin

Fig. 5 Fed-batch fermentation of the engineered strain EIR10 for producing the indirubin

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