Researchers have developed a sustainable biosynthetic platform that produces indican, a water-soluble precursor to indigo, using the probiotic bacterium Escherichia coli Nissle 1917 (EcN). The study, published in Bioresources and Bioprocessing, demonstrates that this engineered strain can synthesize indican from crude glycerol without the use of antibiotics or chemical inducers. This approach addresses the environmental and health concerns associated with traditional chemical indigo synthesis, which relies on aniline and formaldehyde feedstocks and generates carcinogenic byproducts. By leveraging the native high-copy plasmids of EcN, the team created a robust system for green indigo production that avoids the cytotoxicity issues often linked to the accumulation of hydrophobic indigo crystals in microbial cells.
The central challenge in microbial indigo production is the physical damage caused by indigo precipitation. Indigo is highly hydrophobic and spontaneously crystallizes within cells, disrupting membranes and inhibiting growth. Indican, however, is a glycoside derivative of indoxyl that remains water-soluble and chemically stable. This property allows it to serve as an ideal intermediate, as it can be easily extracted from fermentation broth and subsequently converted into high-purity indigo through controlled hydrolysis and oxidation. The research team focused on optimizing the biosynthetic pathway to maximize indican accumulation while maintaining the safety and stability of the probiotic chassis.
The engineering strategy involved the constitutive expression of two key enzymes: the flavin-containing monooxygenase FMOK223R and the glycosyltransferase PtUGT1. To further enhance production, the researchers blocked competing metabolic pathways by inactivating the genes pheA, tyrA, pykA, and ppc. Additionally, the disruption of the trpR gene alleviated feedback inhibition on the tryptophan biosynthetic pathway, ensuring a steady supply of the core precursor L-tryptophan for indican synthesis.
A critical bottleneck in the pathway was the low catalytic efficiency of the native PtUGT1 enzyme. To overcome this, the team employed a semi-rational mutagenesis strategy guided by molecular docking and computational protein design tools such as FireProt, PoPMuSiC, and PROSS. These algorithms identified residue S112 as a critical hotspot for functional optimization. Site-saturation mutagenesis at this site revealed that the S112A mutation significantly improved catalytic performance. The S112A variant exhibited a specific activity of 1.235 U/mg, which was 17.4% higher than the wild-type enzyme, primarily by accelerating the substrate turnover rate.
Further optimization involved the design of fusion proteins to facilitate substrate channeling between the coupled enzymes FMO and PtUGT1. The team tested various linkers and promoters, finding that a flexible linker combined with the Ptac promoter yielded the highest initial indican titers. This configuration allowed for efficient transfer of the intermediate indoxyl, minimizing its leakage and subsequent oxidation into unwanted byproducts. The integration of these genetic modifications resulted in the construction of the high-performance strain EG14, which represented a 553.1% improvement in indican titer compared to the initial parental strain.
In shake-flask cultivations, the optimized EG14 strain achieved a maximum indican titer of 449.3 mg/L after 24 hours. To evaluate the scalability and industrial applicability of this system, the researchers conducted fed-batch fermentations in a 2-L bioreactor using crude glycerol as the sole carbon source. Crude glycerol, a low-cost byproduct of biodiesel manufacturing, was selected to reduce raw material costs and promote sustainable biorefinery practices. The fermentation process was monitored for cell growth, product accumulation, and byproduct formation over a 32-hour period.
The fed-batch fermentation results demonstrated a peak indican titer of 1923.2 mg/L at 32 hours, with an average productivity of 60.1 mg/L/h. Cell growth reached a stationary phase at 28 hours, with a maximum optical density of 16.2. Throughout the fermentation, a low level of indigo byproduct, measured at 101.4 mg/L, was detected. This byproduct resulted from the spontaneous oxidation of indoxyl and accounted for approximately 5.3% of the total indican titer. The presence of this byproduct suggests that further optimization of oxygen levels and enzyme expression ratios could potentially enhance the overall yield.
The study highlights the distinct advantages of using EcN as a chassis for industrial biomanufacturing. Unlike conventional E. coli strains, EcN possesses inherent anti-phage properties and the ability to persistently colonize complex intestinal microenvironments. These characteristics make it a safe and versatile platform for the production of high-value chemicals.
The metabolic engineering approach also provided insights into the regulatory mechanisms governing indican biosynthesis. Exogenous tryptophan supplementation was found to inhibit indican accumulation in earlier strains, indicating that the pathway is primarily constrained by carbon source availability rather than precursor provision. The optimization of glucose and crude glycerol concentrations revealed that 4.0 g/L of glucose or 20.0 g/L of crude glycerol served as optimal carbon dosages. These findings underscore the importance of balancing metabolic flux to prevent cellular stress and maximize product output.
The use of computational tools in the enzyme engineering phase demonstrated the efficacy of multi-algorithm screening in identifying beneficial mutations. The integration of FireProt, PoPMuSiC, and PROSS allowed the team to pinpoint mutations that favored folding energy changes and catalytic activity. While the precise allosteric signaling pathways remain to be fully elucidated, the functional enhancement observed in the mutant enzyme provides a strong foundation for future structural studies.
This research contributes to the broader goal of replacing chemical synthesis with sustainable biological alternatives for natural pigment production. The ability to produce indican at high titers from waste biomass like crude glycerol aligns with the principles of green chemistry and circular economy. The engineered EcN strain offers a cost-effective and environmentally friendly solution for the textile, food, and pharmaceutical industries, which are major consumers of indigo and its derivatives.
Future work will focus on implementing adaptive laboratory evolution to improve the environmental tolerance of the strains and to optimize high-density fermentation processes. The team also plans to investigate strategies for mitigating the formation of indigo byproducts, such as maintaining microaerobic conditions or supplementing with antioxidants. By continuing to refine this platform, researchers aim to establish a robust industrial standard for the biomanufacturing of natural products using probiotic microbial chassis.
The study concludes that endogenous plasmid engineering in EcN provides a viable and scalable route for the production of indican. The elimination of antibiotics and inducers not only reduces production costs but also enhances the safety profile of the fermentation process. This work represents a significant step forward in the application of synthetic biology to the sustainable production of valuable natural compounds, offering a practical alternative to traditional chemical methods.
Subject of Research: Antibiotic- and inducer-free biosynthesis of indican from crude glycerol using engineered probiotic Escherichia coli Nissle 1917
Article Title: Antibiotic- and inducer-free biosynthesis of indican from crude glycerol using engineered probiotic Escherichia coli Nissle 1917
Article References: Mao, L.-J., Su, J.-Q., Mo, Y.-L., Wang, J., He, F.-C., Xiong, M., Liu, Y.-J., Wang, F.-Q., & Xiong, L.-B. (2026). Antibiotic- and inducer-free biosynthesis of indican from crude glycerol using engineered probiotic Escherichia coli Nissle 1917. Bioresources and Bioprocessing, 13(1), Article 135. https://doi.org/10.1186/s40643-026-01131-3
Image Credits: AI Generated
DOI: 10.1186/s40643-026-01131-3
Keywords: Antibiotic-, inducer-free, biosynthesis, indican, crude, glycerol, engineered, probiotic, Escherichia, coli, Nissle, scientific research
