Overview

In inflammatory bowel disease (IBD), damage to intestinal epithelial cells leads to excessive immune activation, which in turn causes further tissue destruction—a vicious cycle that hinders recovery. In addition, non-adherence to medication can make achieving remission more difficult.

To address these challenges, we developed Xylego, an engineered E. coli strain equipped with a system composed of the following three modules:

1. A system that allows long-term colonization of the intestine by utilizing xylitol as a prebiotic, thereby reducing the required frequency of medication.
2. A system that promotes the healing of damaged epithelial cells and induces remission.
3. A system that self-destructs after treatment to minimize potential side effects.

Colonization support

To address the issue that one-third of patients fail to take their medication as prescribed, we developed a xylitol assimilation system that enables long-term colonization in the intestine, thereby reducing the frequency of administration. For more details, please refer to the Description section.

Probiotics are microorganisms that exert beneficial effects within the human gut. One such probiotic, Escherichia coli Nissle 1917 (EcN), has been studied as a potential treatment for IBD and is recognized as a safe strain [1].

When aiming to promote the growth of specific microbial populations, a prebiotic approach can be used. Prebiotics are substances that selectively support the growth of targeted microbes, thereby expanding only the intended population upon administration [2].

Therapies that combine probiotics and prebiotics to enhance colonization of beneficial microbes are called synbiotic therapies [2]. Administering probiotics and prebiotics together enables stable and long-term colonization of the probiotics.

Because EcN lacks a suitable prebiotic substrate for synbiotic therapy, we engineered it to metabolize xylitol, allowing EcN to use xylitol as a prebiotic and enabling its application in synbiotic therapy. This is a novel approach, making it possible for Xylego to establish itself in the intestine without losing the nutritional competition against native gut bacteria.

This system consists of the following two modules:

Module 1: Xylitol Uptake

E. coli retains a system for xylose uptake but not one specialized for xylitol. Therefore, expression levels of the transporter are low unless induced by xylose, resulting in insufficient xylitol uptake [3].

To overcome this limitation, we expressed a xylitol transporter on the E. coli cell membrane, enabling enhanced uptake of xylitol.

The transporter we introduced originates from Pantoea ananatis, a close relative of E. coli, which possesses the xyt operon containing xyB, xytC, xytD, and xytE, responsible for xylitol transport and metabolism [4].

Module 2: Integration of Xylitol into the Pentose Phosphate Pathway

The imported xylitol is converted into D-xylulose by xylitol dehydrogenase, and subsequently phosphorylated to xylulose-5-phosphate by D-xylulokinase, which then enters the pentose phosphate pathway (PPP).

The PPP is an endogenous metabolic pathway in E. coli, producing NADPH and protons from glucose-6-phosphate for ATP synthesis and generating ribose-5-phosphate, a nucleotide precursor [5].

Through this pathway, E. coli can utilize various carbon sources, such as xylose, by converting them into intermediates of the PPP.

Native E. coli metabolizes D-xylose by converting it to D-xylulose via xylose isomerase, and then to xylulose-5-phosphate through D-xylulokinase, thereby using it as a carbon source.

Building on this pathway, we introduced a xylitol dehydrogenase to enable the utilization of xylitol as a carbon source via the route illustrated in the figure.

The xylitol dehydrogenase gene was derived from [6], while the D-xylulokinase is encoded by the endogenous xylB gene of E. coli [7].

We ensured overexpression of these enzymes to prevent metabolic bottlenecks.

Because the reaction catalyzed by xylitol dehydrogenase consumes NAD+ as an oxidizing agent, excessive flux through this pathway could lead to NAD+ depletion, impairing xylitol metabolism.

To counter this, we introduced noxE, an enzyme from Lactococcus lactis that regenerates NAD+, catalyzing the following reaction.

Through these modules, xylitol taken up by the cells is metabolized to xylulose and xylulose-5-phosphate, and further processed via the pentose phosphate pathway.

Mucosal healing

In recombinant probiotic systems, several strategies exist for expressing and delivering therapeutic proteins.

1. Suppressing inflammation

   Interleukins (ILs) are a class of cytokines categorized as either pro-inflammatory or anti-inflammatory, depending on type.

   1. Anti-inflammatory cytokines such as Interleukin-10
   2. Antibodies targeting inflammatory cytokines such as Tumor Necrosis Factor-α [8]

2. Promoting tissue repair

   Epidermal Growth Factor (EGF) promotes epithelial cell proliferation [9].

In our interview with Professor Kiichiro Tsuchiya, he emphasized the need for therapeutic agents with mechanisms distinct from immunosuppression.

EGF is a growth factor naturally secreted from membrane-bound precursors and binds to the Epidermal Growth Factor Receptor (EGFR) on the cell surface, triggering proliferation signaling [10].

While EGF is endogenous, its receptor EGFR is a well-known target in cancer therapy [11] .

Because our engineered E. coli colonizes the gut, EGF must be secreted extracellularly to reach the inflamed regions. Since E. coli is a Gram-negative bacterium, several secretion mechanisms can be employed [12]:

1. A two-step pathway, where the protein is first transported from the inner membrane to the periplasm, then across the outer membrane.
2. A one-step pathway, where the protein passes through both membranes simultaneously via a tubular complex (e.g., hemolysin system).

The two-step pathway often results in protein accumulation in the periplasm and inefficient secretion, so we adopted the one-step pathway.

In this system, a signal sequence is fused to the C-terminus of the target protein. The EGF–LARD3 fusion protein (Lipase ABC transporter recognition domain 3) is secreted by the PrtD, E, F transporter complex derived from Erwinia chrysanthemi, a close relative of E. coli.

This system enabled wound recovery in human intestinal epithelial HCT-8 and non-transformed IEC-18 cells [13].

Unlike the TliD, E, F system, which functions best at 25 °C, the PrtD, E, F secretion system operates efficiently at 37 °C, making it suitable for intestinal secretion [18][18].

Kill Switch (Self-Destruction System)

The use of genetically modified E. coli in the human body poses risks of unintended release into the environment, which could disrupt ecosystems or promote the emergence of antibiotic-resistant strains.

Furthermore, if administered E. coli were to survive after inflammation subsides, excessive EGF signaling could lead to uncontrolled epithelial proliferation and increased cancer risk [9].

To prevent these risks, we introduced a Kill Switch that triggers bacterial self-destruction once inflammation has resolved.

Kill Switch

This system is modeled on a restriction–modification (RM) mechanism analogous to toxin–antitoxin (TA) systems [14].

It detects tetrathionate, a molecule elevated during inflammation, to control the activity of restriction and modification enzymes.

The system consists of four genes:

• TtrS – A tetrathionate sensor derived from Shewanella baltica, located in the cell membrane. Upon sensing tetrathionate, it phosphorylates TtrR [15].
• TtrR – A response regulator from Shewanella baltica that activates the promoter to induce expression of BsaIM and BsaI upon phosphorylation [15].
• BsaIM – A DNA methyltransferase from Geobacillus stearothermophilus (BsaIM1, BsaIM2). It methylates DNA to protect it and is tagged with ssrA for rapid degradation once inflammation subsides [16].
• BsaI (BsaIR) – A restriction enzyme from Geobacillus stearothermophilus, which cleaves DNA. It is more stable than BsaIM and remains active after BsaIM is degraded [16].

By adding the ssrA degradation tag immediately after BsaIM, we ensured that BsaIM is preferentially degraded [17].

During Inflammation

When tetrathionate is present, TtrS phosphorylates TtrR, which then activates the promoter to express both BsaIM and BsaI [15].

DNA is methylated by BsaIM and therefore protected from cleavage by BsaI, allowing bacterial survival.

After Inflammation

When inflammation subsides, tetrathionate concentration decreases and TtrS no longer activates TtrR.

As a result, expression of BsaIM and BsaI stops.

Because BsaIM is unstable, it degrades first, while BsaI persists.

During DNA replication, newly synthesized DNA is unmethylated, leaving it vulnerable to cleavage by BsaI.

Consequently, both plasmid and genomic DNA are cleaved, leading to bacterial death.

Removal of Antibiotic Resistance

This system also prevents the horizontal transfer of antibiotic resistance genes contained in the plasmid.

The plasmid used includes an ampicillin resistance gene, which could otherwise spread to other gut microbes during cell lysis.

By using BsaI, which can cleave the resistance gene sequence, the kill switch simultaneously destroys the plasmid and resistance genes during cell death, preventing their dissemination.

References

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