Design

Epilepsy is a common neurological disease characterized by spontaneous recurrent seizures, which can occur throughout the age group. Although anti-epileptic drugs (ASDs) can effectively control a small number of patients with spontaneous recurrent seizures, at least 1 / 3 of patients still show persistent seizures and subsequent negative health consequences, such as cognitive impairment and comorbid mental health problems. High-fat and low-carbohydrate ketogenic diet (KD) is one of the effective alternatives for the treatment of drug-resistant epilepsy. KD was designed to simulate fasting in the 1920 s and has been reported to control seizures for several years. KD was mainly manifested as generalized ketosis, and the concentrations of ketone bodies (KB),β-hydroxybutyrate (BHB), acetoacetate (ACA) and acetone were significantly increased.

Ketone bodies, including β-hydroxybutyrate (BHB), acetone (Ac), acetoacetate (AcAc), etc., as an indispensable alternative metabolic fuel source, are recognized to play an important role in various fields of life and have attracted the attention of many researchers. These ketone bodies are thought to be involved in systemic energy metabolism and energy supplementation in extrahepatic tissues.

Originally, it was believed that the benefits of the ketogenic diet were due to the decrease in glucose metabolism; however, many recent studies have shown that some of these benefits are instead caused by an increase in ketone body concentrations in the blood. A variety of synthetic ketogenic compounds have been shown to increase ketone body concentration in the blood to the point where dietary ketosis can be maintained without a change from a normal diet.

In recent years, elevated blood ketones concentration by endogenous and exogenous interventions has been widely studied in experimental and clinical research, showing great application potential, including applications in relieving epilepsy, Parkinson’s disease, gout flares, respiratory tract influenza virus infections, and obesity.

However, it is important to realize that elevated circulating BHB has complex physiological consequences and is considered as an attractive strategy to treat multiple diseases by some researchers, which is why the ways to elevate circulating ketones exogenously have been developed, such as oral administration of exogenous BHB supplements and intravenous injection of BHB.

However, relying solely on exogenous supplementation of BHB to increase the level of ketone body circulation in the body makes it difficult to achieve long-term stable maintenance. Moreover, it often fails to fully account for differences in physiological metabolism among individuals, which can easily lead to blood ketone accumulation and trigger complications such as ketoacidosis. In addition, it is obviously not easy to increase blood ketone levels solely through a low-carbohydrate, high-fat ketogenic diet. This dietary structure is inconsistent with people’s normal eating habits, resulting in extremely low adherence and a significant reduction in quality of life.

In order to avoid the side effects and low compliance of ketogenic diet, we use synthetic biology method to synthesize ketone body to treat epilepsy. A pathway capable of producing ketone body β-hydroxybutyric acid (BHB) in E.coli Nissle 1917 was designed and a dynamic regulation module was added by us. The engineered bacteria were wrapped in hydrogel and made into capsules. After oral administration, the engineering bacteria were colonized in the intestinal tract to synthesize BHB for the treatment of epilepsy.

Referring to a variety of existing BHB synthesis pathways, in our project, the BHB biosynthesis pathway was successfully constructed when genes of β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) from Ralstonia eutropha, and propionyl-CoA transferases (pcT) from Megasphaera elsdenii were introduced into Escherichia coli. In this pathway, BHB is excreted by PCT catalytic reaction coupled with the regeneration of acetyl-CoA, the starting material for the synthesis of BHB. Considering the reaction balance of PCT, since acetic acid is the receptor of CoA and is rich in human intestine, the production of BHB catalyzed by PCT is expected to use acetic acid in the intestine to promote BHB production and reduce the metabolic burden of engineering bacteria.

According to the existing studies, the medical reference range (mean±1.96 standard deviation) of blood BHB concentration was 1.1-4.9 mmol/L when receiving classical ketogenic diet treatment. It is suggested that the lower limit of blood BHB concentration should be maintained above 1.1 mmol/L to ensure the efficacy of ketogenic diet. In order to control the amount of ketone bodies produced by intestinal engineered bacteria and prevent the diseases and side effects caused by the accumulation of ketone bodies in the human body, we tried a variety of regulatory pathways to make the modified E.coli produce an appropriate amount of BHB stably.

Initially, considering that there is no sensor that can be used in E.coli to directly respond to BHB, we envisaged the establishment of a negative feedback regulation system that responds to intermediate product feedback in BHB production. The transcriptional expression of PhaB is coupled with Luxl, the synthase of the self-inducing molecule 3-oxohexanoylhomoserine lactone (AHL). When the amount of PhaB enzyme is more, Luxl synthesizes more AHL. The accumulated AHL is recruited by the transcription factor LuxR, and then dimerizes into an activator form. The transcription of downstream PhaA shRNA starts after the activator binds to the lux-box region of the QS Plux promoter. The specific shRNA is processed into siRNA (small interfering RNA) by the intracellular mechanism after transcription, silencing PhaA to cut off the BHB production pathway and redirect the carbon flow to the TCA cycle. Furthermore, we used a transcription regulator that responds to the PhaA product acetoacetyl-CoA. The increase of acetoacetyl-CoA promotes the expression of PhaB and Luxl, and sensitively activates the inhibition of PhaA expression, thereby controlling the production of BHB. By selecting promoters with different sensitivity to Luxl-LuxR to control the transcription of PhaA interfering RNA, we can control the range of BHB production allowed by the entire feedback loop.

However, acetoacetyl-CoA is involved in many metabolic pathways of E.coli.Its concentration is difficult to estimate and control during normal metabolism of bacteria, and it is impossible to construct accurate feedback regulation for BHB production pathway according to the concentration of acetoacetyl-CoA.

In the next step of improvement, we down-regulated the entire intermediate-enzyme negative feedback control loop as a whole. According to the reaction product BHB-CoA in the next step, the transcription regulator responding to BHB-CoA was used to control the expression of LuxI and realize the expression regulation of PhaB.

However, after reviewing the literature, we did not find a BHB-CoA-responsive transcription factor. Therefore, we changed our thinking and used quorum sensing to construct a BHB yield regulation pathway.

A quorum sensing (QS) was designed to form a negative feedback to silence BHB synthesis-related enzymes by RNAi and autonomously regulate BHB production. The QS gene circuit mainly synthesizes self-induced AHL through the constitutive AHL synthase Luxl, and regulates shRNA expression in a cell density-dependent manner. In the case of high cell density,the accumulated AHL is recruited by the transcription factor LuxR and then dimerized into an activator form. Downstream transcription begins after the activator binds to the lux-box region of the QS Plux promoter. The concentration of AHL increased with cell growth, resulting in an increase in the transcription level of specific shRNA. The two specific shRNAs were processed into siRNA (small interfering RNA) by intracellular mechanism after transcription, which inhibited the synthesis of BHB synthesis-related enzymes, closed the BHB production pathway, and formed a negative feedback regulation of BHB production.

In another more refined regulatory circuit, we used CRISPRi to silence BHB synthesis-related enzymes and TCA cycle key enzymes to autonomously regulate BHB production. The AHL accumulated in the QS gene circuit with the increase of bacterial density is recruited by the transcription factor LuxR, and then dimerizes into an activator form. Downstream transcription begins after the activator binds to the lux-box region of the QS Plux promoter. The concentration of AHL increased with cell growth, resulting in an increase in the transcription level of two specific gRNAs. Two specific gRNAs bind to dCas9 and inhibit the translation of BHB synthesis-related enzymes and TCA cycle key enzymes, respectively. When the BHB production pathway is shut down, the cell survival is inhibited by inhibiting the TCA cycle, and the cell density is reduced, that is, the AHL concentration is reduced, forming a negative feedback regulation to ensure the stable production of BHB. In comparison to RNAi, CRISPRi appears to produce a more consistent and robust knockdown given the same number of effector RNAs. Therefore, the loop is expected to produce better control effect.

After referring to the official iGEM white list, we decided to abandon RNAi technology and Cas protein-mediated CRISPRi technology for greater biosafety. At the same time, we simplified the regulatory circuit and used the common bacterial toxicity protein ccdB to control bacterial density. When the bacterial density is too high, the AHL synthesized by the constitutive synthetase Luxl is recruited by the transcription factor LuxR, which activates the expression of ccdB and reduces the bacterial density, thus forming a concise and efficient negative feedback to control BHB production.

We designed such a regulatory pathway and expect to select an appropriate strength promoter through a large amount of experimental data to ensure that a certain size of the bacterial population produces an appropriate amount of BHB, thereby better achieving our goal.

To ensure that engineered E. coli colonizing the intestine initiates suicide after leakage into the extraintestinal environment, and survives in the non-intestinal environment before taking the drug, we designed the following oxygen concentration suicide system with additional pre-dose precaution. The system consists of three modules: PhlF repressor protein controlled by PfdhF promoter, ssrA-tag modified CI repressor controlled by PhlF promoter, operon formed by PVHb promoter and OR operator, and ccdB toxic protein controlled by it. The Tet R family repressor regulatory promoter PhlF from B.subtilis promotes the expression of Cl repressor with ssrA-tag, specifically binds to the OR operator, and acts as an precaution to prevent the activation of the microaerobic promoter PVHb before medication and initiates the suicide mechanism. After administration, the hypoxia promoter PfdhF is activated by the anaerobic environment in the intestine, and PhlF repressor protein is expressed and specifically binds to PhlF and inhibits its function, that is, Cl repressor is expressed. The ssrA-tag ensures that once the induction signal stops the synthesis of CI protein, any pre-existing CI molecules in the cell will be rapidly degraded, relieving its inhibition of the micro-oxygen promoter PVHb. Since then, once the engineered bacteria leaked from the anaerobic environment of the intestine and came into contact with a higher oxygen concentration environment, PVHb would activate ccdB and cause the engineered bacteria to kill themselves.

To mitigate the potential safety risks of engineered bacteria in the intestinal tract, we have considered a series of strategies, including the design of auxotrophic engineered bacteria, the splitting and modification of genetic elements, and physical isolation. The design of auxotrophic bacteria relies on bacterial metabolic pathways; however, the metabolic pathways of commonly used engineered bacteria have not yet been fully characterized, posing a risk of uncharacterized metabolic pathways compensating for metabolites. Additionally, the intestinal environment is complex with a rich diversity of microorganisms, so this approach was ruled out. The splitting and modification of genetic elements involve genetic engineering and are relatively complex—transferring multiple plasmids increases the metabolic burden on E. coli. In contrast, hydrogels operate on a relatively simple principle with direct effects. Therefore, this project proposes encapsulating engineered bacteria within hydrogels to form a stable physical barrier between the engineered bacteria and the external environment. This not only prevents horizontal gene transfer between the engineered bacteria and other intestinal microorganisms but also leverages the hydrogel’s adhesiveness to facilitate the colonization of engineered bacteria in the intestine.

In a broad sense, hydrogels refer to three-dimensional polymeric networks formed via chemical crosslinking (through covalent bonds), physical crosslinking (through non-covalent interactions),or composite crosslinking (a combination of both). They exhibit unique properties such as softness, flexibility, porosity, permeability, biocompatibility, and similarity to soft living tissues. Hydrogels are classified into various types based on common criteria: their source (natural, synthetic, and hybrid), synthesis method (homopolymerization, copolymerization, semi-interpenetrating polymer networks, and interpenetrating polymer networks), crosslinking mechanism (physical and chemical), and ionic charge of functional groups (neutral/non-ionic, ionic, and amphoteric). With the development and application of hydrogels in biomedicine, a new classification method has emerged based on their responsiveness to environmental changes. Hydrogels with "sensor-like" properties, which undergo changes in physicochemical properties in response to minor environmental fluctuations, are collectively termed stimuli-responsive hydrogels, environment-sensitive hydrogels, or "smart" hydrogels.

Probiotics typically exert their effects through the digestive system after oral administration, which includes the oral cavity, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, and ileum), and large intestine (cecum, colon, and rectum). During this process, the viability of probiotics faces severe challenges from the digestive system, such as gastric acid and digestive enzymes. Beyond the survival rate of probiotics in the external environment, the transit time and physiological conditions of different parts of the digestive tract also determine whether probiotics can successfully reach the colon and maintain their functional activity. Saliva contains mucins, mineral ions, and amylase. After oral administration, probiotics inevitably undergo digestion by saliva, and salivary amylase may impair their survival rate. Approximately 20 seconds after mixing with saliva, probiotics pass through the esophagus (a process taking about 10–14 seconds) and reach the stomach. Probiotics generally thrive in a neutral environment (pH ~6–7), so the high acidity of gastric juice (pH ~1–3.5) significantly impairs their activity. This is because the highly acidic conditions in the stomach lower the cytoplasmic pH of probiotics, leading to increased intracellular hydrogen ion levels and reduced glycolytic enzyme activity. Such changes affect the F1F0-ATPase proton pump, which is closely associated with the survival of probiotics under acidic conditions. Furthermore, probiotics in the stomach are exposed to mechanical churning, high ionic strength, digestive enzymes, and other potentially adverse factors, which cause inactivation or death—these are critical issues that need to be addressed for oral probiotic administration.

Hydrogels have emerged as promising targeted drug delivery systems (TDDS) due to their versatility, including pH sensitivity, biodegradability, thermosensitivity, and mucoadhesive properties. In recent years, hydrogels have been widely used in drug delivery owing to their excellent drug protection capabilities and biocompatibility, with their administration routes evolving from topical dressings and in situ injections to oral delivery. For example, Liu et al. described pharmaceutical formulations based on tough liquid-to-gel in situ forming hydrogels, which solidify directly in the stomach to protect encapsulated drugs, therapeutic enzymes, and beneficial bacteria from gastric acid degradation. Additionally, hydrogels can be used to deliver natural and synthetic cytotoxic drugs in the breast cancer microenvironment, thereby enhancing the efficacy of chemotherapeutic agents for breast cancer treatment. Engineered E. coli encapsulated in hydrogels have also been employed as non-invasive diagnostic tools to assess the progression of intestinal inflammation.

Sodium alginate is a derivative of alginic acid, a natural polymer composed of β-1,4-D-mannuronic acid (designated as M units) and α-1,4-L-guluronic acid (designated as G units) linked via glycosidic bonds; its chemical structure is shown in Figure 2. The unique spatial conformation of G units endows sodium alginate with strong gelling ability, while the abundance of carboxyl groups on the uronic acid monomer units confers pH sensitivity. Due to its biocompatibility, low toxicity, relatively low cost, and mild gelation induced by the addition of divalent cations (e.g., Ca²⁺), sodium alginate has been extensively studied and applied in numerous biomedical fields.

Polydopamine (PDA) is a synthetic material inspired by mussel adhesive proteins. Owing to the strong adhesion of its catechol groups, it has been widely used as a coating for various surfaces. The polymerization of dopamine occurs under cytocompatible conditions, and the resulting PDA exhibits negligible cytotoxicity. Due to the inherent presence of amines, thiols, and hydroxyl groups on cell membranes, PDA can be deposited onto cell surfaces through the formation of covalent bonds or hydrogen bonds. It is hypothesized that a robust coating with multimodal motifs can be achieved via the co-deposition of functional molecules and dopamine.

By reviewing the literature, we designed a hydrogel primarily composed of sodium alginate (SA) and polydopamine (PDA), adopting a three-layer structure. This structure consists of an outer pH-responsive layer, a middle adhesive layer, and an inner nutrient layer.

The calcium alginate gel film of the outer pH-responsive layer is formed via the frozen inverse spheroidization method, featuring an uneven pore size distribution. Its outermost part is a dense surface layer with the highest crosslinking degree, formed by the initial contact with a high-concentration SA solution; the pore size here can be as small as several tens of nanometers, serving as the main functional barrier. In contrast, the inner part is a relatively loose transition layer formed by the inward diffusion of Ca²⁺. This pore structure enables it to effectively block the passage of microorganisms while allowing small-molecule substances to diffuse in a controlled manner. The SA backbone contains a large number of carboxyl groups, leading to reversible ionization and deionization processes in solutions with different pH values. Therefore, hydrogels based on SA generally exhibit pH responsiveness. In gastric juice, SA shrinks to form a dense, insoluble film, preventing the release of encapsulated drugs; upon reaching the intestine (with a high pH value), the SA film dissolves, thereby exposing the middle layer.

The middle adhesive layer was developed with improvements based on the methods of Tang T C and Su. By virtue of the Michael addition and Schiff base reactions, a covalently grafted PDA-SA network is formed. Subsequently, free radical polymerization of acrylamide occurs, creating an extremely dense nanoscale microporous network. This network allows water, ions, small-molecule nutrients, and metabolites to diffuse in and out while significantly impeding the passage of macromolecules and microorganisms, thus preventing gene leakage. Additionally, due to the inherent properties of PDA—its high catechol group content—it can form strong non-covalent and covalent bonds with various organic and inorganic surfaces through multiple adhesion mechanisms, such as hydrophobic interactions, bidentate chelation, and π-π stacking. The introduction of PDA in the preparation of the middle layer enhances adhesiveness, enabling the exposed middle-layer hydrogel to adhere to the intestine for colonization.

The inner nutrient layer uses a combination of SA and E. coli culture medium to form the hydrogel core. It has a loose and porous structure, which can effectively encapsulate E. coli, ensure the encapsulated bacteria receive sufficient nutrients and oxygen, facilitate waste exchange, and provide the necessary space for their initial growth and proliferation.