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Obesity is a chronic health issue caused by excessive fat accumulation in the body, often accompanied by metabolic syndrome. It has now become a major global public health challenge. The global number of overweight or obese individuals increased from 929 million in 1990 to 2.6 billion in 2021. Also, approximately 16 million adults died prematurely from noncommunicable diseases related to obesity[1]. Among these conditions, type 2 diabetes mellitus (T2DM)，accounting for 90–95% of all diabetes cases，is a metabolic disorder caused by insulin resistance and β-cell damage resulting from nutritional excess and physical inactivity. Among individuals with T2DM, 55% of premature deaths are associated with obesity[2]. In addition to surgical procedures such as laparoscopic Roux-en-Y gastric bypass and laparoscopic sleeve gastrectomy, GLP-1-based drugs have become a primary pharmacological treatment option in current mainstream obesity treatments due to their high patient compliance and active therapeutic efficacy. Glucagon-Like Peptide-1 (GLP-1), the core component of these drugs,is secreted by L cells in the small intestine. As an incretin hormone with dual hypoglycemic and weight-loss effects, it is a key substance for treating obesity and T2DM[3]. However, most GLP-1 receptor agonists are chemically synthesized drugs. Due to their short in vivo half-life, they require frequent injections and are prone to causing severe side effects. Therefore, developing ultra-long-acting hypoglycemic and weight-loss solutions is of significant importance. To this end, we are exploring the development of an oral probiotic formulation capable of long-term colonization in the intestines and inducing the production of modified GLP-1.
Most marketed GLP-1-based drugs rely on sequence modification or heterologous introduction to extend their in vivo circulation half-life, yet they still require regular and frequent injections. This project focuses on intestinal probiotics, leveraging their ability to flexibly respond to environmental signals and synthesize long-acting GLP-1 modified with unnatural amino acids. From the dual dimensions of "quality" (enhancing long-acting properties through unnatural amino acid modification) and "quantity" (regulating expression levels via genetic circuits) , we aim to develop live biotherapeutic products for ultra-long-acting blood glucose reduction and weight loss based on human-derived GLP-1, providing a solution with high patient compliance. The core technical modules of the project include genetic circuit design, protein modification, and delivery & testing hardware. (Return to design Page for detailed introduction)
With the coupled orthogonal split intein-transcription factor of intein trans-splicin as the core of AND logic,the regulation of GLP-1 synthesis and secretion integrates spatiotemporal signals through operons responsive to protocatechuic acid (PCA) and bile salts (BS), ensuring precise alignment between expression timing and physiological demand.
We have adopted a modular Amp30E signal amplification system to significantly enhance the expression level of GLP-1. Simultaneously, GLP-1 synthesis is regulated through a feedback control loop mediated by the glucose uptake negative-response promoter GURB3-2, thereby adapting to feeding and fasting states,which also prevents excessive activation of downstream signaling and reduces the metabolic burden of the bacterial strain.
Drawing on the design idea of semaglutide, we perform natural amino acid substitution and unnatural amino acid introduction at the cleavable sites of human-derived GLP-1. Through computer simulation methods including semi-rational design and molecular docking, the stable binding sites of GLP-1 with NEP-24.11 and GLP-1R were obtained. Biosynthesizable unnatural amino acids were selected and introduced site-specifically by combining genetic codon expansion (GCE) technology, which solves the core problem of its short in vivo circulation half-life and significantly improves the enzyme cleavage resistance and stability of GLP-1 as well.
An "OR-gate kill-switch" is constructed with arabinose and temperature as dual input signals, serving as the core part for active and passive regulation of the engineered bacteria for intestinal safety, respectively. It not only prevents the escape of engineered bacteria into the natural environment and the subsequent occurrence of horizontal gene transfer (HGT) but also provides operational flexibility for terminating the live bacterial therapy, ensuring the safety of clinical applications.
We have identified high biocompatibility hydrogels as the oral delivery carrier that enables engineered bacteria to tolerate gastric acid and digestive enzymes while colonizing the intestine. Meanwhile, the adhesion of the carrier is optimized to enhance the long-term colonization ability of engineered bacteria.In addition, we have designed an in vitro "turbidostat" to simulate the abundance of E.coli in the human intestine. This system aims to optimize the concentration of inducers for genetic circuits and induction time parameters, thereby providing data support for in vivo applications.
Figure 1. Schematic diagram of GlucoXpert including four core modules
The genetic circuits of live bacteria in vivo are highly complex. Plasmids of different modules need optimized integration, easily leading to excessive overall metabolic burden on the bacterial strain. Moreover, the human intestinal tract lacks an antibiotic selection mechanism, making plasmids prone to loss. Therefore, in practical applications, to enhance genetic stability and metabolic adaptability, key functional elements are better integrated into the genome of the chassis strain.
The currently used Escherichia coli Nissle 1917 (EcN) has a low abundance in the human intestinal tract, and a relatively short colonization time, making it difficult to exert a sustained effect. Further refinement of the selection of chassis strains such as Bacteroides and Lactobacillus with greater adaptability to the intestinal microenvironment and stronger colonization capacity is needed.
The metabolic duration of GLP-1 modified with unnatural amino acids in the human body, as well as the relationship between GLP-1 dosage and gastrointestinal side effects such as nausea and vomiting, remains unclear, requiring validation through subsequent clinical studies. Semi-quantitative determination technology should be employed to precisely measure the secretion levels, avoiding overdose risks and ensuring medication safety while enhancing stability and efficacy .
Differences in intestinal pH, bile salt concentration, and gut flora composition exist among different patients, which may lead to inconsistencies in the quantitative relationship between GLP-1 synthesis amount and inducers such as green tea, thereby affecting the universal efficacy. Although a feedback circuit has been initially designed, it still needs to be optimized to a more precise negative feedback regulation system to reduce the interference of individual variations on drug synthesis and ensure the stability of therapeutic efficacy.
Currently, the in vivo circulation stability of GLP-1, the activity after modification, and the colonization effect of the bacterial strain rely solely on in vitro modeling analysis, which cannot fully simulate the human homeostatic environment. Further in vivo studies such as animal experiments are needed for verification to provide a scientific basis for developing standardized medication regimens.
As a peptide drug, GLP-1 features low transmembrane absorption efficiency and is easily degraded by intestinal enzymes, leading to loss of activity. A more promising future therapeutic approach may be "in situ human cell editing". That is through external induction, intestinal cells directly produce and release modified GLP-1 into the blood circulation, addressing absorption and degradation issues form the source.
We aim to produce GlucoXpert as an oral live bacterial agent, with the final product being in the form of lyophilized powder or granules. We mix hydrogels encapsulating probiotics with glycerol protectant to avoid significant damage to probiotic viability during the lyophilization process. This not only enhances transport stability but also prevents strain death from premature triggering of the biosafety circuit before entering the body's 37°C environment, ensuring normal function.
After receiving the oral bacterial agent, users only need to follow the four steps below:
① Bacterial Agent Reactivation: Brew the bacterial agent with warm water at approximately 37°C (do not exceed 40°C to prevent heat-induced strain damage) to restore the lyophilized hydrogel to its natural form.
② Oral Administration: Directly take the reactivated bacterial agent . The hydrogel will protect EcN from gastric acid and digestive enzymes, allowing them to successfully reach the intestine for colonization.
③ Daily Induction: Drink one cup of low-sugar green tea before meals. PCA in green tea will induce the engineered bacteria to synthesize GLP-1, thereby inhibiting appetite, regulating metabolism, and achieving blood sugar reduction and weight loss effects.
④ Flexible Termination: If satisfied with the weight loss effect, drink an appropriate amount of arabinose to trigger the safety mechanism, eliminate the engineered bacteria in the body, and flexibly terminate the treatment.
Figure 2. Schematic diagram of the usage of GlucoXpert
Based on the above treatment regimen, patients can also develop the healthy habit of drinking green tea. The simple process of "Reactivation-Administration-Induction-Termination" provides a live biotherapeutic products option with both high compliance and usage safety that could replace frequent injections.
We use the SWOT business analysis model to evaluate the internal and external comprehensive conditions of oral bacterial agents from four perspectives: Strengths, Weaknesses, Opportunities, and Threats, clarifying GlucoXpert's core competitiveness and development direction.
① High Technological Innovation and Distinct Differentiation: We employed the well-recognized safe EcN as the chassis strain combining with the "AND-gate sensing circuits" to achieve precise and on-demand expression of GLP-1 in the intestine. At the same time, through the modification of the GLP-1 sequence with unnatural amino acids, the core problem of natural GLP-1's short half-life in vivo can be resolved, forming a unique competitive advantage in the technical route of oral GLP-1.
② Safety, Controllability, and Tolerability: EcN possesses functions of intestinal colonization, anti-inflammation, and gastrointestinal mucosal repair, which can significantly reduce the risk of immune rejection of genetically engineered bacteria. Additionally, the designed "temperature-arabinose OR-gate suicide system" enables both active and passive prevention, avoiding bacterial escape into the natural environment and gene contamination, thus meeting the clinical translation standards for live bacterial drugs.
③ Strong therapeutic synergy: It simultaneously achieves the dual effects of "blood glucose reduction" and "weight loss". Moreover, GLP-1 has the "glucose-dependent glucose-lowering" property, which can fundamentally avoid the risk of hypoglycemia. Meanwhile, oral administration exerts local intestinal effects of delaying gastric emptying and suppressing appetite, reducing side effects associated with injectable administration, and meeting the treatment needs of patients with obesity and T2DM.
④ High Administration Convenience and Compliance: With hydrogel as the oral delivery carrier, and the daily induction of production by green tea drinks, it solves the issue of frequent injections with traditional GLP-1 drugs. The operation is simple and easy to adhere to, making it particularly suitable for patients requiring long-term blood glucose control and weight loss, significantly improving treatment compliance.
① Need for Breakthroughs in Technological Maturity: Currently, the project is still in the proof-of-concept stage, and the core technical indicators have not been verified through large-scale preclinical studies. These include the actual colonization efficiency of the engineered EcN in the human intestine, the secretion amount of GLP-1, and its in vivo circulation utilization rate, which need to be further confirmed through animal experiments and in vitro intestinal simulation tests. Meanwhile, the tolerance and protective ability of the hydrogel carrier against gastric acid and digestive enzymes still need to be optimized to ensure the bacterial strains can successfully reach the intestine and achieve long-term colonizaztion.
② Individual Differences Increase the Difficulty of Efficacy Control: There are significant differences in the intestinal microenvironment and intestinal flora composition among different patients, which may lead to inconsistent signal activation efficiency of the "AND-gate sensing module". This makes it difficult to ensure the individual consistency of GLP-1 expression, complicating efforts of efficacy standardization and precise regulation.
③ Complex Large-Scale Production Process: As a genetically engineered live bacterial preparation, the high-density fermentation of engineered EcN requires strict control of parameters such as temperature, dissolved oxygen, and nutrient ratio to maintain bacterial activity. The biosynthesis of unnatural amino acids requires supporting customized metabolic regulation processes, leading to issues such as high costs and insufficient production stability during large-scale production.
④ High Threshold for Regulatory Approval: Currently, the global regulatory framework for "genetically engineered live bacterial drugs" has not been fully clarified, and there is a lack of unified standards for approval criteria. This may result in a more complex approval process and a longer cycle compared to traditional small-molecule drugs, delaying the clinical translation process.
① Surge in Global Demand for Metabolic Disease Treatments: According to data from the World Health Organization, in 2022, the global obese population accounted for 1/8 of the total. 55% of patients with T2DM died prematurely due to obesity, and the incidence rates of both diseases continue to rise with sedentary lifestyles and aging populations, providing a a vast target market for Glucoxpert.
② Policy Support for Innovative Biotherapies: Countries around the world have shown obvious policy inclinations towards "precision medicine", "prevention and treatment of metabolic diseases", and "live bacterial drugs". Relying on these policies, applications can be made for accelerated clinical approval and scientific research fund subsidies.
③ Strong Demand for Alternatives to Injectable GLP-1: Currently, mainstream GLP-1-based drugs still primarily rely on subcutaneous injection, resulting in insufficient long-term compliance among patients. The convenience of oral bacterial agents helps to enhance market competitiveness.
① Competitive Product Technology Iteration Pressure: Domestic and international pharmaceutical companies are accelerating the R&D of oral GLP-1 products, multi-target glucose-lowering and weight-loss drugs, and small-molecule GLP-1 receptor agonists. The technological iteration of these competitive products will continuously erode Glucoxpert's market space.
② Market Acceptance Risks of Genetic Engineering Products: Some members of the public have safety concerns about "genetically modified probiotics," which may drive up market education costs or trigger negative public opinion.
① Upgrade of Strains and Functional Modules: Modify the fimbrial or adhesion-related genes of EcN to enhance its adhesion to the intestinal mucosa and improve colonization efficiency.
② Preclinical and Clinical Verification: Complete long-term toxicity tests in animal models such as mice, monitoring changes in intestinal flora, liver and kidney function within 6 months, subsequently conduct Phase I clinical trials to verify the safety of the oral bacterial agent and the expression level of GLP-1, laying the foundation for Phase II efficacy trials.
Hydrogel and Bacterial Agent Composite Preparation Workshop: Build a "37 °C constant temperature production line" to realize uniform mixing and embedding of hydrogel raw materials and EcN. Equip with "lyophilization equipment" to quickly process the composite preparation into lyophilized powder for transportation and storage.
Patient Education and Brand Building: Develop "popular science manuals on blood glucose control and weight loss" and "guidelines for the use of oral bacterial agents", and reach patients through hospitals, pharmacies, and online health platforms. Provide professional personlized medication advice based on patients' real-time monitored blood glucose and weight data and free trial quotas for poor patients so as to enhance the social influence of the brand.
[1] GBD 2019 Risk Factors Collaborators (2020). Global burden of 87 risk factors in 204 countries and territories, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet (London, England) , 396(10258), 1223–1249.
[2] World Obesity Federation. (2025). World Obesity Atlas 2025. London: World Obesity Federation. https://data.worldobesity.org/publications/?cat=23.
[3] Zeng, Y. T., Wang, Y., & Zhang, H. L. (2024). Expert consensus on clinical application of glucagon-like peptide-1 receptor agonists (GLP-1RA). Today Pharmacy , 721-735.