Construction of Engineered Probiotics
With the prevalence of high-purine diets, hyperuricemia has gradually become younger and more widespread, with its incidence increasing year by year. Currently, most drugs for treating hyperuricemia have toxic and side effects, and their therapeutic effects are highly limited. Wild strains capable of degrading purine substances have been isolated and screened from the human body, food, and environment, and their application in hyperuricemic mouse models has shown a certain therapeutic effect. This further indicates that the direction of treating hyperuricemia through probiotics is feasible and has promising application prospects.
In recent years, the use of genetically engineered bacteria based on synthetic biology to reduce intestinal uric acid has become a major research focus, as they can be more specifically and effectively customized to address hyperuricemia and related diseases. This approach enhances the degradation capacity of strains by introducing specific metabolic pathways or genes, thereby more effectively reducing uric acid production and minimizing uric acid accumulation in the body.
Therefore, our team has conducted research on constructing engineered probiotics for the prevention and treatment of hyperuricemia. In the preliminary foundational work, the team used E. coli Nissle 1917 (EcN) as the chassis bacterium, overexpressed the xanthine transporter XanQ, enhanced its activity through rational design, and optimized its expression level in the cytoplasmic membrane—enabling it to efficiently transport xanthine and hypoxanthine.
The construction of the engineered probiotic chassis mainly includes the following two aspects:
(1) Double-Site Mutation of XanQ
Mutating the 88th serine to threonine and the 94th phenylalanine to tyrosine in XanQ significantly improved its xanthine transport capacity. The transport efficiency of XanQ F88T/F94Y was 213.19% that of wild-type XanQ.
The determination of xanthine transport ability for the XanQ double-point mutant compared with the wild-type XanQ
(2) Promoter and RBS Engineering
By selecting the promoter J23100 and RBS B0034, the xanthine transport level of the strain with EcN as the chassis was successfully increased by 8.6-fold, and its recognition ability for hypoxanthine was significantly enhanced (4.0-fold that of the original EcN). This optimized strain was named YES301.
Evaluation of the xanthine transport capacity of XanQF94Y/S88T after the expression level was regulated by different strength promoters (A) and RBS (B)
Experiment on Antibiotic-sensitive Engineered Probiotics
We successfully constructed the antibiotic-sensitive engineered probiotic YES302 by scarless knockout of the asd gene in the EcN1917 genome and introducing a recombinant plasmid carrying both the asd gene and the xanthine transporter XanQ mutant.
Performance Tests of YES302
(A) Plasmid passage experiment; (B) Xanthine transport capacity of YES302 and its 20th passage derivative; (C) Survival rate test under simulated gastrointestinal fluid conditions; (D) Xanthine transport capacity after simulation of gastrointestinal fluids.
Antibiotic Sensitivity of YES302
YES302 exhibited efficient extracellular xanthine transport capacity, with a transport efficiency of approximately 90%. The strain remained sensitive to multiple antibiotics in LB and M9 media and showed high plasmid stability—retaining over 95% of plasmid function even after 20 consecutive passages. In addition, YES302 had a significant survival rate under simulated gastrointestinal fluid conditions.
These results indicate that YES302 can reduce the xanthine concentration in the reaction system through efficient extracellular xanthine transport, demonstrating its potential as a safe and sustainable microbial therapy for hyperuricemia. This strain does not rely on traditional antibiotic selection markers, reducing biosafety risks and laying a foundation for its future clinical application.
Design of Uric Acid Transporter UacT
To enhance the uric acid clearance capacity of engineered probiotics, we expressed three membrane proteins with potential uric acid transport functions in the chassis strain E. coli Nissle 1917 (EcN 1917) and conducted in vitro uric acid transport experiments to compare their transport efficiencies.
The results showed that the strain harboring UacT (1917-UacT) reduced the initial uric acid concentration (100 μM) in the reaction system by more than 40 μM within 1 hour, exhibiting the strongest uric acid transport activity (Figure 1). Therefore, we selected UacT as the core uric acid transport module of the engineered bacterium YES303. Potential optimization targets of UacT were predicted through molecular docking analysis, laying a foundation for subsequent site-directed mutagenesis and directed evolution. In the future, we will further optimize the expression level of UacT through promoter/RBS engineering.
Results of Uric Acid Transport Experiments
(Note: The ordinate represents "Uric Acid Uptake (μM)", and the abscissa represents different strains: 1917, 1917-UacT, 1917-PucK, 1917-PucJ; the culture condition is "LB+UA".)
Development of Engineered Probiotics with Uric Acid Metabolism Ability
To reduce the amount of uric acid already produced in the body, we further superimposed the expression of uricase on the basis of the engineered probiotics, endowing the strain with uric acid degradation capacity. This provides a comprehensive and effective approach for the strain to prevent and treat hyperuricemia.
(A) Xanthine Transport and (B) Uric Acid Degradation of YES301-UricaseT Strain
After coupling with uricase, 1x10⁹ CFU/mL of the engineered bacterium could degrade 48.01% of uric acid when co-incubated with 100 μM uric acid for 60 minutes.
In-depth Dialogue with Stakeholders
Math Model & Suprobiotics
Based on experimental data and the Monod equation, we constructed the "Intestinal Purine-Serum Uric Acid Dynamic Model" and optimized it through three rounds of the Design-Build-Test-Learn (DBTL) cycle: In Cycle 1, we established the basic dynamic relationship among "intestinal purines, serum uric acid, and bacterial quantity"; in Cycle 2, we introduced a Gaussian pulse to fit dietary purine fluctuations and added an intestinal uric acid reabsorption module, reducing the simulation error of serum uric acid from 25% to less than 8%; in Cycle 3, we embedded the intelligent voice assistant UriGuard to enable automatic input of "personal characteristics + dietary data", with the error of personalized dosage calculation being less than 5%.
This work breaks the traditional "one-size-fits-all" medication model, provides a quantitative tool for clinical personalized probiotic dosage guidance, and promotes the transformation of hyperuricemia management from "empirical medication" to "precision calculation". The combination of the model and the voice assistant solves the cumbersome problem of manual input for users, enhancing the convenience and accessibility of medication guidance.
We innovatively built Suprobiotics, a comprehensive science popularization and communication platform in the probiotic field. For the general public, this platform can eliminate barriers to accessing health information and help them identify reliable probiotic products; for researchers, it can facilitate the sharing of synthetic biology technologies and academic cooperation; for industrial development, it provides a "scientific and transparent" development paradigm, promoting the transition of engineered bacteria from the laboratory stage to the market, which is in line with the requirements of "Healthy China 2030" for the transformation of innovative medical resources.
Inclusivity Success
We extended popular science lectures from Shanghai to remote mountainous schools; created Braille and audio brochures for the visually impaired, and developed a multilingual interactive assistant for the hearing impaired. For children with autism, we designed calm tactile-experience lessons, and for young learners, we crafted creative tools such as an intestinal whiteboard and children’s picture books.
Meanwhile, we integrated cultural creativity—combining probiotic knowledge with Tai Chi health classes and co-hosting the national "Light of Life" painting competition. We adopted color-blind-friendly color schemes on our popular science website to ensure a smooth browsing experience for those with visual impairments. Finally, we collaborated with 33 iGEM teams to publish the Smashing Synthetic Biology Myths handbook, addressing public concerns with clear scientific evidence.
These initiatives not only broke down barriers for different groups to access scientific knowledge but also enabled diverse populations to actively engage in the understanding and discussion of synthetic biology. They provide a replicable practical model for global research teams to conduct inclusive science popularization, and also promote the transformation of synthetic biology from a "professional field" to "shared by all", practicing the concept of "science knows no boundaries".
Future Plans
Wet Lab: Further Optimization
For YES302 – Advanced Functional Testing
We will expand the performance evaluation of YES302 by testing
its transport efficiency for mixed purine substrates, simulating the complex purine composition found in real
diets. This will help assess the strain’s effectiveness in physiologically relevant conditions.
For YES303 – Safety and Transport Optimization
To enhance biosafety, we will remove the antibiotic
resistance marker in YES303 using the same auxotrophy-based strategy applied in YES302. Functionally, we plan
to carry out site-directed mutagenesis of UacT combined with promoter/RBS engineering to further improve the
transporter's activity and expression strength, aiming for higher uptake rates of uric acid.
For YES304 – Safety and Degradation Optimization
We will also apply the antibiotic-free safety design to
YES304 by deleting its resistance marker. Meanwhile, we will optimize URIT expression through promoter and RBS
engineering to enhance its secretion and uric acid degradation capacity in vivo.
Toward an Integrated Therapeutic Strain
Finally, we aim to combine the optimized modules into a single
next-generation strain that integrates: Front-end xanthine transport to reduce precursors of uric acid in the
gut, and Back-end uric acid transport and degradation to remove residual uric acid. This integrated engineered
probiotic is expected to provide a comprehensive and efficient microbial therapy for hyperuricemia.
Human Practice: Continuously Evolving
We plan to update the Smashing Synthetic Biology Myths Handbook, adding new topics such as "Safety of Interaction Between Engineered Bacteria and Intestinal Flora" and "Verification of No Side Effects from Long-Term Use"; develop an animated short film on "Intestinal Purine Metabolism" (in multiple languages) and a "Probiotic Puzzle Game" for kindergartens; and collaborate with iGEM teams to conduct global popular science lectures. These efforts aim to specifically address the public's concerns about transgenic technology, lower the cognitive threshold for synthetic biology, achieve "full-age-group" coverage of popular science through diverse educational tools, and enhance the public's awareness of hyperuricemia prevention and trust in engineered bacteria therapy.
We will also establish a "quarterly interview + real-time feedback" mechanism, regularly inviting clinicians, enterprise representatives, and users from special groups to participate in seminars to collect suggestions on strain optimization and platform functions; additionally, a "demand feedback channel" will be added to the Suprobiotics platform to promptly respond to users' needs regarding popular science content and product functions.
Commercialization Blueprint
In accordance with the Classification of Biological Products and Requirements for Application Data, we will compile the safety data (such as antibiotic sensitivity and gene transfer risk tests) and efficacy data of the YES302/YES304 strains, and plan to submit an Investigational New Drug (IND) application within one year. We will collaborate with hospitals to conduct Phase I clinical trials (focused on the tolerance of healthy volunteers) and Phase II clinical trials (focused on verifying efficacy in patients). These efforts aim to promote the transformation of engineered bacteria from "laboratory achievements" to "clinical drugs". Through compliance procedures and clinical data verification, we will ensure the safety and efficacy of the products, providing legal and scientific basis for their market launch.
We also plan to cooperate with enterprises to optimize industrial-scale fermentation processes (expanding fermenter scale and reducing costs) and develop freeze-dried microcapsule formulations that can be stored at room temperature. In the initial stage, the products will be promoted in China through the "hospital prescription + online platform" model, with differentiated pricing (high-end customized version and basic version). This will address the issues of "high cost and difficult transportation" in the industrialization of engineered bacteria and expand the product coverage. The differentiated pricing and regional adaptation design will meet the needs of different populations, promoting engineered bacteria therapy to become a safe treatment option for hyperuricemia patients worldwide, and creating both economic value and public health value.