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Gut Microbes
Gut Microbes
Gut Microbes
1949-0976
1949-0984
Taylor & Francis

39288233
10.1080/19490976.2024.2399213
2399213
Version of Record
Research Article
Research Paper
Empowering probiotics with high xanthine transport for effective hyperuricemia management
Z.-P. ZOU ET AL.
GUT MICROBES
https://orcid.org/0000-0002-1110-0803
Zou Zhen-Ping *
Li Ju-Ling *
Zhang Yi-Fan
https://orcid.org/0000-0001-9241-1293
Zhou Ying
https://orcid.org/0000-0002-5555-5359
Ye Bang-Ce
Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, Institute of Engineering Biology and Health, East China University of Science and Technology , Shanghai, China
CONTACT Ying Zhou zhouying@ecust.edu.cn
Bang-Ce Ye bcye@ecust.edu.cn Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
* These authors contribute equally to this work.

17 9 2024
2024
17 9 2024
16 1 2399213Integra17 9 2024
Integra17 9 2024
06 5 2024
24 8 2024
28 8 2024
© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Hyperuricemia, a prevalent metabolic disorder, poses a susceptibility to various complications. The conventional pharmacotherapeutic approaches for hyperuricemia often entail notable adverse effects, posing substantial clinical challenges. Hence, the imperative lies in the development of novel, safe and effective strategies for preventing and treating hyperuricemia. Here, we developed a probiotic Escherichia coli Nissle 1917 strain, designated as YES301, which contains a rationally designed xanthine importer XanQ, enabling efficient uptake of xanthine and hypoxanthine, consequently leading to reduced serum uric acid concentrations and amelioration of renal impairments in a murine model of hyperuricemia. Importantly, YES301 exhibited a therapeutic efficacy comparable to allopurinol, a conventional uric acid-lowering agent, and manifesting fewer adverse effects and enhanced biosafety. These findings highlight the promising potential of engineered probiotics in the management of hyperuricemia through reducing intestinal purine levels.

KEYWORDS

Engineered probiotics
Hyperuricemia
intestinal purine
National Key Research and Development Program of China 10.13039/501100012166 2023YFF1204500 the National Natural Science Foundation of China 22134003 China Postdoctoral Science Foundation 10.13039/501100002858 2023M741176 This work was sponsored by the National Key Research and Development Program of China [2023YFF1204500], the National Natural Science Foundation of China [22134003], and China Postdoctoral Science Foundation [2023M741176].
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pmcIntroduction

Hyperuricemia is a metabolic disorder caused by disturbances in purine metabolism, and it is associated with various common chronic diseases including gout, chronic kidney disease, cardiovascular diseases, and diabetes.1,2 Currently, pharmacotherapeutic interventions such as allopurinol, probenecid, and rasburicase predominantly address hyperuricemia via three mechanisms: firstly, by inhibiting xanthine oxidase activity to reduce uric acid production; secondly, by impeding uric acid transporter function to enhance uric acid excretion; and thirdly, by promoting uric acid metabolism to allantoin to diminish uric acid levels.3 Nonetheless, these pharmacological agents predominantly target renal pathways, thereby frequently precipitating adverse effects of considerable severity,4–6 Approximately two-thirds of the total uric acid present in the human body undergoes renal excretion, with the remaining one-third being eliminated via the gastrointestinal tract.7 This underscores the substantial contribution of intestinal processes to the overall metabolism of uric acid. Although the uricase gene is silent in humans,8 the gut microbiota maintains close relevance to disorders in uric acid metabolism due to its potential engagement in purine and uric acid transportation or metabolism.9 However, the intricate and opaque nature of the physiological behavior of natural microorganism poses challenges to control, leading to a limited capacity to mitigate serum uric acid levels.

In recent years, advances in genetically engineered probiotic therapies, based on synthetic biology strategies, have yielded promising outcomes in the prevention and treatment of metabolic diseases,10–12 inflammatory bowel diseases,13–15 tumors,16–18 and diverse pathological states.19 Several engineered bacteria expressing uricase have been developed to augment uric acid catabolism within the intestinal milieu.20,21 Nonetheless, their efficacy in diminishing serum uric acid concentrations appears circumscribed, conceivably attributable to the anaerobic environment of the intestines, which may attenuate uricase activity. To address this, Zhao et al. enhanced uricase activity under anaerobic conditions by co-expressing hemoglobin VHb and catalase KatG in engineered bacteria expressing uricase.22 However, despite these efforts, oral administration of these engineered bacteria failed to ameliorate hyperuricemia in murine models. Subsequently, He et al. proposed that the subcellular localization of uricase in bacteria profoundly influences its activity. In pursuit of this hypothesis, they engineered recombinant bacterial strains to express periplasmic uricase, aiming to alleviate hyperuricemia in mice.23 Although these studies offer promising insights into the potential of engineered bacterial therapies for hyperuricemia, it remains apparent that current iterations of such interventions primarily target the degradation of preexisting uric acid within the gastrointestinal tract, thus posing challenges in curtailing de novo uric acid production. It is widely recognized that dietary purines, once ingested, are absorbed through the intestinal tract and subsequently enter the circulatory system.24,25 These purines are primarily metabolized by hepatic and renal pathways to produce uric acid, a small fraction of which is excreted into the intestines via the biliary system.26 Consequently, the intestines are not the primary site for uric acid synthesis, which suggests that strategies focusing on uric acid degradation in the intestines might not be the most effective approach. Considering the significant role that dietary purines play as precursors to uric acid synthesis in the human body, it can be hypothesized that reducing purine content in the diet could represent a viable preventive or therapeutic strategy against hyperuricemia.

In this study, we overexpressed the xanthine transporter protein XanQ in the probiotic Escherichia coli Nissle 1917 (EcN) strain, substantially augmenting the bacterium’s proficiency in xanthine uptake. Further rational design and directed evolution of the XanQ protein significantly improved its efficiency in transporting both xanthine and hypoxanthine. Subsequent optimization of the mutated XanQ protein’s expression levels via promoter and RBS engineering culminated in the development of a genetically engineered probiotic variant denoted as YES301, which exhibited an 8.6-fold and 4.0-fold increase in xanthine and hypoxanthine transport capacity compared to the wild-type strain. Upon oral administration of YES301 to a mouse model of hyperuricemia, a significant reduction in serum uric acid levels to within physiological ranges ensued, concomitant with alleviation of kidney damage in the mice. Importantly, YES301 demonstrated efficacy comparable to the clinical drug allopurinol, with fewer side effects and higher biocompatibility.

Results

Screening and rational design of xanthine transporters

To enhance the transport ability of extracellular xanthine by probiotic EcN, we separately incorporated two xanthine transport genes, xanP and xanQ, derived from E. coli MG1655, into the EcN chassis. Xanthine uptake experiments demonstrated that the overexpression of XanP and XanQ proteins can greatly enhance EcN’s transport of extracellular xanthine. Notably, XanQ exhibiting a higher transport efficiency to xanthine compared to XanP (Figure 1a). Consequently, we chose the XanQ transporter for further research. Figure 1. Screening, optimization, and characterization of xanthine transporter proteins. (a) XanQ and XanP transports enhance the uptake of xanthine by EcN. The EcN (OD600 = 0.6) harboring empty plasmids, XanP, and XanQ were suspended in M9 medium supplemented with 100 μM xanthine, and incubate for 60 min before measuring the xanthine content in the supernatant (mean ± SEM, n = 3). (b) The structural model of XanQ docking with xanthine. (c) Determination of the transport capacity of xanthine by different mutations of F94 (mean ± SEM, n = 3). (d) Compared to wild-type XanQ, XanQF94Y exhibit enhanced xanthine transport capabilities (mean ± SEM, n = 3). (e) Assessment of xanthine transport capacity through dual site mutations (mean ± SEM, n = 3). (f) Compared to wild-type XanQ, XanQF94Y/V150C, XanQF94Y/T72C, XanQF94Y/D276E and XanQF94Y/S88T exhibit enhanced xanthine transport capabilities (mean ± SEM, n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test, ***p ≤ 0.001, ****p ≤ 0.0001.

In light of the unavailability of an X-ray crystal structure for the xanthine transporter XanQ, AlphaFold was employed for predictive modeling. Subsequently, multiple docking simulations were conducted using AutoDock Vina to derive the docking structure model with the lowest energy state. The ensuing interaction between xanthine and XanQ was scrutinized through PLIP (Protein Interaction Learning Platform) analysis and visualized using Pymol (Figure 1b). Our investigation showed that the xanthine binding pocket within XanQ contained TM 3, TM 8, and TM 9. Notably, a π-stacking interaction was discerned between the benzene ring of F94 and the imidazole ring of xanthine, resulting in a stable non-covalent interaction at a distance of 5.14Å. Subsequent saturation mutations were introduced at the F94 site, and the resultant mutants were integrated into the EcN chassis to evaluate xanthine transport capacity. Remarkably, the F94Y mutation exhibited a pronounced enhancement in xanthine transport capacity (Figure 1c,d). Molecular docking analyses indicated that the F94Y mutation led to a reduction in the distance of π-stacking to 4.84 Å, thereby fostering tighter substrate-protein binding and potentially augmenting substrate recognition, binding, and transport efficacy (Supplementary Figure S1). In pursuit of further refining transport efficiency, a dual-site combination mutation library predicated on F94Y was constructed. Intriguingly, combinations such as F94Y/L86M, F94Y/D276E, and F94Y/S88T manifested improved xanthine transport capabilities in XanQ (Figure 1e). Particularly noteworthy is the observation that XanQF94Y/S88T displayed a 2.1-fold increase in xanthine transport capability relative to XanQ (Figure 1f).

Moreover, studies have reported that the rearrangement of the XanQ F94 site can broaden its substrate spectrum, allowing it to recognize and transport hypoxanthine. Hence, we examined the transport capacity of XanQF94Y/S88T for hypoxanthine. The findings revealed that, in comparison to the wild-type, this mutation notably enhanced XanQ’s capability to transport hypoxanthine (Supplementary Figure S2).

Development and performance evaluation of YES301 strain

To optimize the expression level of XanQF94Y/S88T in bacterium, we tested standardized promoter and ribosome binding site (RBS) sequences of varying strengths upstream of the r xanQ gene. Xanthine uptake experiments demonstrated that the use of the J23100 promoter and B0034 RBS to regulate the expression levels of XanQF94Y/S88T in EcN strain could further enhance xanthine transport, resulting in the engineered EcN strain named YES301. When the initial concentration of xanthine in the medium is 150 µM, YES301 can uptake 119.55 ± 11.15 μM of extracellular xanthine within 60 min (Figure 2a, b). Figure 2. Generation and characterization of YES301. (a) Regulate the expression of the rationally designed xanQ gene using promoters of varying strengths, and assess their ability to transport xanthine. The bacteria were suspended in M9 medium supplemented with 150 μM xanthine and incubated for 60 min before measuring the xanthine content in the supernatant (mean ± SEM, n = 3). (b) Regulate the expression of the rationally designed xanQ gene using RBS of varying strengths, and assess their ability to transport xanthine (mean ± SEM, n = 3). (c) Determined the temporal kinetics of xanthine uptake by YES301, EcN-XanQ, and EcN-pWT. The strains were suspended in M9 medium supplemented with 150 μM xanthine and incubated for 60 min. Samples were taken at regular intervals to determine the xanthine content in the culture supernatant (mean ± SEM, n = 3). (d) Uptake kinetics determination. The strains were suspended in M9 medium supplemented with 0–400 μM xanthine and incubated for 20 min before measuring the xanthine content in the supernatant (mean ± SEM, n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test, ns p > 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

Next, we investigated the temporal kinetics of extracellular xanthine uptake by YES301. The results showed that within 30 min, YES301 absorbed nearly 80% of extracellular xanthine, a significantly higher rate compared to strains expressing wild-type XanQ (32%, EcN-XanQ) and strains harboring empty plasmids (9%, EcN-pWT) (Figure 2c). The kinetic uptake results revealed that the maximum velocity (Vmax) at which YES301 transports xanthine is 7.48 μM/min. In comparison, the Vmax of EcN XanQ is 3.85 μM/min (Figure 2d). In summary, YES301 demonstrated efficient xanthine transport ability, with a transport capacity for xanthine that is 8.6-fold that of EcN-pWT and 2.4-fold that of EcN-XanQ.

YES301 uptake xanthine in the gut of mice and reduce serum uric acid levels

To evaluate the capacity of engineered bacteria to uptake xanthine in the gastrointestinal tract, YES301 was incubated with simulated gastrointestinal fluid (SGF) for 5 min, then transferred to simulated intestinal fluid (SIF) containing 100 μM of xanthine and continued to be incubated for 60 min, with extracellular xanthine concentrations detected every 15 min. The control group was cultured in M9 medium for 5 min, and then transferred to M9 medium containing 100 μM of xanthine for further incubation for 60 min. The results showed that despite continuous exposure to gastric and intestinal fluids, YES301 can still efficiently transport extracellular xanthine (Supplementary Figure S3a). Although the transport speed of YES301 group slightly decreased compared to the control group, this may be attributed to a reduction in the bacterial population (Supplementary Figure S3b). Furthermore, the transport capacity of YES301 for xanthine remains unaffected by oxygen conditions (Supplementary Figure S4).

To assess the xanthine uptake potential of YES301 in the gastrointestinal tract, we investigated the time-dependent changes in serum xanthine and uric acid levels in mice orally administered with 20 mM xanthine, with a gastric lavage volume of 200 μL. Figure 3a shows that that serum xanthine levels in the control group mice peaked 1 hour after administration, while serum uric acid levels peaked 2 hours after administration. Mice administered YES301 exhibited significantly reduced serum xanthine (Figure 3a) and uric acid (Figure 3b) levels compared to control group mice, with serum xanthine levels returning to baseline levels by 2 hours. Additionally, we assessed xanthine content in the feces of both groups of mice. Figure 3c demonstrates that fecal xanthine levels in control mice peaked 2 hours after administration and remained stable for 2–8 hours, whereas mice administered with YES301 showed significantly lower fecal xanthine levels compared to control mice, returning to pre-xanthine administration levels by 4 hours. These results indicate that YES301 efficiently absorbs xanthine in the intestines, thereby reducing host absorption of xanthine and consequently decreasing uric acid production. We also explored the ability of YES301 to transport hypoxanthine in the intestine, and the results demonstrated that YES301 could uptake hypoxanthine in vivo, thereby reducing fecal hypoxanthine levels (Supplementary Figure S5). Figure 3. Activity of YES301 in mice. Effects of YES301 on serum xanthine (a) and serum uric acid (b) in mice after administration of 20 mM xanthine. (c) Fecal xanthine levels collected from mice described in (a). Mice were orally administered with 2 × 109 CFU of YES301 along with 20 mM xanthine, or with 20 mM xanthine alone. Serum and fecal samples were collected from mice before administration (0 hour), and at 1, 2, 4, and 8 hours post-administration, for analysis of xanthine and uric acid levels (mean ± SEM, n = 5). (d) The effect of YES301 (2 × 109 CFUs) on xanthine induced hyperuricemia in mice. Blood samples were collected from the mice one hour after the final administration to assess serum UA (uric acid) levels (mean ± SEM, n = 5). (e) Serum xanthine levels in in each group of mice (mean ± SEM, n = 5). (f) Effects of YES301 and allopurinol on hyperuricemia mice weight. (g) Histopathological scores of liver tissue in each group of mice (mean ± SEM, n = 5). (h) Histopathological scores of kidney tissue in each group of mice (mean ± SEM, n = 5). (i) The H&E staining slices of liver (top) and kidney (bottom) tissue of each group of mice. For liver tissue slices, the tissue sections from the PBS group and the YES301-treated group show hepatocytes that are round and full, with clear nuclear structure. Hepatocytes are arranged in cord-like formations known as liver plates, with regular and tight arrangement. There is no obvious dilation or compression of hepatic sinusoids, and no apparent abnormalities are observed. There is no apparent infiltration of inflammatory cells. The hyperuricemia group and the allopurinol treatment group exhibited significant histopathological features. Red arrows: hepatocyte necrosis. Black arrows: mild lymphocyte infiltration. For kidney tissue slices, In the PBS group and the YES301 treatment group, the glomeruli exhibit uniform cellularity and matrix distribution. The renal tubular epithelial cells appear round and full, with regular brush borders. White arrows: eosinophilic material. The hyperuricemia group and the allopurinol treatment group exhibited significant histopathological features. Green arrows: glomerular congestion. Orange arrows: tubular atrophy. Blue arrows: tubular epithelial cell edema with pale and loose cytoplasm. Gray arrows: a few tubular epithelial cells show signs of necrosis, with pyknotic nuclei, fragmentation of cytoplasm. Black arrows: intratubular debris. Red arrows: lymphocytic infiltration. Scale bar: 100 μm. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test, ns p > 0.05, *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

Subsequently, we initiated a study utilizing a mouse model of hyperuricemia to investigate the potential of YES301 in reducing serum uric acid levels induced by high purine intake. Mice were randomly allocated into five groups, with hyperuricemia induced groups receiving xanthine (600 mg/kg) and potassium oxonate (600 mg/kg) for 14 consecutive days. EcN-pWT (2 × 109 CFUs), YES301 (2 × 109 CFUs), and allopurinol (5 mg/kg) were orally administered to each group 30 min before the administration of xanthine and potassium oxonate, respectively. Blood samples were collected from the mice one hour after the final administration to assess serum uric acid levels. The findings demonstrated a significant increase in serum uric acid and xanthine levels in mice administered xanthine and potassium oxonate orally (from 125.52 ± 32.49 µM to 273.84 ± 35.86 µM) (Figure 3d). However, both YES301 and allopurinol exhibited notable reductions in serum uric acid levels, restoring them to normal levels, while EcN-pWT did not yield similar effects (Figure 3d). Furthermore, YES301 demonstrated efficacy in mitigating kidney damage in hyperuricemic mice, as evidenced by reductions in serum creatinine and urea nitrogen levels (Supplementary Figure S6). Building upon YES301‘s demonstrated proficiency in hypoxanthine transport in the intestine, we also established a mouse model of hyperuricemia induced by hypoxanthine (300 mg/kg) and potassium oxonate (300 mg/kg), where YES301 displayed efficacy in mitigating elevated blood uric acid levels induced by hypoxanthine and potassium oxonate administration (Supplementary Figure S7).

To further validate the efficacy of YES301 in reducing serum uric acid levels via xanthine transport, we assessed the xanthine content in the serum and feces across experimental groups. As shown in Figure 3e and Supplementary Figure S8, administration of xanthine and potassium oxonate led to elevated serum (Figure 3e) and fecal (Supplementary Figure S8) ×anthine levels in hyperuricemic mice, whereas treatment with YES301 resulted in a significant reduction, similar to healthy mice. Conversely, allopurinol did not attenuate xanthine excretion in mice. This disparity arises from allopurinol’s mechanism of action, which entails inhibition of xanthine oxidase, thereby impeding the conversion of xanthine to uric acid. Given the pronounced inhibitory effects of allopurinol on host enzymes, we propose that the strategy of transporting uric acid precursors with engineered bacteria offers a milder and potentially lower side effect alternative for preventing and treating hyperuricemia. This notion is supported by a comparative analysis of the effects of allopurinol and YES301 on mouse body weight and kidney tissue (Figure 3f-i). Histopathological scoring of the liver and kidney tissues from each group of mice indicate that hyperuricemic mice treated with YES301 did not show significant tissue damage in the liver and kidney. In contrast, mice treated with Allopurinol exhibited notable tissue damage in these organs (Figure 3g, h).

In summary, we demonstrate that YES301 efficiently transports the uric acid precursors, xanthine and hypoxanthine, in the intestine, effectively reducing serum uric acid levels and alleviating kidney damage caused by high uric acid levels.

Pharmacokinetics and biosafety of YES301 in healthy mice

To assess the pharmacokinetics of YES301 in mice, 2 × 109 CFUs of YES301 were administered via gavage, and fecal samples were analyzed for bacterial quantification using serial dilution plating. The findings indicated rapid clearance of YES301 from the intestinal tract of mice (Supplementary Figure S9). Subsequently, the biosafety profile of YES301 was evaluated. Following oral administration of YES301 (2 × 109 CFUs) to mice, blood samples were collected on the 1st, 3rd, and 5th days for assessment of serum inflammatory factors and transaminases. Comparative analysis with the control group (administered PBS) revealed no significant alterations in body weight (Figure 4a), serum inflammatory factor levels (Figure 4b,c), or transaminase levels (Figure 4d) in mice. Upon sacrifice on the final day, histopathological examination of the jejunum, ileum, and rectum showed no evidence of intestinal tissue damage attributable to YES301 (Supplementary Figure S10). Furthermore, 16S ribosomal RNA (rRNA) genome sequencing analysis indicated that oral administration of YES301 did not induce significant changes in the composition or diversity of the mouse gut microbiota (Figure 4e-i), affirming the high level of biosafety associated with YES301. Figure 4. Biosafety evaluation of YES301 in healthy mice. (a) The effect of YES301 on the weight of healthy mice (mean ± SEM, n = 5). (b-c) The effect of YES301 on the inflammatory cytokine levels of healthy mice (mean ± SEM, n = 5). (d) The effect of YES301 on the transaminase levels of healthy mice (mean ± SEM, n = 5). (e-g) Alpha diversities of the gut microbiota were compared between two groups of mice using the Chao, Shannon, and Simpson indices (n = 5). (h) Rank abundance curve of the microbial community (n = 5). (i-k) Box plot showing sample distances (i), Principal Coordinate Analysis (PCoA) plot (j), and ANOSIM between-group similarity analysis box plot (k) (n = 5). (l) Bar chart illustrating the relative abundance of species in the two groups (n = 5). Student’s T-test was used to analyze significant differences between two groups, One-way ANOVA with Tukey’s multiple comparisons test was employed for data analysis involving three groups or more, ns p > 0.05.

Biosafety of YES301 in hyperuricemia mice

We further evaluated the effects of YES301 on inflammatory cytokine and transaminase levels in hyperuricemic mice under both single-dose and multiple-dose oral administration conditions. To induce hyperuricemia, mice received doses of xanthine (600 mg/kg) and potassium oxonate (600 mg/kg) daily for 14 days. Subsequently, the mice were administered YES301 (2 × 109 CFUs) via gavage. Serum samples were collected prior to the YES301 administration (0 day) and on the third and fifth days following gavage to measure levels of serum inflammatory cytokines and transaminases. Our findings suggest that YES301 does not increase inflammatory cytokine or transaminase levels in hyperuricemic mice (Figure 5a-c). We administered YES301 to hyperuricemic mice via gastric gavage for five consecutive days to evaluate its biosafety of its multiple administration. After the fifth administration of YES301, mouse serum samples were collected on days 5, 10 and 15 to measure levels of inflammatory cytokines and transaminases. Our results indicate that compared to healthy mice (control), even after multiple administrations, YES301 does not increase the levels of inflammatory cytokines or transaminases in hyperuricemic mice (Figure 5d-f). Figure 5. Biosafety evaluation of YES301 in hyperuricemia mice. Changes in inflammatory factors IL-6 (a), TNF-α (b), and transaminase levels (c) in hyperuricemic mice after a single gavage of YES301. The mice were administered YES301 (2 × 109 CFUs) via gavage. Serum samples were collected prior to the YES301 administration (0 day) and on the third and fifth days following gavage to measure levels of serum inflammatory cytokines and transaminases. Changes in inflammatory factors IL-6 (d), TNF-α (e), and transaminase levels (f) in hyperuricemic mice after multiple gavage of YES301. Hyperuricemic mice were orally administered YES301 (2 × 109 CFU) consecutively for 5 days, once daily. Serum samples were collected on days 5, 10 and 15 after the final administration of YES301 to measure levels of serum inflammatory cytokines and transaminases, with healthy mouse serum samples used as controls (mean ± SEM, n = 5). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test, ns p > 0.05, *p ≤ 0.05.

In summary, our results demonstrate that YES301 exhibits biological safety when administered to both healthy mice and hyperuricemic mice.

Conclusion

In summary, our study presents a promising avenue for preventing and treating hyperuricemia through the development of the engineered probiotic YES301. Through the overexpression of the xanthine transporter protein XanQ in EcN strain, we significantly enhanced the bacteria’s capacity to uptake xanthine. Subsequent rational design and directed evolution of XanQ further improved its efficiency in transporting xanthine and hypoxanthine. YES301, the resultant engineered probiotic with finely-tuned expression levels of mutated XanQ (XanQF94Y/S88T), exhibited substantial increases in xanthine (8.6-fold) and hypoxanthine (4.0-fold) transport capacity compared to the wild-type strain. Oral administration of YES301 to hyperuricemic mouse models resulted in a significant reduction of blood uric acid levels and alleviation of kidney damage, comparable to the clinical drug allopurinol but with fewer side effects and higher biocompatibility. These findings underscore the potential of YES301 as a novel therapeutic approach for hyperuricemia, warranting further exploration in clinical settings and potential translation to human healthcare applications. However, the gastrointestinal survival ratio of YES301 using EcN as the chassis is inferior to that of Lactobacillus. Therefore, for future investigations, we can choose Lactobacillus as the chassis to improve the potential applicability of engineered bacteria, and use isotope-labeled internal standards for more precise quantify the levels of xanthine and hypoxanthine in the sample.

Given the presence of various purine compounds in human food, future endeavors could involve developing and optimizing transporter proteins for purine compounds other than xanthine and hypoxanthine (such as guanine, adenine), aiming to efficiently absorb a wide range of purine compounds from food and reduce their absorption in the intestine, thereby decreasing uric acid production. Moreover, coupling uricase with YES301 could enhance the efficacy of engineered bacteria by degrading uric acid already generated in the body.

Methods and materials

Strains, plasmids, culture conditions and regents

The strains and plasmids utilized in this research are detailed in Supplementary Table S1. Plasmid construction employed E. coli DH5α, and engineered strains for xanthine and hypoxanthine transportation were developed using EcN. Cultures were maintained at 37°C in lysogeny broth (LB) or M9 Minimal Medium, with antibiotics added as necessary. Streptomycin (100 µg/mL) was the sole antibiotic utilized in this study. LB medium was purchased from Sangon Biotech (Shanghai, China). Xanthine and hypoxanthine were purchased from Macklin Reagent (Shanghai, China). Potassium oxazinate and allopurinol were purchased from Aladdin Biochemical Technology (Shanghai, China). Streptomycin was obtained from Solarbio (Beijing, China).

DNA manipulations

The XanQ (Protein ID: AAC75920.2) and the XanP (Protein ID: AAC76678.1) were obtained through the design of primers for polymerase chain reaction (PCR) and amplified from E. coli str. K-12 substr. MG1655 genomic DNA. The sequences of genetic protein are detailed in Supplementary Table S2. The mutagenesis was also obtained by PCR. Subsequently, XanP, XanQ, and their mutants were cloned into pWT-021a under the control of promoters and RBS with varying strengths (pEASY-Basic Seamless Cloning and Assembly Kit, TransGen Biotech, Beijing, China). Following plasmid preparation in E. coli DH5α, they were introduced into EcN via electroporation using MicroPulserTM, employing a 3-kV, 4-ms pulse.

The HPLC detection method for hypoxanthine and xanthine

An HPLC method, adapted with slight modifications, was employed to determine the content of xanthine/hypoxanthine in complex media. We used an external standard method to quantify the levels of xanthine and hypoxanthine in the samples. A Shimadzu HPLC system with a column (a Diamonsil Plus 5 μm C18-A, 250 × 4.6 mm, DiKMA) was pre-equilibrated with 95% Solvent A (50 mM NH4COOCH3 buffered to pH of 4.6 with glacial acetic acid) and 5% Solvent B (100% acetonitrile). An equal gradient elution (0–15 min, 5% B) was performed using a 10 μL injection volume at a flow rate of 1.0 mL/min. The column temperature was maintained at 30°C with an absorption of 254 nm. All samples were filtered through a 0.22 μm microporous membrane before detection. Standard solutions of xanthine/hypoxanthine were prepared at concentrations of 10 μM, 20 μM, 25 μM, 50 μM, and 100 μM, and analyzed by HPLC. Standard curves were established based on the relationship between peak area and concentration of xanthine/hypoxanthine at different concentrations.

The transport experiment of xanthine and hypoxanthine by YES301

The strains were cultured in LB medium and then transferred to M9 medium overnight. After centrifugation at 6000 rpm for 10 min at room temperature, the cells were collected and resuspended in fresh M9 solution. Subsequently, the xanthine/hypoxanthine transport capacity of YES301 was determined. The OD600 of the whole-cell reaction system was adjusted to a certain value, and a certain amount of xanthine/hypoxanthine solution was added to achieve the specified final concentration in the system. Incubation was carried out at 37°C and 220 rpm for designated time on a shaker, followed by centrifugation at 6000 rpm for 2 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous phase membrane and further analyzed by HPLC to determine the hypoxanthine/xanthine content in the samples.

The methods for determining the transport efficiency at different time points under the same hypoxanthine concentration and for determining under the same concentration but different incubation conditions (aerobic, microaerobic, and anaerobic) are as described above.

Molecular simulation

Using the AlphaFold structure model of XanQ to simulate docking with substrate molecules, and performing docking using AutoDock Vina. Finally, the interactions between substrate and protein were examined using the PLIP (Protein Interaction Learning Platform) web tool, the images were generated using PyMOL software.

Animals

Five weeks old (20–23 g) male C57BL/6 mice (Ziyuan, Hangzou, China) were housed in room (22 ± 2℃) with 65% humidity and 24-hour light-dark cycle. Mice were fed a standard diet for a week to habituate the environment before the experiments were executed. Make sure the mice were in good health in the beginning. Mice were randomly divided into groups for the following experiments.

Detection of xanthine/hypoxanthine in mouse feces

After collection, fecal samples were weighed, followed by grinding in pre-chilled PBS buffer using a grinder pre-set to 4°C. Centrifugation at 6000 rcf for 10 minutes was performed to collect the supernatant, which was subsequently filtered using a 0.22 μm filter. Unless immediately analyzed, samples were flash-frozen in liquid nitrogen and stored at −80°C for later use. The filtered samples were analyzed for xanthine/hypoxanthine using HPLC and quantified based on external standards.

Detection of xanthine in mouse serum

The collected blood was equilibrated at room temperature for 30 minutes, followed by centrifugation at 4°C and 3000 rpm for 20 minutes to obtain serum. Use ELISA kits for xanthine detection in mouse serum (Yuanmu Bio-Technology, Shanghai China). In short, add 10 μL of the sample to be tested followed by 40 μL of sample diluent into each sample well. Then, add 100 μL of horseradish peroxidase (HRP) conjugated detection antibody to each well, seal the plate with a plate seal, and incubate at 37°C for 60 minutes. Discard the liquid, pat dry on absorbent paper, fill each well with wash buffer, let it sit for 1 minute, then flick away the wash buffer and pat dry on absorbent paper. Repeat this washing process five times. Add 50 μL of each chromogenic substrate to each well and incubate at 37°C in the dark for 15 minutes. Finally, add 50 μL of stop solution to each well and measure the absorbance of each well at a wavelength of 450 nm within 15 minutes.

Detection of uric acid in mouse serum

The collected blood was equilibrated at room temperature for 30 minutes, followed by centrifugation at 4°C and 3000 rpm for 20 minutes to obtain serum. Use colorimetric methods for uric acid detection in mouse serum (Nanjing Jiancheng Bioengineering Institute, Nanjing China). The colorimetric principle is as follows:Uricacid+2H2O+O2−−⟶uricaseAllantion+CO2\break+2H2O2

H2O2+C11H13N3O+C12H18NNaO4S−−−−−⟶peroxidase\breakQuinonepigments+5H2O

The formula for calculating uric acid concentration is as follows (detection is performed at a wavelength of 510 nm):Uricacidconcentration(μ mol/L)\break=Absorbancesample−AbsorbanceblankAbsorbancestandard−Absorbanceblank\break\break×Calibrationstandard concentration

Hyperuricemia mouse models

Based on a modified version of the method by Wang27 and Guo.28 Hyperuricemia was induced in mice by daily oral administration for fourteen consecutive days of 600 mg/kg xanthine and 600 mg/kg potassium oxonate, or 300 mg/kg hypoxanthine and 300 mg/kg potassium oxonate (total gavage volume of 200 μL).

The effect of YES301 on reducing uric acid was evaluated using a mouse model of hyperuricemia induced by 600 mg/kg xanthine and 600 mg/kg potassium oxazinate. Mice were randomly divided into five groups, with hyperuricemia-induced groups receiving xanthine (600 mg/kg) and potassium oxonate (600 mg/kg) treatment for 14 consecutive days. EcN-pWT (2 × 109 CFUs/200 μL), YES301 (2 × 109 CFUs/200 μL), and allopurinol (5 mg/kg, 200 μL) were orally administered to each group 30 minutes prior to the administration of xanthine and potassium oxonate, respectively. Blood samples were collected from the mice one hour after the final administration to assess serum uric acid and xanthine levels.

Hematoxylin and eosin (H&E) staining and histopathological analysis

Mice were sacrificed, and their liver, kidney, and intestinal tissues were collected. The tissues were fixed in 4% paraformaldehyde for 24 hours, followed by histological processing (dehydration, paraffin embedding, sectioning, and H&E staining). Images were captured at 40× or 200× magnifications for histopathological analysis. The histopathological scoring was completed by Servicebio Technology (Wuhan, China).

Analysis of serum inflammatory factors, transaminase, creatinine, and urea nitrogen

The contents of IL-6 and TNFα in the serum were analyzed using a cytokine detection kit according to the manufacturer’s instructions (Servicebio Technology, Wuhan, China).

The serum levels of transaminase, creatinine, and urea nitrogen were measured using a biochemical analyzer, commissioned to Servicebio Technology (Wuhan, China).

Analysis of gut microbiota

Fecal samples were collected for 16S rRNA sequencing to determine the composition and relative abundance of the microbiota. After collection, fecal samples were flash-frozen in liquid nitrogen and stored at −80 degrees Celsius until analysis. This experimental work was commissioned to Sangon Biotech (Shanghai, China).

Pharmacokinetic analysis

Mice adapted to the environment, then gavaged with YES301 at 2 × 109 CFUs. Fecal samples were collected from the mice at 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h after gavaged. These samples were weighed and placed into grinding tubes containing 1 mL PBS and grinding beads, then processed in a grinder. After centrifugation, the diluted samples were subjected to colony counting for Smr resistance on plates, followed by incubation at 37°C for 12 hours for colony enumeration at each time.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0.2. Student’s T-test was used to analyze significant differences between two groups, One-way ANOVA with Tukey’s multiple comparisons test was employed for data analysis involving three groups or more. Data fitting was also conducted using this software. Results are presented as mean ± SEM, with significance denoted as follows: ns p > 0.05, *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

Supplementary Material

Supplemental Material

Acknowledgments

This work was sponsored by the National Key Research and Development Program of China (2023YFF1204500), the National Natural Science Foundation of China (22134003), and China Postdoctoral Science Foundation (2023M741176).

Disclosure statement

No potential conflict of interest was reported by the author(s).

CRediT authorship contribution statement

Zhen-Ping Zou: Conceptualization; Data acquisition; Methodology; Funding acquisition; Investigation; Writing – review & editing. Ju-Ling Li: Data acquisition; Methodology; Writing – original draft. Yi-Fan Zhang: Data acquisition. Ying Zhou: Conceptualization; Funding acquisition; Writing – review & editing. Bang-Ce Ye: Conceptualization; Funding acquisition; Project administration; Writing – review & editing.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2024.2399213
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