Intestinal probiotic engineering bacterium and application thereof in uric acid metabolism

Abstract

The invention relates to the technical field of biology, and particularly discloses a construction method and application of escherichia coli engineering bacteria for overexpressing uric acid degradation gene clusters. According to the invention, a mixed promoter box is inserted into Escherichia coli, overexpression coding is carried out on genes of regulatory protein YgeV and key genes in a uric acid degradation pathway, and the Escherichia coli engineering bacteria which can overcome inhibition of intestinal glucose on uric acid degradation and enable the Escherichia coli engineering bacteria to overexpress uric acid degradation gene clusters are successfully constructed. The coliform engineering provided by the invention provides a new strategy for prevention and treatment of hyperuricemia and gout.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a probiotic engineered intestinal bacteria and its application in uric acid metabolism. Background Technology

[0002] Uric acid is a key intermediate product of purine degradation in many organisms, and its accumulation in the human body can lead to inflammation and gout. In most mammals, uricase converts uric acid into allantoin, which is more water-soluble and facilitates excretion. However, humans and other higher primates lack functional uricase and primarily excrete uric acid directly through urine and the intestines. Moderate amounts of uric acid have antioxidant and neuroprotective effects, but high uric acid levels can lead to various diseases. Due to its high solubility and crystallinity, uric acid easily forms sodium urate crystals, which can deposit in the kidneys leading to kidney stones or trigger gout in the joints. The key to uric acid metabolism in the body is the balance between uric acid produced by the liver and uric acid excreted through the kidneys and intestines. Approximately two-thirds of the uric acid in the body is primarily excreted through the kidneys, while the remaining one-third is excreted through the intestines. For patients with renal insufficiency, intestinal secretion of uric acid is also an important pathway, and transport proteins that promote uric acid excretion are present in various parts of the intestine. Intestinal dysbiosis can lead to elevated serum uric acid concentrations, impaired intestinal barrier, increased permeability, and excessive lipopolysaccharide entering the bloodstream. Lipopolysaccharide binds to the TLR4 receptor, causing upregulation of pro-inflammatory cytokines. When kidney function is impaired due to inflammation, it leads to abnormal uric acid metabolism in the kidneys.

[0003] Hyperuricemia is one of the most common metabolic diseases caused by high uric acid levels. It has a high incidence rate in many countries and is showing a clear trend towards affecting younger people. The increasing intake of high-fat, high-purine foods (seafood, beer, and red meat) and sub-optimal health under excessive stress can easily lead to elevated uric acid levels in the body. Disorders of purine metabolism, excessive uric acid production, or reduced uric acid excretion can result in abnormally high uric acid levels in the blood, causing hyperuricemia and eventually developing into gout. Gout is a crystal-related joint disease caused by the deposition of uric acid monosodium crystals in or around the joints. Furthermore, hyperuricemia and gout increase the risk of cardiovascular disease, chronic kidney disease, diabetes, metabolic syndrome, and neurodegenerative diseases.

[0004] In the human body, endogenous and exogenous nucleotides are converted into nucleosides, which are further converted into purine bases. Uric acid is the final metabolic product of purine compounds. Adequate uric acid levels are beneficial; hyperuricemia without gout is usually asymptomatic. However, various factors, including genetic diversity, environmental exposure, gene-environment interactions, and intrinsic risk factors (including age, sex, and weight), increase the risk of developing gout. Currently, the main drugs used to treat hyperuricemia clinically include allopurinol, colchicine, and benzbromarone. Allopurinol is a selective xanthine oxidase inhibitor that can treat gout by lowering blood urate concentration. Benzbromarone mainly lowers blood uric acid levels by inhibiting the reabsorption of uric acid in the renal tubules; however, all of these drugs have significant side effects (Zhang, Mengjie, et al. "Atavistic strategy for the treatment of hyperuricemia via ionizable liposomal mRNA." Nature Communications 15.1:1-16.). In terms of non-pharmacological interventions, low-purine diets, as a clinical nutritional strategy to reduce serum uric acid levels and the risk of gout, have limited effectiveness and poor adherence.

[0005] Current uric acid degradation drugs are mainly divided into xanthine oxidation inhibitors, uric acid excretion promoters, and uricase inhibitors. These drugs suffer from limitations in metabolic pathways and the disruption of the gut microbiota by antibiotic use, failing to directly target specific uric acid metabolism pathways. Therefore, the development and application of new drugs for the prevention, intervention, and / or treatment of gout is urgently needed in this field. Summary of the Invention

[0006] This invention specifically studies the biochemical characteristics of the uric acid degradation gene cluster in *Escherichia coli*, elucidates its specific pathway for uric acid degradation, and provides a probiotic based on this uric acid degradation pathway as well as a fluorescent marker for uric acid detection. This pathway includes eight related enzymes. In this pathway, uric acid is first reduced to isoxanthine (2,8-dioxanone, IsoX) by xanthine dehydrogenase (XdhD), followed by dearomatization and ring-opening of the purine core catalyzed by flavin / iron-sulfur cluster-dependent oxidoreductase (YgfK) and amide hydrolase (SsnA). The pyrimidine and imidazole rings are progressively cleaved by D-phenylhydantoinase (HyuA) and carbamoyltransferase (YgeW) to generate 2,3-diuretopropionic acid, which is then hydrolyzed by ureoyl hydrolase (YgeY) to generate 2,3-diaminopropionic acid, and finally broken down into pyruvate and ammonia by diaminopropionic acid-dependent lyase (YgeX). Figure 14As shown in the figure, this pathway proposes a novel diagnostic marker for uric acid, isoxanthine. Simultaneously, the parent strain EcNc, derived from the probiotic Escherichia coli Nissle strain, was engineered. Specifically, the constitutive promoter of this probiotic pathway was replaced to overcome the inhibition of uric acid degradation by intestinal glucose, resulting in overexpression of the uric acid degradation gene cluster. Oral administration in a uricase knockout hyperuricemia mouse model showed a significant reduction in serum uric acid levels and alleviated associated kidney damage, providing a potential pathway for the development of uric acid-degrading probiotics.

[0007] This invention provides an intestinal probiotic engineered bacterium that, based on Escherichia coli, overexpresses a gene encoding the regulatory protein YgeV (transcription factor) and a uric acid degradation gene cluster in the uric acid degradation pathway. The uric acid degradation gene cluster includes a gene encoding xanthine dehydrogenase (XdhD), a gene encoding a flavin / iron-sulfur cluster-dependent oxidoreductase (YgfK), a gene encoding an amide hydrolase (SsnA), a gene encoding D-phenylhydantoinase (HyuA), a gene encoding carbamoyltransferase (YgeW), a gene encoding a urease (YgeY), and a gene encoding diaminopropionic acid lyase (YgeX).

[0008] Specifically, the overexpression involves inserting a mixed promoter cassette into the spacer region between the gene encoding the transcription factor YgeV and the gene encoding the carbamoyltransferase YgeW in the genome of the E. coli originating strain. The spacer region is the gene interval from 3005786 to 3006262 in the EcNc genome. The Uniprot number of YgeV is Q46802, and the Uniprot number of the amino acid sequence of YgeW is Q46803. The insertion of the mixed promoter cassette upregulates the expression of the endogenous uric acid degradation gene cluster in the E. coli originating strain. The mixed promoter cassette contains a constitutive promoter and an anaerobic promoter. Preferably, the mixed promoter cassette contains a strongly constitutive promoter and an anaerobic promoter.

[0009] Specifically, the originating strain of Escherichia coli is Escherichia coli K12, Escherichia coli BL21, Escherichia coli Nissle1917 strain or its derived parent strain, such as EcNc. Preferably, the originating strain of Escherichia coli does not contain free plasmids, such as pMUT1 and / or pMUT2.

[0010] Specifically, the hybrid promoter cassette comprises a strong constitutive promoter and an anaerobic promoter. The strong constitutive promoter is gapA, tufB, lpp, or rrnB, and the anaerobic promoter is anaerobic nirB, narG, narK, dmsA, or frdA. Preferably, the anaerobic promoter nirB contains only an anaerobic-responsive FNR region and no nitrate / nitrite-responsive Nar region. Preferably, the strong constitutive promoter has 1-2 non-conserved base substitutions. Preferably, there is a spacer sequence between the strong constitutive promoter and the anaerobic promoter. Preferably, the nucleotide sequence of the hybrid promoter is shown in SEQ ID NO.1.

[0011] Specifically, the unirot number encoding xanthine dehydrogenase is Q46814, the unirot number encoding flavin-dependent reductase is Q46811, the unirot number encoding amide hydrolase is Q46812, the unirot number encoding D-phenylhydantoinase is Q46806, the unirot number encoding carbamoyltransferase is Q46803, the unirot number encoding urease is P65807, and the unirot number encoding diaminopropionic acid lyase is P66899.

[0012] Specifically, the engineered bacteria can overcome the inhibition of uric acid degradation pathway by intestinal glucose. Preferably, after culturing in an anaerobic LB system containing 0.4% (w / v) glucose for 72 h, the engineered bacteria can reduce the residual concentration of the initial 3 mmol L-1 uric acid to ≤30% (the control strain has a residual concentration of ≥90%).

[0013] The present invention also provides the application of the above-mentioned intestinal probiotic engineered bacteria in the preparation of drugs for reducing host serum uric acid levels.

[0014] The present invention also provides the application of the above-mentioned probiotic engineered bacteria in the preparation of drugs for improving hyperuricemia.

[0015] The present invention also provides the application of the above-mentioned intestinal probiotic engineered bacteria in the preparation of a drug for treating gout.

[0016] This invention also provides a method for constructing probiotic engineered bacteria for the gut, comprising the following steps:

[0017] S1. The hybrid promoter box is introduced into the Escherichia coli starting strain using gene editing technology;

[0018] S2. Screening to obtain intestinal probiotics that constitutively overexpress uric acid degradation gene clusters, wherein the intestinal probiotics overcome the inhibition of uric acid degradation by intestinal glucose.

[0019] Specifically, the importation involves inserting a hybrid promoter cassette into the gene region 3005786 to 3006262 of the E. coli originating strain EcNc genome, while simultaneously replacing the binding sites of the natural promoter and crp in the gene region.

[0020] The present invention also provides a fluorescent marker for uric acid metabolism, wherein the fluorescent marker is used to detect the uric acid degradation pathway, and the fluorescent marker is isoxanthine, wherein the CAS ID of isoxanthine is 13230-99-4.

[0021] Specifically, the fluorescence characteristics of the fluorescent marker are λex / em = 308 / 363nm.

[0022] Specifically, the fluorescent marker is used in the preparation of biomarkers for the auxiliary diagnosis of gout.

[0023] The present invention also provides a method for detecting the fluorescent marker, the method comprising using fluorescence spectroscopy or LC-MS to detect the content of the fluorescent marker in a biological sample, the biological sample including serum, urine or intestinal contents.

[0024] This invention also provides a urate-degrading enzyme, which is a xanthine dehydrogenase with Uniprot number Q46814, wherein the 606th amino acid is glutamic acid. Specifically, the urate-degrading enzyme can directionally recognize uric acid, reducing its carbonyl group at position 6.

[0025] Beneficial Effects: This application analyzes the uric acid degradation pathway and reveals that Xadh's recognition and conversion of uric acid depends on glutamic acid residue at amino acid position 606. Simultaneously, it develops an engineered intestinal probiotic based on this uric acid degradation pathway and a fluorescent marker for uric acid detection. This effectively improves the efficient expression and utilization of uric acid degradation proteins in the intestinal hypoxic environment, overcomes the inhibition of uric acid degradation by intestinal glucose, and has significant therapeutic effects in uricase-deficient (UOX- / -) hyperuricemia, significantly reducing serum uric acid and improving renal function indicators. It has potential application value in reducing uric acid levels in vivo and treating gout. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the gene cluster in the uric acid degradation pathway of Escherichia coli. Figure 1 The "a" indicates that multiple enzymes work together to catalyze the conversion of the central C3 portion of uric acid to diaminopropionic acid, a process requiring a net reduction of four electrons and multiple hydrolysis steps. Figure 1 In the middle b, it represents the uric acid degradation gene cluster in other strains;

[0027] Figure 2These are purified gel images of proteins related to the uric acid degradation pathway, where ah represents purified gel images of EcYgeY, EcYgeX, CdHyuA, CdUacY, EcYgeW, EcYgfK, EcSsnA, and CdUacX, respectively.

[0028] Figure 3 This is a gel image of XdhD protein purification;

[0029] Figure 4 This is the mass spectrum of the XdhD-YgfM reaction;

[0030] Figure 5 This involves the detection of the YgfK and SsnA coupling activity and the CdUacX and SsnA coupling reaction. Figure 5 In the figure, 'a' represents the reaction process in which isoxanthine is reduced and hydrolyzed into products 5UDU and UMH under the coupling action of YgfK and SsnA. Figure 5 In the figure, b indicates that the fragment ions in the secondary mass spectrometer are 85.1, 101.1, and 130.0. Figure 5 In the image, 'c' represents the mass spectrum of the coupling reaction between E. coli strains YgfK and SsnA. Figure 5 In the image, d represents the mass spectrum of the coupling reaction between UacX and SsnA proteins from the Clostridioides difficile strain.

[0031] Figure 6 This includes the detection of HyuA activity alone and its activity when coupled with CdUacY or YgeW, respectively. Figure 6 Figure a shows a schematic diagram of the HyuA hydrolysis of 5UDU reaction and the reaction mass spectrometry results. Figure 6 Figure b shows a schematic diagram of the HyuA hydrolysis UMH reaction and the reaction mass spectrometry results. Figure 6 Figure c shows the results of the catalytic reduction of UMH to 3-ureo-2-aminopropionic acid (Albizziin) by coupling HyuA with CdUacY or YgeW.

[0032] Figure 7 It is an activity detection method for YgeY and YgeX conjugation;

[0033] Figure 8 This is a schematic diagram of the XdhD structure;

[0034] Figure 9 It is the detection of isoxanthine content in clinical samples. Figure 9 Image a shows the mass spectrum of isoxanthine content in a clinical sample from a hospital. Figure 9 Figure b shows a comparison of isoxanthine levels in gout patients and healthy individuals.

[0035] Figure 10 This is a schematic diagram of engineered bacteria modification, in which... Figure 10In the text, 'a' represents the process of constructing engineered bacteria. Figure 10 In the image, b represents the ECN1917 genome sequence map with the insertion of a mixed promoter sequence, where the nirB promoter sequence marks the FNR region;

[0036] Figure 11 This is a graph of RNA-seq results;

[0037] Figure 12 This is a schematic diagram illustrating how E. coli overexpressing urate-degrading enzymes overcomes glucose inhibition. Figure 12 In Figure a, xanthine consumption was observed in wild-type MG1655 and gene knockout (ΔXdhA-MG1655 and ΔXdhD-MG1655) Escherichia coli strains cultured anaerobically in xanthine-supplemented M9 medium. Figure 12 Figure b shows the uric acid accumulation of wild-type MG1655 and gene knockout (ΔXdhA-MG1655 and ΔXdhD-MG1655) Escherichia coli strains anaerobically cultured in xanthine-supplemented M9 medium. Figure 12 In the figure, c represents the uric acid consumption of wild-type EcN strain, engineered strain CBT2.0, and gene knockout (ΔXdhA-CBT2.0 and ΔXdhD-CBT2.0) Escherichia coli strains anaerobically cultured in uric acid-supplemented LB medium. Figure 12 In the middle section (d), isoxanthine accumulation was observed in wild-type EcN strain, engineered strain CBT2.0, and gene knockout (ΔXdhA-CBT2.0 and ΔXdhD-CBT2.0) Escherichia coli strains anaerobically cultured in uric acid-supplemented LB medium. Figure 12 In Figure 'e', ​​the uric acid consumption of wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strains cultured anaerobically in LB medium supplemented with uric acid and glucose is shown. Figure 12 The value of f in the figure represents the isoxanthine accumulation of wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strains cultured anaerobically in LB medium supplemented with uric acid and glucose. Figure 12 The value of 'g' indicates the accumulation of UMH in wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strains cultured anaerobically in LB medium supplemented with uric acid and glucose. Figure 12 In the figure, 'h' represents the isoxin consumption of wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strains cultured anaerobically in LB medium with added isoxanthine and glucose. Figure 12In the figure, 'i' represents the accumulation of UMH in wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strains cultured anaerobically in LB medium supplemented with isoxanthine and glucose. Figure 12 In the figure, j represents the accumulation of Albizziin in wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strain anaerobic cultured in LB medium supplemented with isoxanthine and glucose.

[0038] Figure 13 The effectiveness of engineered probiotics in animal models of gout. Figure 13 The experimental design of the mouse shown in Figure a, Figure 13 In the figure, b represents the plasma uric acid (UA) levels in mice in the CBT2.0 group, WT group, and PBS group at different time points. Figure 13 The plasma urea nitrogen (BUN) levels of mice in different groups at different time points are shown in Figure c. Figure 13 The plasma creatinine (CRE) levels of mice in different groups at different time points are shown in figure d. Figure 13 The qPCR detection results of engineered bacteria CBT2.0 in the colon contents of mice shown in figure e are as follows. Figure 13 f represents the plasma uric acid (UA) concentration in mice 8 weeks after gavage was stopped;

[0039] Figure 14 The appearance of the kidneys of mice under different treatments ( Figure 14 (as shown in Figure a) and a representative diagram of hematoxylin-eosin staining ( Figure 14 (As shown in b). Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Example 1: Analysis of gene clusters in the uric acid degradation pathway in Escherichia coli

[0044] A schematic diagram of the uric acid degradation pathway is shown below. Figure 1 As shown, the gene clusters in E. coli involved in uric acid degradation include several key enzymes, such as... Figure 1 As shown in Figure a: Xanthine-like dehydrogenase complex (XdhD-YgfM), flavin / iron-sulfur cluster-dependent oxidoreductase (YgfK), PydB-like amide hydrolase (SsnA), D-specific phenylhydantoinase (HyuA), carbamoyltransferase (YgeW), carbamoyl kinase (YqeA), urease (YgeY), and diaminopropionic acid lyase (YgeX) that acts on the L / D isomer. These enzymes collectively catalyze the conversion of the central C3 portion of uric acid to diaminopropionic acid, a process requiring a net reduction of four electrons and multiple hydrolytic steps.

[0045] Comparison of uric acid degradation gene clusters between *Escherichia coli* and *Clostridium difficile* revealed that *Clostridium difficile* replaces *E. coli*'s *YgfK* with a PydA homolog (named UacX); furthermore, in *Clostridium difficile*, *YgeW* and *YqeA* are replaced by a D-aminoacyl amino acid hydrolase homolog (D-aminoacylase, named UacY). Figure 13 (As shown in b).

[0046] In summary, this gene cluster is also present in several known strains capable of anaerobic uric acid degradation, such as *Enterococcus faecalis*, *Peptoniphilus asaccharolyticus*, and *Anaerococcus prevotii*. Figure 1 As shown in Figure b, these analytical results provide important evidence for understanding the key reaction steps in the uric acid degradation pathway.

[0047] Example 2: Preparation of enzymes encoding gene clusters of uric acid degradation pathway

[0048] Based on the analysis of the uric acid degradation pathway, relevant enzymes in this pathway were prepared, including YgfK (flavin / iron-sulfur cluster-dependent oxidoreductase, Uniprot ID: Q46811), SsnA (amide hydrolase, Uniprot ID: Q46812), HyuA (D-phenylhydantoinase, Uniprot ID: Q46806), YgeW (carbamoyltransferase, Uniprot ID: Q46803), YgeY (ureidoyl hydrolase, Uniprot ID: P65807), YgeX (diaminopropionic acid lyase, Uniprot ID: P66899), CdHyuA (D-phenylhydantoinase encoding Clostridioides difficile strain, Uniprot ID: Q181U3), and CdUacX (encoding Clostridioides difficile strain). The flavin / iron-sulfur cluster-dependent oxidoreductase of the difficile strain (Uniprot ID: Q181U4) and the CdUacY gene (encoding the carbamoyltransferase of the Clostridioides difficile strain (Uniprot ID: Q181U6)) were introduced into the pET28a vector to construct a recombinant plasmid, which was then transformed into Escherichia coli BL21(DE3) and cultured overnight at 37°C.

[0049] Single colonies were picked and cultured in 5 mL of LB broth containing kanamycin sulfate. The next day, they were transferred to 1 L of LB broth containing kanamycin sulfate and cultured until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.3 mM and expression was induced at 18°C ​​for 16 hours. The cells were collected by centrifugation at 4000 × g for 10 minutes at 4°C. The cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM PMSF, 0.2 mg / mL lysozyme, 0.03% Triton X-100, 1 μL DNase I). The frozen-thawed cells were thawed and lysed in a 25°C water bath. 5 mM β-mercaptoethanol (BME) and 1% streptomycin sulfate were added to precipitate the nucleic acid. The cells were centrifuged at 8000 × g for 10 minutes at 4°C to remove debris. The supernatant was filtered through a 0.45 μm PES membrane and loaded onto pre-equilibrated TALON Co. 2+ Affinity chromatography columns were used to elute contaminating proteins with buffer A (20 mM Tris-HCl pH 7.5, 200 mM KCl, 5 mM BME) and the target protein with buffer B (buffer A, containing 150 mM imidazole). The eluent was collected and dialyzed against buffer A at 4°C for 3 hours to remove imidazole. The eluent was then concentrated using an Amicon Ultra-15 centrifugal filter (Millipore), flash-frozen in liquid nitrogen, and stored at -80°C.

[0050] Protein purification results are as follows Figure 2 As shown, lane 1 is the protein marker, and lanes 2-4 contain purified 1μg, 2μg, and 4μg proteins, respectively. Figure 2 In the figure, 'a' represents the electrophoresis result of YgeY (i.e., EcYgeY) derived from E. coli. Figure 2 In the image, b represents the electrophoresis result of YgeX (i.e., EcYgeX) derived from E. coli. Figure 2 In the figure, 'c' represents the electrophoresis result of HyuA (i.e., CdHyuA) derived from Clostridioides difficile. Figure 2 In the figure, 'd' represents the electrophoresis result of UacY (i.e., CdUacY) derived from Clostridioides difficile. Figure 2 The 'e' in the text indicates that it originates from Escherichia coli YgeW (i.e., EcYgeW). Figure 2 In the figure, f represents the electrophoresis result of YgfK (i.e., EcYgfK) derived from Escherichia coli. Figure 2 In the figure, g represents the electrophoresis result of SsnA (i.e., EcSsnA) derived from Escherichia coli. Figure 2 In the figure, h represents the electrophoresis result of UacX (i.e., CdUacX) derived from Clostridioides difficile.

[0051] The XdhD (xanthine dehydrogenase, Uniprot ID: Q46814) gene was expressed using the low-copy plasmid pSC101, with a TEV restriction site, a (GGGGS)3 flexible linker, and a tandem Protein A tag (ProtA) introduced at the C-terminus. XdhD expression does not require an inducer. Single colonies were picked and cultured overnight, then transferred 1:100 to 1L of LB globulin containing kanamycin sulfate and cultured at 37°C for 20 h. Cells were collected by centrifugation at 25000×g for 60 min at 4°C. Nucleic acid was precipitated with 2% streptomycin sulfate. The supernatant was filtered through a 0.45 μm PES membrane and loaded onto a pre-equilibrated 1 mL rabbit IgG Beads 4FF column using a peristaltic pump. The ProtA tag was removed by column digestion with 1 mg / mL TEV protease. The lysed protein was collected, concentrated to approximately 500 μL, and stored at -80°C for subsequent enzymatic assays.

[0052] XdhD protein purification gel image as shown Figure 3 As shown, lane 1 is the protein marker, and lanes 2-4 are the purified 1μg, 2μg, and 4μg proteins, respectively; YgfM is the oxidoreductase subunit co-transcribed with XdhD and purified by IgG affinity column, Uniprot ID: P64557.

[0053] Example 3: Detection of uric acid degradation enzyme pathway activity

[0054] 3.1 Activity detection of XdhD

[0055] A solution containing 1 mM uric acid, 10 μM XdhD (Uniprot ID: Q46814) obtained in Example 1, 20 mM Tris-HCl (pH = 7.5), 200 mM KCl, and 1 mM NADH was incubated at room temperature for 1 h to allow the system to react completely. An equal volume of acetonitrile solution was added and mixed thoroughly. The mixture was centrifuged at 14000 × g for 10 min, and the protein precipitate was removed by filtration through a 0.22 μm filter. This group was designated as the reaction group. The enzyme-free control group was the reaction without the addition of the oxidoreductase XdhD. All other experimental conditions were the same for the control group and the reaction group. The isoxanthine standard was used as a positive control.

[0056] The reaction formula and reaction mass spectrum results are as follows: Figure 4 As shown, the left side represents the reaction formula, where uric acid is reduced to isoxanthine (2,8-dioxanone, IsoX) under the catalysis of XdhD and YgfM (an oxidoreductase subunit co-transcribed with XdhD). The right side shows the mass spectrum of the reaction product isoxanthine detected by LC-MS, with the horizontal axis representing retention time (in minutes) and the vertical axis representing signal intensity. The mass spectrometry data include product standards, the reaction group, and the enzyme-free control group. LC-MS detection of the XdhD-catalyzed reduction of uric acid to isoxanthine showed no product peak detected in the enzyme-free control group, while a product peak with a molecular weight of 153 was detected in the experimental group, demonstrating that the oxidoreductase XdhD is the initiating step in the entire uric acid reducing degradation pathway.

[0057] 3.2 Activity detection of YgfK and SsnA

[0058] A solution containing 1 mM IsoX, 10 μM YgfK and SsnA obtained in Example 1, 20 mM Tris-HCl (pH = 7.5), 200 mM KCl, 1 mM ZnCl2, and 1 mM NADH was incubated at room temperature for 1 h to allow the system to react fully. The experiment was conducted in an oxygen-free glove box. All solutions, including protein solutions and reaction buffers, were deoxygenated using a Schlenk line before entering the glove box. An equal volume of acetonitrile solution was added, mixed thoroughly, and centrifuged at 14000 × g for 10 min. The protein precipitate was removed by filtration through a 0.22 μm filter membrane, and this group was designated as the reaction group. Control groups: The YgfK-SsnA-free group was the reaction without the addition of reductase YgfK and hydrolase SsnA; the SsnA-free group was the reaction without the addition of hydrolase SsnA; the substrate-free group was the reaction without the addition of IsoX; and the coenzyme NADH-free group was the reaction without the addition of NADH. The control group and the experimental group had the same experimental conditions. Urimethylhydantoin (UMH, major product) or 5-ureidodihydrouracil (5UDU, minor product) standards were used as positive controls.

[0059] Simultaneously, the solution of CdUacX obtained in Example 1, SsnA, 20mM Tris-HCl (pH=7.5), 200mM KCl, 1mM ZnCl2, and 1mM reducing agent methyl viologen (MV+·) was incubated in a glove box at room temperature for 1 h. An equal volume of acetonitrile solution was added and mixed well, and the mixture was centrifuged at 14000×g for 10 min. The protein precipitate was filtered out through a 0.22 μm filter membrane and recorded as the reaction group. The control groups were: the CdUacX-SsnA-free group (reaction without CdUacX and SsnA hydrolase), the substrate-free group (reaction without IsoX), and the methyl viologen-free group (reaction without methyl viologen). Urimethylhydantoin (UMH, major product) or 5-ureidodihydrouracil (5UDU, minor product) standards were used as positive controls.

[0060] The coupling activity of YgfK and SsnA catalyzed reduction of IsoX to ureomethylhydantoin (UMH, major product) or 5-ureodihydrouracil (5UDU, minor product) was detected by LC-MS. The results are as follows: Figure 5 As shown, the horizontal axis represents retention time in minutes, and the vertical axis represents the intensity of the mass spectrometry response signal. Figure 5 In the figure, 'a' represents the reaction process in which isoxanthine is reduced and hydrolyzed into products 5UDU and UMH under the coupling action of YgfK and SsnA. Figure 5 In the figure, 'b' indicates that the molecular weights of products 5UDU and UMH are both 173, and the fragment ions in the secondary mass spectrometer are 85.1, 101.1, and 130.0, respectively. Figure 5 In the image, 'c' represents the mass spectrum of the coupling reaction between E. coli strain YgfK and SsnA. No product peak was detected in the control group, while product peaks of UMH (molecular weight 173) and 5 UDU were detected in the experimental group. Figure 5 In the figure, d represents the mass spectrum of the coupling reaction of UacX and SsnA, which are proteins from the same family of the Clostridioides difficile strain. No product peaks were detected in the control group, while product peaks of UMH with a molecular weight of 173 and 5UDU were detected in the experimental group.

[0061] It was demonstrated that the reductase YgfK and the hydrolase SsnA are the second step in the entire uric acid reducing degradation pathway. It was also demonstrated that YgfK has the same activity as the CdUacX protein in the Clostridioides difficile species, and can be coupled with SsnA to catalyze the reduction of IsoX to UMH or 5UDU.

[0062] 3.3 Activity detection of HyuA and CdUacY / YgeW

[0063] A solution containing 10 μM of the hydrolase HyuA obtained in Example 1, 20 mM Tris-HCl (pH = 7.5), 200 mM KCl, and 1 mM ZnCl2 was incubated with 10 mM 5 UDU and 10 mM UMH, respectively, at room temperature for 1 h to allow the system to react completely. An equal volume of acetonitrile solution was added, and the mixture was centrifuged at 14000 × g for 10 min. The protein precipitate was removed by filtering through a 0.22 μm filter membrane, and this was designated as the reaction group. The control group consisted of the reaction without the addition of reductase HyuA, with all other experimental conditions remaining the same.

[0064] Simultaneously, solutions containing 10 μM of the hydrolase HyuA obtained in Example 1, 20 mM Tris-HCl (pH = 7.5), 200 mM KCl, and 1 mM ZnCl2 were incubated with 10 mM UMH substrate, CdUacY, and YgeW at room temperature for 1 h to allow the system to react fully. An equal volume of acetonitrile solution was added, mixed thoroughly, and centrifuged at 14,000 × g for 10 min. The protein precipitate was removed by filtration through a 0.22 μm filter membrane, and this group was designated as the reaction group. The control groups were the reactions without HyuA reductase, without CdUacY amide hydrolase, without EcYgeW, without any enzyme substrate, and without substrate, respectively. Albizziin standard was used as a positive control. All other experimental conditions were the same for the control and experimental groups.

[0065] LMC was used to detect the activities of HyuA in catalyzing the reduction of 5UDU to 2-ureidopropionate acid (DUPA), the reduction of UMH to DUPA, and the reduction of UMH to 3-ureido-2-aminopropionate (Albizziin) catalyzed by HyuA coupled with CdUacY or YgeW. CdUacY is an isoenzyme of YgeW in the Clostridioides difficile strain. The results are as follows: Figure 5 As shown, the horizontal axis represents retention time in minutes, and the vertical axis represents the intensity of the mass spectrometry response signal.

[0066] Schematic diagram and mass spectrometry results of the HyuA hydrolysis of 5UDU are shown below. Figure 6 As shown in Figure a, the left side is a schematic diagram of 5UDU being hydrolyzed by HyuA to generate DUPA, and the right side is the mass spectrum of the HyuA reaction. The results show that, compared with the control group, the whole reaction group detected a DUPA product peak with a molecular weight of 191.

[0067] Schematic diagram and mass spectrometry results of the HyuA hydrolysis UMH reaction are shown below. Figure 6As shown in Figure b, the left side is a schematic diagram of UMH being hydrolyzed by HyuA to generate DUPA, and the right side is the mass spectrum of the HyuA reaction. The results show that, compared with the control group, a DUPA product peak with a molecular weight of 191 was detected in the whole reaction group. This proves that the hydrolase HyuA is the third step in the entire uric acid reducing degradation pathway.

[0068] HyuA coupled with CdUacY or YgeW catalyzes the reduction of UMH to 3-ureo-2-aminopropionic acid (Albizziin). Results are as follows: Figure 6 As shown in Figure c, the hydrolysis of UMH by HyuA to generate DUPA, and the hydrolysis of DUPA by YgeW to generate Albizziin are schematic diagrams. CdUacY is an isoenzyme of YgeW. In the mass spectrometry of the reaction of HyuA coupled with CdUacY or YgeW, no product peak was detected in the control group, while an Albizziin product peak with a molecular weight of 148 was detected in the reaction group containing CdHyuA (HyuA from the Clostridioides difficile strain) and CdUacY. A weak Albizziin product peak with a molecular weight of 148 was detected in the reaction group containing CdHyuA and YgeW. YgeW has weaker enzyme activity than CdHyuA, resulting in a weaker product amount. This proves that the hydrolases HyuA and CdUacY / YgeW are the third and fourth steps in the entire uric acid reducing degradation pathway.

[0069] 3.4 Activity detection of YgeY and YgeX

[0070] A solution containing 5 mM albizziin, 10 μM urease YgeY and diaminopropionic acid lyase YgeX obtained in Example 1, 20 mM Tris-HCl (pH = 7.5), 200 mM KCl, and 1 mM CoCl2 was incubated at room temperature for 1 h to allow the system to react completely. An equal volume of acetonitrile solution was added, mixed thoroughly, and centrifuged at 14000 × g for 10 min. The protein precipitate was removed by filtration through a 0.22 μm filter membrane, and this was designated as the reaction group. The control group consisted of the reaction without the addition of YgeY and YgeX, and all other experimental conditions were the same as those in the experimental group. LC-MS was used to detect the reduction of L / D-albizziin to pyruvate catalyzed by YgeY and YgeX. The results are as follows: Figure 7 As shown, the horizontal axis represents retention time in minutes, and the vertical axis represents the intensity of the mass spectrometry response signal. The left side shows a schematic diagram of the reaction, and the right side shows the reaction mass spectrum. No product peak was detected in the control group, while a pyruvate product peak with a molecular weight of 87 was detected in the reaction group. This proves that hydrolase YgeY and lyase YgeX are the fifth and sixth steps in the entire uric acid reducing degradation pathway.

[0071] Example 4: Identification of the active site of urate-degrading enzyme

[0072] After confirming in vitro that XdhD contains urate oxidoreductase activity, the structure of this enzyme was simulated using the Boltz-1 server. Simultaneously, the structure of its homolog XdhA, located in the same gene cluster and also derived from xanthine dehydrogenase, was simulated using the Boltz-1 server. The results are as follows: Figure 8 As shown, the XdhD structure exhibits a catalytic site consisting of a MoCo binding domain, a glutamine (GLN449), and a glutamic acid (GLU606). These spatially close binding sites interact with the substrate uric acid, reducing its C6 carbonyl group. The XdhA structure also shows a catalytic site consisting of a MoCo binding domain and a glutamine (GLN241). These spatially close binding sites also interact with the substrate.

[0073] Structural analysis revealed that XdhD differs from its family member XdhA in substrate specificity because the E606 side chain carboxyl group (a negatively charged glutamate Glu) of XdhD can form a hydrogen bond network, enabling directional recognition of uric acid. The E606 side chain carboxyl group, through dipole repulsion with the C6=O of uric acid, forces the purine ring to bind in a specific direction (C6 towards the active site of molybdenum cofactor). This unique characteristic of XdhD ensures that the reduction reaction occurs at the C6 position, converting uric acid into the intermediate product IsoX, which can be further degraded by E. coli.

[0074] Example 5: Identification of fluorescent markers for the urate-degrading enzyme pathway

[0075] Bioinformatics analysis revealed that isoxanthine (IsoX) is widely present in anaerobic bacteria. Isoxanthine is a product of the first step of uric acid metabolism (XdhD) and a substrate of the second step (YgfK+SsnA). It also exhibits unique fluorescence characteristics at λex / em = 308 / 363 nm. Isoxanthine was detected in clinical samples. Serum samples were collected from 25 gout patients and 43 age / sex-matched healthy volunteers. All procedures were performed according to the research protocol approved by the Institutional Review Committee of Tianjin First Central Hospital. After collection, blood samples were allowed to coagulate at room temperature for 30 minutes and then centrifuged at 1,500g for 10 minutes at 4°C. 50 μL of serum was aliquoted, and 150 μL of an ice-cold acetonitrile-methanol mixture (1:1, v / v) was added to remove proteins. After vortexing for 1 minute, the mixture was centrifuged at 16,000g for 10 minutes. The supernatant was analyzed using an Agilent 1260 HPLC-6420 triple quadrupole mass spectrometer under the aforementioned multiple reaction monitoring (MRM) conditions. Serum uric acid and isoxanthine concentrations were quantified using an external standard calibration curve.

[0076] Test results as follows Figure 9 As shown, with Figure 9 Table a shows the mass spectrometry of isoxanthine levels in clinical samples from a hospital, with the horizontal axis representing retention time in minutes and the vertical axis representing mass spectrometry response signal intensity. Table b compares isoxanthine levels in gout patients and healthy individuals. Isoxanthine was detectable in samples when uric acid levels in the blood of patients reached 737.6 μM or higher, but not when uric acid levels were only 217.1 μM and 326.1 μM. Comparing isoxanthine levels in the blood of gout patients and healthy individuals revealed a significant increase in serum isoxanthine levels in gout patients, with the isoxanthine exhibiting fluorescence at λex / em = 308 / 363 nm, which can serve as a biomarker for gout diagnosis.

[0077] Example 6: Construction of Escherichia coli overexpressing urate-degrading enzyme

[0078] This invention hypothesizes that constitutive overexpression of the uric acid degradation pathway in engineered gut bacteria can enhance the uric acid degradation capacity of the gut microbiome, bypassing the inhibition of energy-rich dietary molecules such as glucose. Based on the parental strain EcNc (DSMZ:Cat#DSM6601) derived from the probiotic Escherichia coli Nissle 1917 (EcN), a hybrid promoter cassette (containing a strong constitutive gapA promoter and an anaerobic nirB promoter, as shown in SEQ ID NO.1) was inserted into the spacer region (3,005,786-3,006,262) between YgeV and YgeW via CRISPR-Cas9 genome editing, replacing the original natural promoter and the natural Crp (cAMP receptor protein) binding site. The resulting strain was named CarBT4gout_2.0 (CBT2.0). The host strain was selected from E. coli hosts that do not contain free plasmids pMUT1 and / or pMUT2 to improve the stability of genome engineering and meet the safety requirements of subsequent probiotic preparations (such as EcNc, or K-12 / BL21 derivatives that do not naturally contain this plasmid). If the starting strain contains the above plasmids, it can be excluded by non-selective passage or CRISPR targeted cleavage. The region 3,005,786-3,006,262 of the EcNc genome is the natural regulatory entry point for the uric acid degradation cluster; replacement can achieve "overall control," allowing the sustained expression of the uric acid degradation gene cluster, including XdhD, YgfK, SsnA, HyuA, YgeW, YgeY, and YgeX; the strong constitutive promoter gapA enhances sustained high transcription, and the addition of nirB further enhances this in the anaerobic intestinal environment, synergistically overcoming fluctuations in dietary carbon sources. Specific construction process:

[0079] S0. Strains and reagents: The strain selected was EcNc, a parent strain derived from wild-type Escherichia coli Nissle 1917. Reagents: LB (Luria-Bertani) medium, M9 medium, glucose, uric acid, L-arabinose, antibiotic (Km), PEG (polyethylene glycol), magnesium chloride and DMSO. CRISPR vector pCas9-gRNA was also prepared.

[0080] S1, EcNc competent cell preparation: EcNc competent cells were prepared using the TSS (Transformation and Storage Solution) method;

[0081] S2. Sequence extraction: Select the spacer region between YgeV and YgeW (specifically located at 3,005,786-3,006,262 in the EcNc genome, which contains the complete Crp binding site);

[0082] S3. Design of a hybrid promoter cassette: A strong constitutive promoter, gapA, requiring no induction, and a nirB promoter with a partially deleted NarL binding sequence (retaining the FNR region, also known as the FNR binding site / FNR regulatory element, containing the FNR consensus sequence TTGAT-N4-ATCAA DNA region; under hypoxia, the FNR protein dimer binds there and activates the transcription of downstream genes, an anaerobic response) are selected. This reduces the promoter's dependence on nitrates, requiring only anaerobic conditions for activation. A 15bp spacer separates the gapA and nirB promoters to avoid structural interference. Two rare restriction enzyme sites are also introduced for easy subsequent replacement. The hybrid promoter nucleic acid sequence is shown in SEQ ID NO.1, where the 5′ end is the strong gapA promoter (TTGACA / TATAAT) located approximately -35bp to -10bp upstream of the transcription start site (TSS), as shown in SEQ ID NO.1. As shown in NO.1, 1-100bp, the 3′-end is a truncated fragment of the nirB anaerobic promoter (64-1bp upstream of the transcription start site TSS, containing the anaerobic response FNR region, and removing the nitrate / nitrite response Nar region of -79-60bp), as shown in SEQ ID NO.1, 116-175bp.

[0083] S4. Construction of donor DNA: The gapA promoter fragment and the nirB promoter fragment were obtained by PCR and constructed in the pUC19T vector to obtain the pUC19T-gapAp-nirBp plasmid.

[0084] S5, pCas9-gRNA-YgeV-gapAp-nirBp construction: Design sgRNA based on the intermediate sequences of YgeV and YgeW to be edited, and ligate it into the pCas9-gRNA vector. At the same time, amplify the gapAp-nirBp sequence from the pUC19T-gapAp-nirBp plasmid to obtain the pCas9-gRNA-YgeV-gapAp-nirBp plasmid.

[0085] S6. Host editing: Electroporate pCas9-gRNA-YgeV-gapAp-nirBp plasmid (KAN resistance) into EcNc; select positive clones; maintain incubation at 30℃;

[0086] S7, induced Cas9 cleavage, and λ-Red homologous recombination: during the logarithmic phase (OD) 600 ≈0.4) Add 0.2% L-arabinose to cells and induce λ-Red at 37℃; simultaneously activate Cas9; after induction for 12–14 h, plate the cells (Kan resistant);

[0087] S8. Initial screening of integrated clones and sequencing verification: Select single clones; perform colony PCR using a pair of outer genome primers and a pair of inner insertion primers; perform Sanger sequencing on the correctly amplified fragments to obtain strains with inserted mixed promoter cassettes;

[0088] S9. Remove editing plasmids, name and preserve: The obtained correctly sequenced strains are cultured at 37℃ for 2-3 rounds without antibiotics, and the colony Km sensitivity is tested; PCR is performed without Cas9; plasmid-free integrated strains are retained; the resulting strains are cryopreserved and named CarBT4gout_2.0 (CBT2.0).

[0089] Construction diagram as follows Figure 10 As shown in Figure a, Figure 10 The image in Figure 'b' represents the EcN1917 genome sequence map with inserted gapA and nirb promoter sequences. The nirB promoter sequence marks the FNR region. RNA-seq analysis confirmed the overexpression of the uric acid degradation gene, as shown in the figure. Figure 11As shown, dots represent individual genes. Red dots indicate significant upregulation (padj < 0.05, log2FC > 1), blue dots indicate significant downregulation (padj < 0.05, log2FC < -1), and gray dots represent genes with no significant difference. The X-axis represents the log2FoldChange value (the log-2 ratio of gene expression change between the CBT2.0 strain group and the wild-type strain group; a positive value indicates gene upregulation in the CBT2.0 strain group, i.e., the experimental group, and a negative value indicates gene downregulation in the experimental group), and the Y-axis represents the -adjustedp value (-log10). The results showed that the expression of the uric acid degradation gene clusters XdhD, YgfK, SsnA, YgeY, YgeX, YgeW, and HyuA was significantly upregulated.

[0090] Example 7: Escherichia coli overexpressing urate-degrading enzyme overcomes glucose inhibition

[0091] To evaluate the role of the above-mentioned uric acid degradation pathway in vivo, we constructed XdhA knockout inhibitors based on the Escherichia coli MG1655 strain. Figure 12 As shown in ΔxdhA-MG1655), knock out XdhD ( Figure 12 The strain ΔxdhD-MG1655 was used to construct a YgfK knockout strain based on Escherichia coli EcN. Figure 12 The strain ΔygfK-EcN was used to construct XdhA (as shown in the image) based on the CBT2.0 strain. Figure 12 As shown in ΔxdhA-CBT2.0), knock out XdhD ( Figure 12 The strain (ΔxdhD-CBT2.0) was used. We added uric acid, xanthine, or isoxanthine to standard LB medium, M9 complete medium containing glucose, and M9 complete medium without glucose, respectively. The culture supernatant was collected every 12 hours, and the amount of downstream products of uric acid metabolism was analyzed by LC-MS. The results of the *E. coli* strain CBT2.0 overexpressing uric acid-degrading enzyme overcoming glucose inhibition are shown below. Figure 11 As shown, the horizontal axis represents different incubation times (in hours); the vertical axis represents the abundance of mass spectrometry response signals. Quantitative analysis was performed, and significance analysis was conducted based on the differences in mass spectrometry abundance.

[0092] Figure 12 In section a: xanthine consumption of wild-type MG1655 strains and gene knockout (ΔXdhA-MG1655 and ΔXdhD-MG1655) Escherichia coli strains anaerobically cultured in xanthine-supplemented M9 medium to supplement xanthine M9 medium; blank medium ( Figure 12As shown in the blank M9 (as a control), the results showed that in M9 glucose medium supplemented with xanthine, wild-type MG1655 strain and ΔXdhD-MG1655 consumed xanthine, while ΔXdhA-MG1655 did not consume it, consistent with the role of XdhA in xanthine oxidation.

[0093] Figure 12 Figure b shows the uric acid accumulation of wild-type MG1655 and gene knockout (ΔXdhA-MG1655 and ΔXdhD-MG1655) Escherichia coli strains anaerobically cultured in xanthine-supplemented M9 medium, compared to blank medium. Figure 12 As shown in blank M9 in the middle, uric acid accumulates in ΔXdhD-MG1655 as a control, while wild-type MG1655 strain and ΔXdhA-MG1655 do not accumulate uric acid, consistent with the role of XdhD in subsequent uric acid degradation. Xanthine consumption is accompanied by uric acid accumulation.

[0094] Figure 12 In the middle c, wild-type EcN strain, engineered strain CBT2.0, and gene knockout (ΔXdhA-CBT2.0 and ΔXdhD-CBT2.0) Escherichia coli strains were anaerobic cultured in uric acid-supplemented LB medium, and the results showed that all tested strains exhibited uric acid degradation in uric acid-supplemented LB medium.

[0095] Figure 12 In Figure d, isoxanthine accumulation was observed in wild-type EcN strain, engineered strain CBT2.0, and gene knockout (ΔXdhA-CBT2.0 and ΔXdhD-CBT2.0) *E. coli* strains anaerobically cultured in uric acid-supplemented LB medium. Fresh LB medium ( Figure 12 Low levels of isoxanthine (IsoX) were detected in the empty LB (as shown in the middle d), but IsoX accumulated further in CBT2.0 and ΔxdhA-CBT2.0, while no IsoX was observed in ΔXdhD-CBT2.0, indicating an increased XdhD flux in these strains.

[0096] Figure 12 In the figure, 'e' represents the uric acid consumption of wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strain anaerobic cultured in LB medium with added uric acid and glucose. Glu- represents the control group inoculated with wild-type EcN strain without added glucose, and empty LB represents the blank control group without added uric acid and glucose.

[0097] Figure 12The f in the figure represents the isoxanthine accumulation of wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strain anaerobic cultured in LB medium with added uric acid and glucose. Glu- represents the control group inoculated with wild-type EcN strain without added glucose, and empty LB represents the blank control group without added uric acid and glucose.

[0098] Figure 12 The 'g' indicates the accumulation of UMH in wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strains cultured anaerobically with uric acid and glucose added to LB medium. Glu- indicates the control group inoculated with wild-type EcN strain without glucose. Empty LB represents the blank control group without uric acid and glucose.

[0099] In LB medium supplemented with uric acid and glucose, uric acid degradation in the wild-type EcN strain was inhibited, while CBT2.0 remained unaffected, indicating that CBT2.0 can bypass glucose inhibition. Glucose inhibition refers to the bacteria preferentially utilizing glucose and inhibiting metabolic pathways that utilize uric acid and other carbon sources. IsoX accumulates in CBT2.0. Figure 12 As shown in f), compared with LB (which does not contain glucose). Figure 12 The observations in (c and d) are similar. UMH was detected only in CBT2.0 and wild-type cultures without glucose, supporting its role as a downstream intermediate.

[0100] Figure 12 In the LB medium, h represents the wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strain anaerobic cultured with isoxanthine and glucose added. Isoxanthine consumption is consumed in these strains. Glu- represents the control strain inoculated with wild-type EcN strain without added glucose. Empty LB represents the blank control strain without added isoxanthine and glucose.

[0101] Figure 12 In the figure, i represents the accumulation of UMH in wild-type EcN strain, engineered strain CBT2.0 and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strain anaerobic cultured in LB medium with added isoxanthine and glucose. Glu- represents the control inoculated with wild-type EcN strain without added glucose. Empty LB represents the blank control without added isoxanthine and glucose.

[0102] Figure 12The value of j indicates the accumulation of albizziin in wild-type EcN strain, engineered strain CBT2.0, and gene knockout type ΔYgfK-CBT2.0 Escherichia coli strain anaerobic cultured in LB medium supplemented with isoxanthine and glucose. Glu- represents the control group inoculated with wild-type EcN strain without glucose, and empty LB represents the blank control group without isoxanthine and glucose.

[0103] Figure 12 The "hj" indicates that in glucose-containing LB medium, all strains showed IsoX degradation, but under glucose-free conditions, the CBT2.0 strain and the wild-type EcN strain degraded IsoX faster (e.g., ...). Figure 12 (As shown in h), consistent with the trend of uric acid. UMH was detected in all strains except ΔYgfK-CBT2.0, consistent with the role of YgfK in the conversion of IsoX to UMH. Low levels of Albizziin were detected in fresh LB medium, but further accumulation occurred only in CBT2.0, reflecting enhanced activity of upstream enzymes (such as YgeW) in the uric acid degradation pathway involved in the CBT2.0 strain.

[0104] This invention employs a dual-promoter substitution method to relieve glucose inhibition. A hybrid promoter consisting of the strong constitutive promoter gapA and the anaerobic promoter nirB replaces the Crp binding site in the YgeV-YgeW spacer region of the natural promoter. The resulting CBT2.0 strain can still degrade and utilize uric acid in the presence of grapes, with a 72-hour UA residue rate of <30% vs. >90% for the wild type. Figure 12 As shown in Figure e), wild-type ECN could not metabolize uric acid within 72 hours in the presence of 0.4% (w / v) glucose, while CBT2.0 preferentially utilized uric acid as a carbon source in the presence of 0.4% (w / v) glucose, degrading uric acid to 70% within 72 hours, demonstrating resistance to glucose inhibition and an increase in the flux of the uric acid degradation pathway.

[0105] Example 8: Escherichia coli overexpressing urate-degrading enzyme for the treatment of gout

[0106] To evaluate the potential of CBT2.0 in lowering uric acid in the host, we used a uricase-deficient (UOX- / -) hyperuricemia mouse model. Mice were randomly divided into three groups and administered CBT2.0, wild-type EcN (WT), or PBS via gavage daily for 6 consecutive weeks as a control. Blood samples were collected weekly to monitor plasma uric acid (UA), blood urea nitrogen (UN), and creatinine (CRE) levels.

[0107] Results of the effectiveness of engineered probiotics in animal models of gout: Figure 13 As shown, CBT2.0 (group 2.0, blue, 1×10) 10CFU), wild-type Escherichia coli Nissle 1917 (WT group, red, 1×10⁻⁶) 10 CFU (cytokine oxidase) or vector control (PBS group, orange, 200 μL), plasma samples were collected weekly. Bar chart data are all mean ± standard deviation (n = 6-7 mice / group), and individual data points are displayed in superimposed form. Statistical analysis was performed using a two-tailed t-test, with the specific p-value indicated above parentheses. The experimental design for mice is as follows: Figure 13 As shown in Figure a, male urate oxidase-deficient (UOX- / -) mice were discontinued from allopurinol one week earlier (week -1) and administered 1×10⁻⁶ mg / day by gavage for six consecutive weeks starting from week 0. 10 CFU's CBT 2.0, 1×10 10 CFU wild-type Escherichia coli Nissle 1917 and PBS. Figure 13 In the figure, b represents the plasma uric acid (UA) levels in mice in the CBT2.0 group, WT group, and PBS group. During the treatment period, the plasma UA level in the CBT2.0 group was significantly lower than that in the PBS control group. At week 6, the mean UA concentration in the CBT2.0 group was 171.63 ± 91.59 μmol / L, while the UA concentration in the PBS group remained at 463.26 ± 70.81 μmol / L (p < 0.001), and the UA concentration in the WT group was 387.73 ± 266.85 μmol / L. The UA level in the WT group was slightly lower than that in the PBS group at some time points, but this was not consistent throughout the study period. These results indicate that CBT2.0 can more reliably reduce systemic uric acid levels in UOX- / - mice compared to WT.

[0108] The physiological effects of hyperuricemia in mice differ slightly from those in humans; uric acid crystals are more likely to deposit in the kidneys than in the joints, leading to potentially fatal acute kidney injury. Impaired kidney function is typically manifested as systemic accumulation of uric acid renal enzymes (CRE) and unresolved uric acid (UN). To assess the renal effects of the three treatments, we monitored plasma UN levels and evaluated CRE levels at weeks 0, 3, and 6. Figure 13 As shown in the figure. Plasma urea nitrogen (BUN) levels in each group of mice are as follows. Figure 13 As shown in Figure c, at week 6, the UN level in the CBT2.0 group (33.40±8.96 mmol / L) was significantly lower than that in the WT group (44.02±10.07 mmol / L, p=0.0718) and the PBS group (48.75±4.70 mmol / L, p=0.004). Plasma creatinine (CRE) levels in each group at different time points are shown below. Figure 13As shown in Figure d, CRE levels showed a similar trend, with the CBT2.0 group (14.61±3.97 μmol / L) significantly lower than the PBS group (34.14±6.66 μmol / L, p<0.0001) and the WT group (23.86±3.81 μmol / L, p=0.0013). The qPCR results of engineered bacteria CBT2.0 in mouse colon contents are shown below. Figure 13 As shown in Figure e, the vertical axis represents the Ct values ​​obtained using primers specific to the CBT2.0 gene; each scatter point represents one mouse (n=6 per group), and the value is the average of three technical replicates. Figure 13 In the figure, f represents the plasma uric acid (UA) concentration in mice 8 weeks after gavage was stopped. The plasma uric acid concentration in mice treated with CBT2.0 was significantly lower than that in wild-type and PBS mice.

[0109] The overall appearance of the kidneys of 15-week-old mice (6 mice each from CBT2.0, WT, and PBS) in the same batch as those used for serum uric acid testing is as follows: Figure 14 As shown in Figure a, scale bar = 2cm. The procedure involved fixation with 4% paraformaldehyde → paraffin embedding → 4μm sectioning → H&E staining → microscopic imaging. A representative image of hematoxylin-eosin (H&E) staining of kidney tissue is shown below. Figure 14 As shown in Figure b, the top row is 40× magnification and the bottom row is 100× magnification. The WT and PBS groups showed significant renal tubular dilation and luminal protein casts; the pathological changes in the CBT2.0 group were significantly alleviated. The overall pathological score of the CBT2.0 group was lower than that of the WT (P<0.05) and PBS (P<0.01). These results indicate that CBT2.0 effectively alleviates kidney tissue damage in hyperuricemic model mice.

[0110] It is noteworthy that the UA level in the CBT2.0 group mice was lower than that in the PBS group in week 1 (although it did not reach statistical significance in weeks 2 and 5, p>0.05), and the significant reduction in BUN in the CBT2.0 group first appeared in week 3, while the CRE level was not significantly different at this time, suggesting that the renal protective effect of CBT2.0 treatment (possibly mediated by UA reduction) may have a time lag.

[0111] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of engineered intestinal probiotic, characterized in that, Based on the Escherichia coli origin, it overexpresses the gene encoding the regulatory protein YgeV and the uric acid degradation gene cluster in the uric acid degradation pathway. The uric acid degradation gene cluster includes the gene encoding xanthine dehydrogenase, the gene encoding flavin / iron-sulfur cluster-dependent oxidoreductase, the gene encoding amide hydrolase, the gene encoding D-phenylhydantoinase, the gene encoding carbamoyltransferase, the gene encoding urease, and the gene encoding diaminopropionic acid lyase. Preferably, constitutive overexpression of the uric acid degradation gene cluster in the uric acid degradation pathway is used.

2. The intestinal probiotic engineered bacteria as described in claim 1, characterized in that, The overexpression involves inserting a mixed promoter cassette into the spacer region between the gene encoding the transcriptional regulatory factor YgeV and the gene encoding the carbamoyltransferase YgeW in the genome of the originating strain of *E. coli*. This spacer region is the gene interval from 3005786 to 3006262 in the *EcNc* genome. The uniplot number of YgeV is Q46802, and the uniplot number of the amino acid sequence of YgeW is Q46803. The insertion of the mixed promoter cassette upregulates the expression of the endogenous gene encoding the regulatory protein YgeV and genes in the uric acid degradation gene cluster within the originating strain of *E. coli*. The mixed promoter cassette contains both constitutive and anaerobic promoters; preferably, it contains both a strongly constitutive promoter and an anaerobic promoter.

3. The intestinal probiotic engineered bacteria as described in claim 1, characterized in that, The originating strain of Escherichia coli is Escherichia coli K12, Escherichia coli BL21, Escherichia coli Nissle 1917 strain or a parental strain derived therefrom, such as EcNc. Preferably, the originating strain of Escherichia coli does not contain free plasmids, such as pMUT1 and / or pMUT2.

4. The intestinal probiotic engineered bacteria according to claim 3, characterized in that, The strong constitutive promoter is gapA, tufB, lpp, rrnB promoter and its equivalents, and the anaerobic promoter is anaerobic nirB, narG, narK, dmsA, frdA promoter and its equivalents. Preferably, the anaerobic promoter contains only the anaerobic-responsive FNR region and no nitrate / nitrite-responsive Nar region. Preferably, the strong constitutive promoter has 1-2 non-conserved base substitutions. Preferably, there is a spacer sequence between the strong constitutive promoter and the anaerobic promoter. Preferably, the nucleotide sequence of the mixed promoter is shown in SEQ ID NO.

1.

5. The intestinal probiotic engineered bacteria according to claim 1, characterized in that, The unirot number encoding xanthine dehydrogenase is Q46814, the unirot number encoding flavin-dependent reductase is Q46811, the unirot number encoding amide hydrolase is Q46812, the unirot number encoding D-phenylhydantoinase is Q46806, the unirot number encoding carbamoyltransferase is Q46803, the unirot number encoding urease is P65807, and the unirot number encoding diaminopropionic acid lyase is P66899; the engineered bacteria can overcome the inhibition of uric acid degradation pathway by intestinal glucose.

6. The use of the intestinal probiotic engineered bacteria according to any one of claims 1-5 in the preparation of a drug for reducing host serum uric acid levels, or in the preparation of a drug for improving hyperuricemia, or in the preparation of a drug for treating gout.

7. A method for constructing intestinal probiotic engineered bacteria according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. The hybrid promoter box is introduced into the Escherichia coli originating bacteria using gene editing technology; S2. Screening to obtain intestinal probiotics that constitutively overexpress uric acid degradation gene clusters, wherein the intestinal probiotics overcome the inhibition of uric acid degradation by intestinal glucose; Preferably, the importation involves inserting the hybrid promoter box into the gene region 3005786 to 3006262 of the Escherichia coli originating strain genome, while replacing the binding sites of the natural promoter and crp in the gene region.

8. A fluorescent marker for uric acid metabolism, characterized in that, The fluorescent label is used to detect the uric acid degradation pathway. The fluorescent label is isoxanthine, and the CAS ID of isoxanthine is 13230-99-4. Specifically, the fluorescence characteristics of the fluorescent label are λex / em = 308 / 363nm.

9. The use of the fluorescent marker according to claim 8 in the preparation of a marker for the auxiliary diagnosis of gout; specifically, the use includes detecting the content of the fluorescent marker in a biological sample, including serum, urine or intestinal contents, using fluorescence spectroscopy or LC-MS.

10. A uric acid-degrading enzyme, characterized in that, The uric acid degrading enzyme is xanthine dehydrogenase, and the Uniprot number of xanthine dehydrogenase is Q46814, wherein the 606th amino acid is glutamic acid, which can achieve targeted recognition of uric acid and reduce the carbonyl group at the 6th position.

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