Regulation and control system for degrading uric acid to reduce uric acid level and application of regulation and control system

Abstract

The invention relates to the technical field of gene engineering, and particularly provides a regulation and control system for degrading uric acid to reduce the uric acid level and application thereof.The system comprises a first functional gene, a second functional gene and a third functional gene, and the first functional gene encodes a first functional protein capable of degrading the uric acid; the first functional protein comprises one or more sequences as shown in SEQ ID NO: 1-10 or having at least 85% identity with the first functional protein; a second functional gene encoding a second functional protein capable of transporting uric acid, the second functional protein comprising one or more sequences as shown in SEQ ID NO: 11-15 or having at least 85% identity therewith; the first promoter is used for regulating and controlling the expression of the first functional gene and / or the second functional gene; the first terminator is used for terminating the expression of the first functional gene and / or the second functional gene. The regulation and control system for degrading the uric acid to reduce the uric acid level, provided by the embodiment of the invention, shows an efficient uric acid degradation function.

Description

Technical Field

[0002] This application relates to the field of genetic engineering technology, specifically to a regulatory system for degrading uric acid to lower uric acid levels and its application. Background Technology

[0003] Uric acid (UA) is an important intermediate product of the purine metabolic pathway. Clinically, normal uric acid levels in the human body are typically considered to be within the range of 2.4-6.0 mg / dL (women) and 3.4-7.0 mg / dL (men). Genetic factors, medications used to treat diseases, excessive alcohol consumption, lack of sleep, and high-purine diets can all increase the risk of uric acid homeostasis imbalance, leading to abnormally high blood uric acid levels and causing hyperuricemia (HUA). With the improvement of socioeconomic level, the enrichment of material life, and the acceleration of the pace of life, the incidence of hyperuricemia has increased significantly. Statistics from 2019 show that the prevalence of hyperuricemia in China was 13.3%, and the overall incidence of gout was 1.1%, showing a trend of patients becoming younger.

[0004] Recently, a series of applied studies have focused on the potential site of action of intestinal uric acid excretion, developing strategies for treating hyperuricemia with gut probiotics. For example, functional elements such as uricase are introduced into the mouse gut to enhance the activity of uric acid metabolism and reduce uric acid levels in humans. However, these gut probiotics vary in quality, with many exhibiting problems such as insufficient uric acid degradation activity, the need for co-administration, potential cytotoxicity, or the use of inducers (such as IPTG and galactose) that are easily absorbed by the human body, leading to a sharp decline in induction effects. Furthermore, the available substrate bacteria for current recombinant probiotic intervention routes are extremely limited.

[0005] Therefore, there is an urgent need to develop beneficial bacterial strains that can efficiently and stably lower uric acid levels to meet the growing demand for the treatment of hyperuricemia and gout. Summary of the Invention

[0006] This application addresses at least one of the problems of the related technology in the following aspects.

[0007] Therefore, embodiments of this application provide a regulatory system for degrading uric acid to lower uric acid levels. The regulatory system includes: a first functional gene encoding a first functional protein capable of degrading uric acid, the first functional protein comprising one or more sequences as shown in or having at least 85% identity with SEQ ID NO: 1-10; a second functional gene encoding a second functional protein capable of transporting uric acid, the second functional protein comprising one or more sequences as shown in or having at least 85% identity with SEQ ID NO: 11-15; a first promoter for regulating the expression of the first functional gene and / or the second functional gene; and a first terminator for terminating the expression of the first functional gene and / or the second functional gene.

[0008] In some embodiments, the regulatory system further includes: a second promoter for regulating the expression of the first functional gene and / or the second functional gene; and a second terminator for terminating the expression of the first functional gene and / or the second functional gene. Optionally, the second promoter and the first promoter are constitutive promoters.

[0009] In some embodiments, the regulatory system specifically includes: a first promoter for regulating the expression of the first functional gene; a first terminator for terminating the expression of the first functional gene; a second promoter for regulating the expression of the second functional gene; and a second terminator for terminating the expression of the second functional gene, wherein the first promoter and the second promoter each comprise any sequence selected from SEQ ID NO: 16-23 or any sequence having at least 85% identity with it, the first promoter preferably comprises the sequence shown in SEQ ID NO: 16 or any sequence having at least 85% identity with it, the second promoter preferably comprises any sequence shown in SEQ ID NO: 17-23 or any sequence having at least 85% identity with it; the first terminator and the second terminator each comprise any sequence selected from SEQ ID NO: 24-26, the first terminator preferably comprises the sequence shown in SEQ ID NO: 24 or SEQ ID NO: 26, more preferably the sequence shown in SEQ ID NO: 26; the second terminator preferably comprises the sequence shown in SEQ ID NO: 25.

[0010] In some embodiments, the regulatory system comprises: a first functional gene encoding the first functional protein, the first functional protein comprising any one of the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 6-10, preferably comprising the sequence shown in SEQ ID NO: 2; a second functional gene encoding the second functional protein, the second functional protein comprising the sequence shown in SEQ ID NO: 15; a first promoter comprising the sequence shown in SEQ ID NO: 16; a first terminator comprising the sequence shown in SEQ ID NO: 24 or SEQ ID NO: 26, preferably comprising the sequence shown in SEQ ID NO: 26; a second promoter comprising any one of the sequences shown in SEQ ID NO: 18-23, preferably comprising the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 23; and a second terminator comprising the sequence shown in SEQ ID NO: 25.

[0011] In some embodiments, the first functional protein comprises the sequence shown in SEQ ID NO: 2; the first terminator comprises the sequence shown in SEQ ID NO: 26; and the second promoter comprises the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 23.

[0012] This application also provides an expression vector comprising a regulatory system for degrading uric acid to lower uric acid levels as described in any of the above embodiments of this application. Optionally, the expression vector is a eukaryotic expression vector.

[0013] This application also provides a recombinant microorganism comprising a regulatory system or expression vector for degrading uric acid to lower uric acid levels as described in any of the above embodiments of this application.

[0014] In some embodiments, the microorganism is a yeast species (Saccharomyces sp.), preferably Saccharomyces cerevisiae, and more preferably Saccharomyces cerevisiae var. boulardii.

[0015] This application also provides a composition for degrading uric acid to lower uric acid levels, comprising recombinant microorganisms and / or their metabolites as described in any of the above embodiments of this application and pharmaceutically or food-acceptable excipients. Optionally, the microorganisms in the composition retain activity. Optionally, the composition is administered orally.

[0016] This application also provides the use of the recombinant microorganisms or compositions described in any of the above embodiments of this application in the preparation of foods, health products, food additives or pharmaceuticals for degrading uric acid to lower uric acid levels.

[0017] This application also provides pharmaceuticals or health products for degrading uric acid to lower uric acid levels, which contain recombinant microorganisms and / or their metabolites or compositions as described in any of the above embodiments of this application.

[0018] In some embodiments, the medicine or health product is in the form of tablets, capsules, granules, pills, powders, gels, or solutions.

[0019] This application also provides a medicament for treating hyperuricemia-related diseases, comprising the recombinant microorganisms and / or their metabolites or compositions as described in any of the above embodiments of this application.

[0020] In some embodiments, the hyperuricemia-related diseases include: hyperuricemia, gout, uric acid stones, acute and chronic uric acid nephropathy.

[0021] The embodiments of this application achieve the following beneficial effects:

[0022] The regulatory system proposed in this application for degrading uric acid to lower uric acid levels identifies a set of regulatory elements capable of achieving high-level uric acid transport and degradation by screening and integrating functional protein sequences such as uricase and uric acid transporter sequences from different species, and further screening their corresponding efficient promoters and terminators. These regulatory elements are highly adaptable to yeast strains. After introducing the regulatory system containing these regulatory elements into Saccharomyces cerevisiae, the recombinant yeast expresses exogenous uricase with high-level uric acid-degrading activity and exhibits high-level whole-cell uric acid-lowering activity. The regulatory system and its application proposed in this application provide a more efficient and reliable option for microbial intervention routes for diseases such as hyperuricemia. Furthermore, the implementation of this solution based on recombinant yeast does not require additional inducers, thus avoiding additional medical costs and potential cytotoxicity. It has low usage costs, wide applicability, and higher safety. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1This is a flowchart illustrating the screening of a regulatory system for degrading uric acid to lower uric acid levels according to embodiments of this application;

[0025] Figure 2 This is a schematic diagram of uric acid degradation by recombinant microorganisms according to an embodiment of this application;

[0026] Figure 3 To determine the uric acid degradation level in Saccharomyces cerevisiae using different control systems according to embodiments of this application;

[0027] Figure 4 This study aims to determine the uric acid degradation level of recombinant yeast according to embodiments of this application. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] This application is based on the inventor's following understanding:

[0030] Currently, in addition to traditional treatment regimens using small molecule inhibitors to suppress uric acid synthesis and promote uric acid excretion, or injecting recombinant macromolecular drugs directly into the circulatory system to degrade uric acid, research is gradually proposing to modify gut probiotics to assist in uric acid metabolism and excretion. However, in practical applications, the unit degradation activity of recombinant probiotics is not stable enough and generally depends on the stimulation of specific natural and non-natural molecules. This dependence increases costs in practical applications and, more importantly, brings additional safety and stability concerns. For example, the non-natural strong initiator molecule isopropyl-β-D-1-thiogalactopyranoside (IPTG), which is widely used in research, is not only expensive to produce but also has potential cytotoxicity; another example is the natural inducible molecule galactose, which is absorbed by the human body in the intestine and faces metabolic competition from other gut microbes, making it difficult to guarantee the induction effect. Furthermore, the available substrate bacteria for current recombinant probiotic intervention routes are extremely limited.

[0031] Based on this, this application proposes a regulatory system for degrading uric acid to lower uric acid levels. This system includes regulatory elements capable of achieving high-level uric acid transport and degradation. These regulatory elements are adaptable to yeast strains. Introducing a regulatory system containing these regulatory elements into *Saccharomyces cerevisiae* can achieve the expression of exogenous uricase with high uric acid-degrading activity and exhibit beneficial effects such as high whole-cell uric acid-lowering activity. The regulatory system proposed in this application and its application provide a more efficient and reliable option for microbial intervention in diseases such as hyperuricemia. Furthermore, its implementation requires no additional inducing agents, thus avoiding additional medical costs and potential cytotoxicity. It has low usage costs, wide applicability, and higher safety.

[0032] The first aspect of this application provides a regulatory system for degrading uric acid to lower uric acid levels, comprising: a first functional gene encoding a first functional protein capable of degrading uric acid, the first functional protein comprising one or more sequences as shown in or having at least 85% identity with SEQ ID NO: 1-10; a second functional gene encoding a second functional protein capable of transporting uric acid, the second functional protein comprising one or more sequences as shown in or having at least 85% identity with SEQ ID NO: 11-15; a first promoter for regulating the expression of the first functional gene and / or the second functional gene; and a first terminator for terminating the expression of the first functional gene and / or the second functional gene.

[0033] In the embodiments of this application, "reducing uric acid levels" refers to reducing the uric acid levels in the intestines and / or the blood uric acid levels of an organism.

[0034] In this application embodiment, the term "percentage of identity" for nucleic acid or polypeptide sequences is defined as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical to a known polypeptide after arranging the sequence to obtain the maximum percentage of identity and introducing gaps (if necessary) to achieve the maximum percentage of homology. N-terminal or C-terminal insertions or deletions should not be interpreted as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using algorithms employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic AcidsRes [Nucleic Acids Research]. 25, 3389-3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 87, 2264-2268), programs tailored for sequence similarity searches.

[0035] The "at least 85% identity" proposed in this application embodiment may include amino acid sequences or nucleotide sequences that have at least 85%, 86%, 87%, 88%, 89%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, or any value between these values, but less than 100% identity with the related sequence. These sequences also have the same biological function as the related sequence.

[0036] In this embodiment, the first functional protein capable of degrading uric acid can be uricase (also known as uricase oxidase, UOX), which rapidly oxidizes uric acid into allantoic acid, thus preventing its reabsorption by the renal tubules and its excretion. In this embodiment, the first functional protein may contain one or more sequences selected from the following: UOX Bsub (SEQ ID NO: 1), UOX Vvul (SEQ ID NO: 2), UOX Cneo (SEQ ID NO: 3), UOX Hvol (SEQ ID NO: 4), UOX Mmus (SEQ ID NO: 5), UOX Nver(SEQ ID NO: 6), UOX Hlfs (SEQ ID NO: 7), UOX Rery (SEQ ID NO: 8), UOX Hlbs (SEQ ID NO: 9), UOX Psim (SEQ ID NO: 10). In some embodiments, the first functional protein may comprise SEQ ID NO: 2 (UOX). Vvul ), UOX Nver (SEQ ID NO: 6), UOX Hlfs (SEQ ID NO: 7), UOX Rery (SEQ ID NO: 8), UOX Hlbs (SEQ ID NO: 9) or UOX Psim The sequence shown in SEQ ID NO: 10. In the embodiments of this application, by introducing the selected sequences shown in SEQ ID NO: 1-10 into the control system, the system can be endowed with a highly efficient uric acid degradation function.

[0037] In this embodiment, the second functional protein capable of transporting uric acid can be a uric acid transporter (UAT), which enables the transcellular transport of uric acid, for example, transporting uric acid from the gut of an organism to microorganisms expressing this protein for breakdown and metabolism, thereby reducing the uric acid level in the gut and / or the blood uric acid level. In this embodiment, the second functional protein may contain one or more sequences selected from the following: UAT YgfU (SEQ ID NO: 11), UAT PucJ (SEQ ID NO: 12), UAT PucK (SEQ ID NO: 13), UAT Xut1 (SEQ ID NO: 14), UAT UapA (SEQ ID NO: 15). In some embodiments, the second functional protein may comprise SEQ ID NO: 15 (UAT) UapA The sequences shown in SEQ ID NO: 11-15 are used in this application embodiment. By introducing the selected sequences shown in SEQ ID NO: 11-15 into the regulatory system, the system can be endowed with efficient and stable uric acid-lowering activity.

[0038] In this embodiment, the regulatory system further includes a first and / or a second promoter and a first and / or a second terminator to regulate the expression of the first functional gene and the second functional gene holistically or separately. The first promoter and / or the second promoter may be a constitutive promoter. In some embodiments, the system may only include a first promoter / second promoter and a first terminator / second terminator to holistically regulate the expression of the first functional gene and the second functional gene. In other embodiments, the system includes first and second promoters and first and second terminators, which are used to regulate the expression of the first functional gene and the second functional gene, respectively.

[0039] In some embodiments, the first promoter and the second promoter may respectively comprise any sequence of pTDH3 (SEQ ID NO: 16), pEFB1 (SEQ ID NO: 17), pPDC1 (SEQ ID NO: 18), pCCW12 (SEQ ID NO: 19), pENO2 (SEQ ID NO: 20), pFBA1 (SEQ ID NO: 21), pTEF2 (SEQ ID NO: 22), pPGK1 (SEQ ID NO: 23) or any sequence having at least 85% identity with it, and the first terminator and the second terminator may respectively comprise any sequence of tPGK1 (SEQ ID NO: 24), tADH1 (SEQ ID NO: 25), tSCW4 (SEQ ID NO: 26) or any sequence having at least 85% identity with it.

[0040] In other embodiments, the first promoter may comprise the sequence shown in SEQ ID NO: 16 (pTDH3), and the second promoter may comprise the sequences shown in SEQ ID NO: 17 (pEFB1), SEQ ID NO: 18 (pPDC1), SEQ ID NO: 19 (pCCW12), SEQ ID NO: 20 (pENO2), SEQ ID NO: 21 (pFBA1), SEQ ID NO: 22 (pTEF2), or SEQ ID NO: 23 (pPGK1), preferably the sequences shown in SEQ ID NO: 18 (pPDC1), SEQ ID NO: 19 (pCCW12), SEQ ID NO: 20 (pENO2), SEQ ID NO: 21 (pFBA1), SEQ ID NO: 22 (pTEF2), or SEQ ID NO: 23 (pPGK1), more preferably the sequences shown in SEQ ID NO: 18 (pPDC1) or SEQ ID NO: 23 (pPGK1); and the first terminator may comprise SEQ ID NO: 16 (pTDH3). The sequence shown in SEQ ID NO: 24 (tPGK1) or SEQ ID NO: 26 (tSCW4), preferably including the sequence shown in SEQ ID NO: 26 (tSCW4), and the second terminator includes the sequence shown in SEQ ID NO: 25 (tADH1). In the embodiments of this application, by introducing a screened specific promoter and terminator into the regulatory system, a strongly expressed system can be constructed, thereby further enhancing the expression level of uricase and / or uric acid transporter, thus endowing the system with efficient and stable uric acid-lowering activity.

[0041] In some specific embodiments of this application, the regulatory system may include the following elements: a. a first functional gene, which encodes a first functional protein, UOX. Vvul (SEQ ID NO: 2), the first promoter is pTDH3 (SEQ ID NO: 16), the first terminator is tPGK1 (SEQ ID NO: 24) or tSCW4 (SEQ ID NO: 26); and b. a second functional gene, which encodes a second functional protein UAT. UapA If the second promoter is pEFB1 (SEQ ID NO: 15), pPGK1 (SEQ ID NO: 17), pPDC1 (SEQ ID NO: 23), or pPDC1 (SEQ ID NO: 18), and the second terminator is tADH1 (SEQ ID NO: 25), then the control system can be represented as pEFB1 / pPGK1 / pPDC1_UAT. UapA _tADH1 / tSCW4-pTDH3_UOX Vvul_tPGK1 / tSCW4. In some embodiments, the control system may include the following element: pEFB1_UAT UapA _tADH1 / tSCW4-pTDH3_UOX Vvul _tPGK1. In other embodiments, the control system may include the following element: pTDH3_UOX Vvul _tPGK1-pENO2_UAT UapA _tADH1、pTDH3_UOX Vvul _tPGK1-pFBA1_UAT UapA _tADH1、pTDH3_UOX Vvul _tPGK1-pCCW12_UAT UapA _tADH1、pTDH3_UOX Vvul _tPGK1-pTEF2_UAT UapA _tADH1、pTDH3_UOX Vvul _tPGK1-pPDC1_UAT UapA _tADH1、pTDH3_UOX Vvul _tPGK1-pPGK1_UAT UapA _tADH1 or pTDH3_UOX Vvul _tSCW4-pPDC1_UAT UapA _tADH1, preferably pTDH3_UOX Vvul _tPGK1-pPDC1_UAT UapA _tADH1、pTDH3_UOX Vvul _tPGK1-pPGK1_UAT UapA _tADH1 or pTDH3_UOX Vvul _tSCW4-pPDC1_UAT UapA _tADH1. In the embodiments of this application, the screened specific elements included in the regulatory system can form a strongly expressive system, thereby endowing the system with efficient and stable uric acid-lowering activity.

[0042] In this embodiment, the first functional gene may contain any sequence as shown in SEQ ID NO: 27-36; the second functional gene may contain any sequence as shown in SEQ ID NO: 37-41 or any sequence having at least 85% identity with it. In this embodiment, by introducing optimized first and second functional genes that are more suitable for microbial expression, it is beneficial to regulate the efficient expression of the system in transformed microorganisms, thereby achieving efficient and stable uric acid-lowering activity.

[0043] It is understood that the specific sequences of the first and second functional genes listed in the embodiments of this application are merely exemplary. Based on the sequences of the first and second functional proteins, any modifications and alterations made to the first and second functional proteins of this application, and the resulting changes in the sequences of the first and second functional genes, based on principles such as codon degeneracy and amino acid synonymous substitution, fall within the protection scope of this application.

[0044] A second aspect of this application provides an expression vector comprising a regulatory system for degrading uric acid to lower uric acid levels, as described in any of the embodiments of the first aspect above. In some embodiments, the expression vector is a eukaryotic expression vector, such as HcKan_O, HcKan_P, HcKan_T, POT2, POT3, m416B, pCAS, etc. It is understood that the elements in the regulatory system proposed in this application can be constructed on the same expression vector or separately in different vectors. For example, the first promoter, the first functional gene, and the first terminator can be constructed on the same expression vector, while the second promoter, the second functional gene, and the second terminator can be constructed simultaneously on another expression vector. This application does not limit the construction location of each element.

[0045] A third aspect of this application provides a recombinant microorganism comprising a regulatory system for degrading uric acid to lower uric acid levels as described in any embodiment of the first aspect, or an expression vector as described in any embodiment of the second aspect. In some embodiments, the microorganism is an intestinal microorganism, such as a yeast (Saccharomyces sp.), preferably Saccharomyces cerevisiae, more preferably Saccharomyces boulardii or Saccharomyces cerevisiae var. boulardii. This application embodiment, by introducing a regulatory system into yeast, which does not possess a uric acid metabolism pathway, enables the efficient expression of exogenous uricase and uric acid transport proteins with uric acid-degrading activity in yeast cells, achieving a high level of whole-cell uric acid-lowering activity.

[0046] The fourth aspect of this application provides a composition for degrading uric acid to lower uric acid levels, comprising recombinant microorganisms and / or their metabolites as described in any embodiment of the third aspect of this application, and pharmaceutically or food-acceptable excipients.

[0047] In the embodiments of this application, pharmaceutically or food-grade acceptable carriers or excipients may be selected from at least one of glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and physiological saline.

[0048] In some embodiments, the composition may also contain other probiotics and / or prebiotics. It is understood that other probiotics or prebiotics may be added to the composition without affecting the efficacy of the recombinant microorganisms in the compositions presented in this application, thereby enabling the compositions to possess more functions or enhancing their original efficacy. These probiotics or prebiotics may be those reported in existing research and are not specifically limited herein. In some embodiments, the prebiotics in the compositions of this application are selected from at least one of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, and oligosaccharides.

[0049] In some embodiments, the recombinant microorganisms in the composition retain their activity, for example, by maintaining their biological activity in lyophilized or suspension form. The composition may contain substances that help maintain the activity of at least one of the recombinant microorganisms in the composition; these active substances may be those reported in existing studies and are not specifically limited herein. In some embodiments, the substances that help maintain the activity of the strain are selected from at least one of cysteine, glutathione, butylated hydroxyanisole, butylated methyltoluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid or its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C, and vitamin E.

[0050] In some embodiments, the composition is administered orally.

[0051] The fifth aspect of this application provides a pharmaceutical or health product for degrading uric acid to lower uric acid levels, comprising recombinant microorganisms and / or their metabolites as described in any embodiment of the third aspect of this application, or a composition as described in any embodiment of the fourth aspect of this application.

[0052] In this embodiment of the application, the health product may also include excipients acceptable to the health product, wherein the excipients are selected from one or more combinations of fillers, capsule shell materials, solvents, stabilizers, flavoring agents, sweeteners, and pigments.

[0053] In the embodiments of this application, the drug can be in the form of tablets, capsules, granules, pills, powders, gels, or solutions, and can also be used in combination with other uric acid-lowering drugs, as long as they do not affect each other's activity. It is understood that the drug of this application can adopt various existing dosage forms, as long as it does not affect the activity of the recombinant microorganisms in the composition. Furthermore, the drug of this application may further include excipients commonly used in pharmaceuticals or dosage forms, such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, etc.

[0054] The pharmaceutical product of this application can be administered in any of the following forms: oral liquid, tablets, injections, orally disintegrating tablets, lyophilized powder preparations, or enteric-coated formulations. Enteric-coated formulations, such as capsules or enteric-coated tablets, are preferred to facilitate the passage of the active ingredient, i.e., the microorganisms, through the stomach without being destroyed by gastric acid. More preferably, the pharmaceutical product of this application can be formulated into enteric-coated tablets for oral administration.

[0055] The sixth aspect of this application provides a food or food additive comprising recombinant microorganisms and / or their metabolites as described in any embodiment of the third aspect of this application, or a composition as described in any embodiment of the fourth aspect of this application.

[0056] In this application embodiment, food can be any edible item in a broad sense, such as beverages or solid food, and can also be fermented food or animal feed. It should be noted that the key to the food in this application is that it contains the composition of this application. As for the specific form of the food, such as solid or liquid, it can be determined according to different food products or usage needs, and is not specifically limited here.

[0057] The seventh aspect of this application also proposes the use of recombinant microorganisms and / or their metabolites as described in any embodiment of the third aspect of this application, or compositions as described in any embodiment of the fourth aspect of this application, in the preparation of health products or pharmaceuticals for degrading uric acid to lower uric acid levels.

[0058] The eighth aspect of this application also proposes the use of recombinant microorganisms and / or their metabolites as described in any embodiment of the third aspect of this application, or compositions as described in any embodiment of the fourth aspect of this application, in the preparation of food or food additives.

[0059] The ninth aspect of this application also proposes the use of a recombinant microorganism and / or its metabolites as described in any embodiment of the third aspect of this application, or a composition as described in any embodiment of the fourth aspect of this application, in the preparation of a medicament for treating hyperuricemia.

[0060] The tenth aspect of this application also proposes a medicament for treating hyperuricemia, comprising recombinant microorganisms and / or their metabolites as described in any embodiment of the third aspect of this application, or a composition as described in any embodiment of the fourth aspect of this application. It is understood that the embodiments of this application, based on the proposed regulatory system and the uric acid-lowering function of the recombinant microorganisms comprising it, can be effectively used to treat hyperuricemia and related diseases, such as cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease, chronic kidney disease, gout, insulin resistance, hypertension, dyslipidemia, renal insufficiency, obesity, prediabetes, and diabetes. In some embodiments, hyperuricemia-related diseases include hyperuricemia, gout, uric acid stones, and acute and chronic uric acid nephropathy.

[0061] It should be noted that the explanation and description of the embodiment of the regulatory system for degrading uric acid to reduce uric acid levels in the first aspect of this application also applies to the expression vector, recombinant microorganism, composition, health product, drug and its application in the preparation of health product or drug for degrading uric acid to reduce uric acid levels proposed in the embodiments of this application, and they have the same beneficial effects as the regulatory system, which will not be elaborated here.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0063] It should be noted that the plasmids and all engineered microorganisms and their chassis starting bacteria involved in the embodiments of this application are publicly available from the inventors and can only be used to replicate the invention in the laboratory.

[0064] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0065] Example 1 - Screening for exogenous uricase expressing high levels of uric acid-degrading activity in yeast cells

[0066] Based on the National Center for Biotechnology Information (NCBI) database, using the uricase / uricase oxidase orthologous protein cluster 3648 (COG3648) contained in the bifunctional uricase pucL of Bacillus subtilis as the query domain, the conserved functional domain search function was used to search the NCBI protein database to obtain coding sequences with potential UOX functions. Further manual searches were conducted to find genomes containing these potential UOX coding sequences. The selection was limited to genomes containing coding sequences for all three enzymes: UOX (Enzyme Commison Number EC 1.7.3.3), 5-HIUH (5-hydroxyisourate hydrolase, EC 3.5.2.17), and OHCUDC (2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, EC 4.1.1.97), and originating from environments with high-purine nutrition. Representative sequences from mammals, archaea, fungi, Gram-positive bacteria, and Gram-negative bacteria were further selected from the obtained genomes. UOX sequences from Bacillus subtilis and Mus musculus were used as references (UOX sequences from each genome were not specified in the provided text). Bsub UOX Mmus ), and added to the sequence set (Table 1).

[0067] After optimizing candidate sequences for yeast expression preference using bioinformatics analysis, optimized sequences were synthesized. The specific workflow can be found in Patel, JR, Oh, J., Wang, S., Crawford, JM & Isaacs, FJ Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome. Cell 185, 1487-1505.e14 (2022) and Koblan, LW, Doman, JL, Wilson, C., Levy, JM, Tay, T., Newby, GA, Maianti, JP, Raguram, A., Liu, DRI improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol. 36(9), 843–846 (2018). The optimized nucleic acid sequences are shown in Table 1.

[0068] Table 1

[0069] serial number protein Source genome Classification protein sequence Optimized nucleic acid sequence <![CDATA[UOX Bsub ]]> PucL Bacillus subtilis Gram-positive SEQ ID NO: 1 SEQ ID NO: 27 <![CDATA[UOX Vvul ]]> PucL Vibrio vulnificus Gram-negative SEQ ID NO: 2 SEQ ID NO: 28 <![CDATA[UOX Cneo ]]> Uox Cryptococcus neoformans fungi SEQ ID NO: 3 SEQ ID NO: 29 <![CDATA[UOX Hvol ]]> PucL Haloferax volcanii Archaea SEQ ID NO: 4 SEQ ID NO: 30 <![CDATA[UOX Mmus ]]> Uox Mus musculus mammal SEQ ID NO: 5 SEQ ID NO: 31 <![CDATA[UOX Nver ]]> UriC Natrinema versiforme Archaea SEQ ID NO: 6 SEQ ID NO: 32 <![CDATA[UOX Hlfs ]]> UriC Haloferax sp. Archaea SEQ ID NO: 7 SEQ ID NO: 33 <![CDATA[UOX Rery ]]> UriC Rhodococcus erythropolis Gram-positive SEQ ID NO: 8 SEQ ID NO: 34 <![CDATA[UOX Hlbs ]]> Uox Halobacillus sp. Gram-positive SEQ ID NO: 9 SEQ ID NO: 35 <![CDATA[UOX Psim ]]> Uox Peribacillus simplex Gram-positive SEQ ID NO: 10 SEQ ID NO: 36

[0070] UOX Bsub Protein sequences and optimized nucleic acid sequences:

[0071] MFTMDDLNQMDAQTLTDTLGSIFEHSSWIAEEAAALRPFSSLSDLHHKMAGIVKAADRQTQLDLINKHP

[0072] RLGTKKTMSASSVREQQNAGLSTLEQQEYEEFLKLNEHYYKRFGFPFILAVKGKTKQDIHQALLARLKN

[0073] EREAEFQQALEEIDRIARFRLADIITKEGETQMKRTMSYGKGNVFAYRTFLKPLTGVKPIPESSFTGRANT

[0074] VVGVDVTCEIGGDAFLPSFTDGDNTLVVATDSMKNFIQRHLASYEGTTAEGFLHYVAHRFLDTYSHMD

[0075] TITLTGEDIPFEAMPAYEEQELGTSQVVFRRSRNERARSVLKAKRTGDTITIKEQYSEIIDLQLVKVSGNSF

[0076] VGFIRDEYTTLPEDGNRPLFVHLNIGWHYENTNDAYASDPARYVAAEQVRDLASTVFHELETPSIQNLIY

[0077] HIGCRILTRFPQLTDVSFQSQNHTWDTVVEEIPGSKGKVYTEPRPPFGFQRFTVTREDAEKEKQKAAEALGSLKA(SEQ ID NO:1)

[0078] ATGTTCACCATGGATGACTTGAACCAAATGGACGCCCAAACTTTAACCGATACTTTGGGTTCCATCT

[0079] TCGAACACTCCTCTTGGATTGCCGAAGAAGCTGCTGCCTTAAGACCATTCTCTTCTTTGTCTGATTTA

[0080] CACCACAAGATGGCTGGTATTGTTAAAGCTGCTGACAGACAAACCCAATTGGATTTGATCAACAAG

[0081] CACCCACGTTTAGGTACTAAGAAGACTATGTCTGCCAGCTCAGTTAGAGAACAACAAAACGCTGGT

[0082] TTGTCCACTTTGGAACAACAAGAATATGAAGAATTTCTTAAGTTGAACGAACATTACTACAAGAGA

[0083] TTCGGTTTCCCTTTCATTTTAGCTGTCAAGGGTAAGACCAAGCAAGACATCCACCAAGCTTTGTTGG

[0084] CGAGATTGAAAAATGAACGTGAAGCTGAATTTCAACAAGCTTTGGAAGAAATTGACCGTATTGCCC

[0085] GTTTCAGATTGGCTGATATTATTACCAAGGAAGGTGAAACCCAAATGAAACGTACCATGTCTTACG

[0086] GTAAGGGAAATGTTTTCGCCTACAGAACTTTCTTGAAACCTCTCACCGGTGTCAAGCCAATCCCAG

[0087] AATCCTCTTTTACTGGTAGAGCTAACACTGTTGTCGGTGTTGACGTTACCTGTGAAATTGGTGGTGA

[0088] TGCTTTCTTGCCATCTTTCACTGACGGTGACAACACTCTCGTTGTTGCTACCGACTCCATGAAGAAC

[0089] TTCATACAAAGACATTTGGCTTCCTACGAAGGTACTACTGCTGAAGGTTTCTTACATTACGTCGCTC

[0090] ACAGATTTTTGGACACCTACTCACACATGGACACTATTACATTGACAGGTGAAGACATTCCATTCG

[0091] AAGCTATGCCAGCTTACGAAGAGCAAGAATTGGGTACCTCTCAAGTTGTTTTCAGAAGATCCAGAA

[0092] ACGAAAGAGCTAGATCTGTCTTGAAGGCTAAGAGAACCGGTGACACCATCACTATCAAGGAACAA

[0093] TACTCTGAAATCATCGATTTGCAATTGGTTAAGGTTTCTGGTAACTCCTTCGTAGGTTTCATCAGAG

[0094] ATGAATACACCACTTTGCCAGAAGATGGTAACAGACCATTGTTCGTCCACTTGAACATCGGTTGGC

[0095] ATTACGAAAACACCAACGATGCCTATGCTTCTGACCCAGCTAGATACGTCGCTGCCGAACAAGTCA

[0096] GAGATTTGGCCAGTACCGTCTTCCACGAATTGGAAACTCCATCTATCCAAAACTTGATTTACCACAT

[0097] TGGTTGTAGAATCTTGACCCGTTTCCCACAACTAACTGATGTCTCTTTCCAATCCCAGAACCACACT

[0098] TGGGACACTGTGGTTGAAGAAATCCCAGGCTCTAAGGGTAAGGTTTACACTGAACCAAGACCACCA

[0099] TTTGGTTTCCAAAGATTTACCGTTACTAGAGAGGATGCCGAAAAGGAAAAGCAAAAGGCCGCTGAAGCTTTGGGTTCCCTAAAGGCT(SEQ ID NO:27)

[0100] UOX Vvul Protein sequence and optimized nucleic acid sequence:

[0101] MTIQNENRTMFYGKGDVFVYRTFVQPLTGLRKIPESSFTERDNTIFGFNCQISLKGEAFFSSFTEGDNSLV

[0102] VATDSMKNIIHRQAANYQGNTAEGFLKFICEVFLDKYSHIDAVELTATEVPFDHVLVPKGEGHENSDVV

[0103] YRCSRNERATTTIEVKRTPTGSKIVKHSSGIVDVHLIKVKGSSFYGFIQDEYTTLPEAQDRPLFIYLDFDW

[0104] EYSRWEDGTGANPEKYVAAEQICDIANTVFHELNNRSIQQLIYHIGIRILERFPQLANVQFKTNNRTWETVVETIPNSEGSVYQEPRPPFGFQGFVVTQEDVKQKKAADSTVGASR(SEQ ID NO:2)

[0105] ATGACAATCCAAAACGAAAACAGAACCATGTTCTATGGTAAGGGTGATGTCTTTGTGTACAGAACT

[0106] TTCGTCCAACCATTGACCGGTTTTGAGAAAGATCCCAGAATCTTCTTTTTACCGAAAGAGACAACACT

[0107] ATTTTCGGTTTCAACTGCCAAATCTCTTTGAAAGGTGAAGCTTTCTTCTCCTCTTTCACCGAAGGTGA

[0108] CAACAGTTTGGTTGTTGCTACCGACTCCATGAAGAACATCATTCACAGACAAGCTGCCAACTACCA

[0109] AGGTAACACCGCTGAAGGTTTCTTAAAATTCATTTGTGAAGTCTTCTTAGACAAGTATTCTCATATC

[0110] GATCCGTCGAACTAACTGCAACTGAAGTACCTTTCGATCACGTTTTGGTTCCAAAGGGTGAAGGT

[0111] CACGAAAATTCCGATGTCGTTTACAGATGTAGCAGAAACGAAAGAGCTACTACTACCATTGAAGTT

[0112] AAGCGTACCCCCAACTGGTTCCAAGATTGTCAAGCACTCTTCTGGCATCGTTGACGTCCATTTGATCA

[0113] AGGTTAAAGGTTCCTCTTTCTACGGTTTCATCCAAGATGAATACACCACTTTGCCAGAAGCTCAAGA

[0114] CAGACCATTGTTCATCTACTTGGACTTTGACTGGGAATACTCTAGATGGGAAGATGGTACTGGTGCC

[0115] AACCCAGAAAAGTACGTTGCTGCTGAACAAATCTGTGATATTGCTAATACTGTTTTCCACGAATTGA

[0116] ACAACCGTTCTATTCAACAATTGATTTACCACATTGGTATCAGAATCTTGGAAAGATTCCCACAATT

[0117] AGCTAACGTTCAATTCAAGACTAACAACAGAACCTGGGAAACTGTTGTTGAAACTATTCCAAATTC

[0118] AGAAGGTTCTGTCTACCAAGAACCAAGACCACCATTCGGTTTCCAAGGTTTTGTTGTCACTCAAGAAGACGTCAAGCAAAAGAAGGCTGCCGACTCCACTGTTGGTGCTTCCCGTTGA(SEQ ID NO:28)UOX Cneo Protein sequence and optimized nucleic acid sequence:

[0119] MSQPGYLSAACYGKDLVKVARVVRDGEQHHIVEYTLRVLLEGEIEASYTKADNSCVVATDTVKNTCNI

[0120] FAKTSPYVLDAPVFALHLGLHFVTKYKHIHKCFVDIIGLKWSRISVDGKPHKWSFVRDGDEKAIVECAV

[0121] DGTAGPDAATADLKIGIKDLLVLKTSGSAFENFHRDENTTLADVADRIFSTSVSVQCSISLPPNTPLTIGN

[0122] LASIAKDLDFPKMKELILKDVLDTFATDESASVQATLYLTLQKILTSCPAVKDASMQLPNKHYIPVNLSAFDLDNNLGYEGGAEVFYPAADPSGYITATVTRK(SEQ ID NO:3)

[0123] ATGTCTCAACCAGGTTACTTATCCGCTGCTTGTTACGGTAAGGACTTGGTCAAGGTTGCCAGAGTTG

[0124] TCAGAGATGGTGAACAACACCACATTGTCGAATACACTTTGAGAGTGTTGTTGGAAGGTGAAATTG

[0125] AAGCTTCTTACACCAAAGCAGATAACTCTTGCGTCGTTGCTACTGACACCGTCAAGAACACTTGTA

[0126] ACATCTTTGCTAAGACTTCTCCATACGTTTTGGATGCTCCAGTTTTCGCTTTGCACCTAGGTTTGCAT

[0127] TTCGTCACCAAGTACAAGCACATCCACAAGTGTTTCGTCGACATTATTGGTTTGAAGTGGTCTAGAA

[0128] TCTCTGTTGATGGTAAGCCACACAAATGGTCATTCGTTAGAGATGGTGACGAAAAGGCTATCGTTG

[0129] AATGTGCTGTCGACGGTACTGCTGGTCCAGATGCTGCTACCGCCGATTTGAAGATTGGTATCAAGG

[0130] ATTTATTGGTTTTAAAGACCAGTGGTTCCGCTTTCGAAAACTTCCACAGAGATGAAAACACCACCCT

[0131] GGCCGATGTTGCTGACAGAATCTTCTCCACTTCTGTTTCTGTCCAATGTTCTATTAGCTTGCCACCAA

[0132] ACACTCCATTGACTATCGGTAACTTGGCTTCCATCGCCAAGGATCTTGACTTCCCAAAGATGAAGG

[0133] AATTGATCTTGAAAGACGTTTTGGACACTTTTGCCACAGATGAATCTGCTTCTGTTCAAGCTACTTT

[0134] ATACTTAACTTTGCAAAAGATCTTGACCTCTTGTCCTGCTGTTAAAGACGCCTCCATGCAATTGCCT

[0135] AACAAGCATTACATTCCAGTCAATTTGTCTGCTTTCGACTTGGACAACAACTTGGGTTACGAAGGTG

[0136] GTGCTGAAGTTTTCTACCCAGCTGCTGACCCATCCGGTTATATTACCGCCACTGTTACCAGAAAGTGA(SEQ ID NO:29)

[0137] UOX Hvol protein sequence and optimization nucleic acid sequence:

[0138] MTAQQSPVDGDAPDSGEQRTMNYGKENVAVYRTYATPLEGVRTIPESSFDGRDNVLFGLDVRVQVEG

[0139] EEFLPSFSEGDNTKVATDSMKNFILHHAGEYDGATLEGFLEFVGSGFLDTYSQMSAVEVSADEIRFDEL

[0140] PVPEDDGDGGYEASDLVFRVSDNESGYGSISLTRDDGTPVITDQTSGVTGLELVKVEGSSFTGYVQDEYT

[0141] TLPEREDRTLYISLDIFWSYDDPEDALGEDPERYVPSEQVRDIAHVVFDEVDSNSIQDLIYQIGLRVLERYPQLASVRFEANNRTWLSVRDDLDGDASVLREPPAPTGFQQFSMDRGDLDEQ(SEQ IDNO:4)ATGACTGCTCAACAATCTCCAGTTGACGGTGACGCTCCAGACAGTGGTGAACAAAGAACCATGAACTACGGTAAGGAAAACGTTGCCGTCTACAGAACCTACGCTACCCCATTGGAAGGTGTTAGAACTATTCCAGAATCCTCTTTTGATGGTAGAGACAACGTCTTGTTCGGTTTGGATGTTAGAGTCCAAGTTGAAGGTGAAGAATTTTTGCCATCCTTCTCCGAAGGTGACAATACCAAAGTTGTTGCCACTGATTCCATGAAGAACTTCATCTTGCATCACGCTGGTGAGTATGACGGTGCTACTTTGGAAGGTTTCTTGGAATTCGTTGGTTCTGGTTTCCTTGACACTTACTCTCAAATGTCTGCTGTCGAAGTTTCCGCTGACGAAATCAGATTCGACGAATTACCTGTTCCAGAAGACGACGGTGACGGCTACGAAGCCTCAGATTTGGTTTTCAGAGTTTCTGACAACGAATCTGGTTATGGATCCATTTCCTTGACCAGAGATGATGGTACTCCAGTCATTACTGACCAAACATCCGGTGTCACTGGTTTGGAATTGGTCAAGGTTGAAGGTTCTTCTTTCACCGGTTACGTTCAAGATGAATACACCACCTTGCCTGAAAGAGAAGACAGAACTTTATACATCTCTTTGGATATCTTCTGGTCTTACGATGACCCAGAAGATGCTTTGGGTGAAGATCCAGAAAGATACGTCCCATCAGAACAAGTCCGTGACATTGCTCACGTCGTCTTCGATGAAGTCGATTCCAACTCTATCCAAGACTTGATTTACCAAATCGGTCTACGTGTATTAGAACGTTACCCACAATTGGCTTCTGTCAGATTTGAAGCTAACAACAGAACCTGGTTGTCTGTTAGAGATGACTTAGATGGTGATGCCTCCGTTTTGAGAGAACCACCAGCTCCAACTGGTTTCCAACAATTCTCTATGGACAGAGGTGATTTGGACGAACAATGA(SEQ IDNO: 30) UOX Mmus Protein sequence and optimized nucleic acid sequence:

[0142] MAHYHDNYGKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLRSKKDYLHGDNSDIIP

[0143] TDTIKNTVHVLAKLRGIRNIETFAMNICEHFLSSFNHVTRAHVYVEEVPWKRFEKNGIKHVHAFIHTPTG

[0144] THFCEVEQMRNGPPVIHSGIKDLKVLKTTQSGFEGFLKDQFTTLPEVKDRCFATQVYCKWRYQRRDVD

[0145] FEAIWGAVRDIVLQKFAGPYDKGEYSPSVQKTLYDIQVLSLSQLPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLPSRL(SEQ ID NO: 5)

[0146] ATGGCTCACTACCACGATAACTATGGTAAGAACGATGAAGTCGAATTCGTCAGAACTGGTTACGGT

[0147] AAAGATATGGTTAAGGTTTTACACATTCAAAGAGATGGTAAGTACCATAGTATAAAGGAAGTTGCC

[0148] ACCTCCGTTCAATTAACTTTACGTTCTAAGAAGGACTACTTGCACGGTGACAATTCTGACATTATTC

[0149] CAACTGACACCATCAAGAACACTGTTCACGTTTTGGCTAAGTTGAGAGGTATCAGAAATATTGAAA

[0150] CCTTTGCTATGAACATCTGCGAACACTTTTTGTCTTCTTTCAACCATGTTACCAGAGCCCATGTCTAC

[0151] GTTGAAGAAGTTCCATGGAAGAGATTCGAAAAGAACGGTATTAAGCACGTCCACGCTTTCATTCAC

[0152] ACTCCAACCGGTACTCACTTCTGTGAAGTTGAACAAATGAGAAACGGCCCTCCAGTTATCCACTCT

[0153] GGTATCAAAGATTTGAAAGTGTTGAAGACCACTCAATCCGGTTTCGAGGGTTTCTTGAAGGACCAA

[0154] TTCACCACATTACCTGAAGTCAAAGACAGATGTTTCGCTACCCAAGTTTACTGTAAGTGGAGATAC [[ID=1十一]]

[0155] CAAAGACGTGACGTTGATTTTGAAGCTATCTGGGGTGCTGTCCGTGATATTGTTTTGCAAAAGTTCG

[0156] CCGGTCCATACGACAAGGGTGAATACTCTCCATCCGTCCAAAAGACTTTGTACGATATCCAAGTCTT

[0157] GTCCCTATCACAACTGCCAGAAATTGAAGACATGGAAATCTCTTTGCCAAACATCCATTACTTCAAC

[0158] ATTGACATGTCCAAGATGGGTTTGATCAACAAGGAAGAAGTCTTGTTGCCATTGGACAACCCATATGGTAAGATCACTGGTACCGTCAAGAGAAAGTTGCCATCTAGATTGTGA(SEQ ID NO:31)UOX Nver Protein sequence and optimized nucleic acid sequence:

[0159] MNYGKEKIAVYRTYASSLEGVRTIPESSFDGRDNTLFGLDVRVQVDGEAFLPSFSEGDNSMVVATDSM

[0160] ​

[0161] SAFGEIYLDRSEDGPVIADHISGVTDIELVKVKGSSFTDYVQDEYTTLPEREDRALHITLDIFWTYAESAD

[0162] ALGTDPERYVPAEQVRDIAQVVFHEVDSNSIQDLIYQIGLRVLERFPQLESVSFEANNRTWIQERDLDGDAKVLKEPPRPTGFQQFSMDHSDLEEMSE(SEQ ID NO:6)

[0163] ATGAACTACGGTAAGGAAAAGATCGCTGTCTACAGAACATACGCTTCTTCTTTGGAAGGTGTTAGA

[0164] ACCATCCCAGAATCCTCTTTCGATGGTAGAGATAACACTTTGTTTGGTTTGGATGTCAGAGTCCAAG

[0165] TTGACGGTGAAGCTTTCTTGCCATCTTTTTCCGAAGGTGACAACTCTATGGTCGTTGCTACTGATTCC

[0166] ATGAAGAACTTCGTCTTGCACCAAGCTGGTGAATACGATGGTGCTACCATTGAAGGTTTCTTGGAA

[0167] TTCGTTGGTTCTCAATTTTTGGACACCTACTCCCAAATGGAATCAATCCAAATGTCCGCAGACGAAA

[0168] TTCCATTCGAAGAACGTGATGTTCCAGGTGAAAACGACTTCGAAGACTCCGGTTTAGTTTTCAGAC

[0169] AATCTCACAACGAAAGTGCCTTCGGCGAGATCTACTTGGACCGTTCTGAAGATGGTCCAGTTATTG

[0170] CCGACCATATTTCTGGTGTCACTGACATCGAATTGGTCAAAGTCAAGGGTAGCTCCTTCACCGACTA

[0171] CGTTCAAGATGAATACACCACTTTACCTGAAAGAGAAGACAGAGCTTTGCACATCACTTTGGATAT

[0172] CTTCTGGACTTATGCTGAATCTGCCGATGCTTTGGGTACTGACCCAGAAAGATACGTCCCAGCTGAA

[0173] CAAGTTAGAGACATTGCTCAAGTTGTGTTCCACGAAGTTGATTCTAATTCCATCCAAGACTTAATTT

[0174] ACCAAATCGGTTTGAGAGTTTTGGAAAGATTCCCACAATTGGAATCTGTTTCTTTTGAAGCCAACAA

[0175] CAGAACCTGGATTCAAGAACGTGATCTAGATGGTGACGCTAAGGTTTTGAAGGAACCACCAAGACCAACCGGTTTCCAACAATTCTCCATGGACCACTCTGACCTGGAAGAAATGTCTGAA(SEQ ID NO:32)UOX Hlfs Protein sequence and optimized nucleic acid sequence:

[0176] MDGDAPDSGEQRTMNYGKENVAVYRTYATPLEGVRTIPESSFDGRDNVLFGLDVRVQVEGEEFLPSFS

[0177] EGDNAKVVATDSMKNFILHHAGEYDGATLEGFLEFVGSEFLDTYSQMNAVEVSADEIRFDELPVPDDD

[0178] GDGFEASDLVFRVSDNESGFGSVSLSREDDEPVITDQTSGVTGLELVKVKGSSFTGYVQDEYTTLPERED

[0179] RTLYISLDIFWTYDGPEDALGEDPEQYVPSEQVRDIAHVVFDEVDSNSIQDLIYQLGLRVLERYPQLASVQFEANNRTWLSVRDDLDGDASVLREPPAPTGFQQFSMDRGDLDEQ(SEQ ID NO:7)

[0180] ATGGATGGTGACGCCCCAGACTCTGGTGAACAAAGAACCATGAACTACGGTAAGGAAAACGTTGC

[0181] TGTCTACAGAACCTACGCTACTCCATTGGAAGGTGTCAGAACAATTCCAGAATCTTCCTTCGATGGT

[0182] AGAGACAACGTCTTGTTCGGTTTAGACGTTCGTGTCCAAGTCGAAGGCGAAGAATTCTTGCCATCTT

[0183] TTAGTGAAGGTGACAATGCTAAGGTTGTCGCCACTGATTCCATGAAGAACTTCATTTTGCATCACGC

[0184] TGGTGAATACGATGGTGCTACCTTAGAAGGTTTCTTGGAATTCGTGGGTTCTGAATTTTTAGACACT

[0185] TACTCTCAAATGAACGCTGTTGAAGTCTCTGCTGATGAAATCAGATTCGACGAATTGCCAGTCCCA

[0186] GATGACGACGGTGACGGTTTCGAAGCCTCCGATTTAGTCTTCAGAGTTTCTGACAACGAATCCGGTT

[0187] TCGGTTCTGTTTCATTGTCCCGTGAAGATGATGAACCAGTTATCACTGACCAAACTTCTGGTGTTAC

[0188] TGGTTTGGAGTTGGTTAAAGTCAAGGGTTCCTCTTTCACCGGATACGTCCAAGATGAATATACCACC

[0189] CTACCTGAAAGAGAAGACAGAACTTTGTACATTTCTTTGGATATCTTCTGGACTTACGATGGTCCAG

[0190] AAGATGCTTTGGGTGAAGACCCAGAACAATACGTTCCATCCGAACAAGTTAGAGACATTGCTCACG

[0191] TTGTTTTCGACGAAGTTGACTCCAACTCTATCCAAGACTTGATCTACCAATTAGGTTTGCGTGTCTT

[0192] GGAAAGATATCCACAATTGGCCAGCGTTCAATTTGAAGCTAACAACAGAACCTGGTTGTCTGTTAG

[0193] AGATGATTTGGACGGTGATGCTTCTGTCTTGAGAGAACCTCCAGCTCCAACCGGTTTCCAACAATTCTCCATGGACAGAGGTGACTTGGACGAACAA(SEQ ID NO:33)

[0194] UOX Rery Protein sequence and optimized nucleic acid sequence:

[0195] MTTENITTGNITTGVTSTDRLSGSIELTGHQYGKAENRVVRIYRDTPRHEIHDINVSTCLRGDFADAYLT

[0196] GDQSKVLPTDTQKQTAYSYAKEKGLISIETYGLELARHFVDDIEPVSGARIEIEEYAWERAIVDGSEHDY

[0197] TWIRKGQEVRTASITISDTGTADAGTWVIGGLKDLVILKSTGSEFSGFLEDPYTILEPTDDRVMATTLVA

[0198] QWRFVTTDVDWESVYVGIKAQIVKQFAQVHSLALQQTLFHIGKAILEEFPIIAEVRLSAPNKHHFEYALGRFGVENDNEVFHAADRPYGLIQAAVSRSDAPDAGPSWTPYSGWLS(SEQ ID NO:8)

[0199] ATGACGACTGAAAATATCACCACCGGAAACATCACTACCGGTGTTACCTCTACCGATAGATTGTCT

[0200] GGTTCCATTGAATTGACTGGTCACCAATATGGTAAAGCTGAAAACAGAGTTGTTCGTATTTACAGA

[0201] GATACTCCAAGACACGAAATTCACGATATTAACGTTTCCACTTGTTTGAGAGGTGATTTTGCTGACG

[0202] CTTACTTGACCGGTGACCAAAGTAAGGTTTTGCCAACCGACACTCAAAAGCAAACCGCTTACTCTT

[0203] ACGCTAAGGAAAAGGGTCTGATTTCCATCGAAACCTACGGTTTAGAATTGGCTCGTCATTTCGTTGA

[0204] TGACATTGAACCAGTTTCTGGTGCTAGAATCGAAATCGAAGAATACGCTTGGGAAAGAGCCATTGT

[0205] CGATGGTTCTGAACACGACTACACTTGGATCAGAAAAGGTCAAGAAGTTAGAACTGCCTCTATCAC

[0206] CATCTCTGACACAGGTACTGCTGATGCCGGTACTTGGGTTATTGGTGGTCTAAAGGACTTGGTTATC

[0207] TTGAAGTCCACTGGTAGCGAATTCTCCGGTTTCTTGGAAGACCCATACACCATCTTGGAACCAACTG

[0208] ACGACAGAGTCATGGCTACCACCTTAGTTGCTCAATGGAGATTTGTCACTACCGATGTTGACTGGG

[0209] AATCTGTCTACGTTGGTATCAAGGCTCAAATCGTCAAGCAATTCGCCCAAGTCCACTCTTTGGCTTT

[0210] GCAACAAACTTTGTTCCACATTGGTAAGGCCATTTTGGAAGAATTCCCAATTATTGCTGAGGTCCGT

[0211] CTATCAGCCCCAAACAAGCACCACTTCGAATACGCTTTGGGTAGATTCGGTGTCGAAAACGACAAC

[0212] GAAGTCTTCCATGCCGCGGATAGACCATACGGTTTAATCCAAGCTGCTGTCTCTAGATCCGATGCTCCAGACGCTGGTCCATCTTGGACTCCTTATTCCGGTTGGTTGTCC(SEQ ID NO:34)

[0213] UOX Hlbs Protein sequence and optimized nucleic acid sequence:

[0214] MDNDRLMYYGKKDVFAYRTYMKPLEGVKEIPESDFSGRDNVVYGLNASVSVGGKEFLSSFTEGDNSK

[0215] VVATDSMKNFILRHLATYEGATTEGFLAYAAKAFLDRYPHFERVEMSGEEIPFEQTSARSLSDGRSKSD

[0216] VVFKHSRNERVSASTEFIRTADGGTVINKQRSSLTDLQLIKVKGNSFTGYIRDEYTTLPEDSNRPLFIYLNI

[0217] HWGYENVADAYGWKPPLYVAAEQIRDIASSVFHELQTPSIQNLIYRIGCRILERFPQLKDVTFESQNRTWEAVVEEIEGSEGKVYTEPRPPYGFQVFHVTKADLGSEETTVSEARMHT(SEQ ID NO:9)

[0218] ATGGACAATGACAGATTGATGTACTACGGTAAGAAGGACGTTTTCGCTTACAGAACTTACATGAAA

[0219] CCACTCGAAGGTGTTAAGGAAATTCCAGAATCCGATTTCTCCGGAAGAGACAATGTCGTTTATGGT

[0220] TTGAACGCTTCCGTTTCTGTTGGTGGTAAGGAATTTTTGTCATCTTTCACCGAAGGTGACAACTCCA

[0221] AAGTTGTTGCTACTGATTCCATGAAGAACTTCATCCTACGTCACTTGGCAACCTACGAAGGTGCTAC

[0222] CACTGAGGGTTTCTTGGCCTACGCTGCCAAGGCTTTCTTGGACAGATACCCACACTTCGAACGTGTC

[0223] GAAATGTCTGGTGAAGAAATCCCATTCGAACAAACCTCTGCTAGATCCTTATCCGACGGTCGTTCTA

[0224] AGTCTGATGTCGTCTTCAAGCACTCTAGAAACGAAAGAGTTAGCGCCAGTACTGAATTCATTCGTA

[0225] CTGCTGATGGTGGTACTGTTATCAACAAGCAAAGATCTTCTTTGACTGACTTACAATTAATCAAGGT

[0226] CAAGGGTAACTCTTTCACCGGTTACATCAGAGATGAATACACTACTTTGCCAGAAGATTCTAACAG

[0227] ACCATTATTCATCTACTTGAACATTCATTGGGGTTACGAAAACGTTGCCGATGCTTATGGTTGGAAG

[0228] CCACCATTGTACGTCGCTGCTGAACAAATCAGAGACATTGCTTCATCCGTCTTTCACGAATTGCAAA

[0229] CACCATCTATCCAAAACTTGATTTACAGAATTGGTTGTAGAATCTTGGAAAGATTCCCACAATTGAA

[0230] GGATGTTACTTTTGAATCTCAAAACAGAACCTGGGAAGCTGTCGTTGAAGAAATTGAAGGTTCTGA

[0231] AGGTAAAGTCTACACCGAACCAAGACCTCCATACGGTTTCCAAGTTTTCCACGTTACCAAGGCTGACTTGGGTTCCGAAGAAACCACTGTTTCTGAAGCCAGAATGCATACT(SEQ ID NO:35)

[0232] UOX Psim Protein sequence and optimized nucleic acid sequence:

[0233] MTAQNENRTMFYGKGDVFVYRTFVQPLTGLRKIPESSFTERDNTIFGFNCQISLKGEAFLSSFTEGDNSL

[0234] VVATDSMKNIIHRQAANYQGNTAEGFLKFICEVFLDKYSHIDAVELTATEVPFDHVLVPKGEGHENSDV

[0235] VYRCSRNERATTTIEVKRTPTGSKIVKHSSGIVDVHLIKVKGSSFYGFIQDEYTTLPEAQDRPLFIYLDFD

[0236] WEYSRWEDGTGADPEKYVAAEQICDIANTAFHELNNRSIQQLIYHIGIRILERFPQLANVQFKTNNRTWETVVETIPNSEGSVYQEPRPPYGFQGFVVTQEDVKQKKAADSTVGASR(SEQ ID NO:10)

[0237] ATGACTGCCCAAAACGAAAACAGAACCATGTTCTATGGTAAGGGCGACGTCTTTGTCTACAGAACT

[0238] TTTGTTCAACCATTGACTGGTTTGAGAAAGATCCCAGAATCCTCATTCACCGAAAGAGACAATACC

[0239] ATCTTCGGTTTCAACTGCCAAATCTCTTTGAAGGGTGAAGCCTTCTTGTCTTCCTTCACTGAAGGTG

[0240] ACAACTCTCTAGTCGTTGCCACTGACTCCATGAAGAACATTATCCACAGACAAGCTGCCAACTACC

[0241] AAGGTAACACCGCCGAAGGTTTTTTAAAATTCATCTGTGAAGTTTTCTTGGACAAGTACTCTCACAT

[0242] CGATGCTGTTGAATTGACCGCTACTGAAGTCCCATTCGATCATGTTTTGGTTCCAAAGGGTGAAGGT

[0243] CACGAAAACTCCGATGTTGTCTACAGATGTTCTAGAAACGAAAGAGCTACCACCACTATTGAAGTC

[0244] AAGAGAACCCCAACCGGTTCCAAGATTGTTAAGCACTCTTCTGGTATTGTCGATGTCCATTTGATCA

[0245] AGGTTAAAGGTTCGTCCTTCTACGGTTTCATCCAAGATGAATACACCACTTTGCCAGAAGCTCAAG

[0246] ACAGACCTTTATTCATTTATTTGGATTTTGACTGGGAGTACTCCAGATGGGAAGATGGTACTGGTGC

[0247] TGACCCAGAAAAGTACGTCGCTGCTGAACAAATCTGTGACATTGCTAACACTGCTTTCCACGAATT

[0248] GAACAATCGTTCTATTCAACAATTAATATACCACATTGGTATCAGAATCTTGGAAAGATTCCCACA

[0249] ATTGGCTAACGTTCAATTCAAGACCAACAACCGTACATGGGAAACTGTTGTCGAAACCATTCCAAA

[0250] The DNA coding sequence of the candidate UOX listed in Table 1 was artificially synthesized as CTCTGAAGGTTCTGTTTACCAAGAACCAAGACCACCATACGGTTTCCAAGGTTTCGTTGTTACTCAAGAAGACGTTAAGCAAAAGAAAGCTGCTGATTCTACTGTCGGTGCTAGTCGT (SEQ ID NO: 36). Following the YeastFab method, the optimized coding sequence was constructed into a standardized expression plasmid (refer to Guo, Y. et al. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res 43, e88 (2015).) to characterize the activity of the candidate UOX in the yeast system. Specifically, firstly, BsaI restriction endonuclease sites with standardized sticky end sequences were added to the 5' and 3' ends of the synthesized coding sequence by PCR, using primers shown in Table 2 (SEQ ID NO: 42-61). Then, the PCR products and HcKan_O plasmid were digested with BsaI enzyme (NEB, Cat#R3733L) and ligated with T4 DNA ligase (NEB, M0202L) before being transformed into Escherichia coli DH5α strain. The plasmids contained in the transformants were confirmed by selecting non-red transformants and performing colony PCR and Sanger sequencing, ultimately obtaining a series of HcKan_UOX coding sequence element plasmids (Table 3).

[0251] The constitutive promoter pTDH3 (SEQ ID NO: 16) and terminator tPGK1 (SEQ ID NO: 24) were amplified by PCR using the same method. The primers used are shown in Table 2 as SEQ ID NO: 72-85 and SEQ ID NO: 86-91, respectively. Based on the different normalized sticky end sequences, they were assembled into HcKan_P and HcKan_T plasmids, respectively, to obtain HcKan_pTDH3 and HcKan_tPGK1 plasmids (Table 3). Then, each of the obtained HcKan_UOX plasmids was combined with the HcKan_pTDH3 and HcKan_tPGK1 plasmids in the order of "promoter-UOX coding sequence-terminator," and together with the POT2 transcription unit vector, they were digested with BsmBI restriction endonuclease (NEB, R0739L) and then ligated with T4 DNA ligase. The specific sticky end sequences of "promoter, UOX coding sequence, terminator" were joined end-to-end to form a eukaryotic microbial gene expression transcription unit, which was then transformed into *E. coli* DH5α strain. The sequences of the plasmids containing the transcription units in the transformants were confirmed by selecting non-red transformants and performing colony PCR and Sanger sequencing, ultimately obtaining a series of POT2_TU_UOX expression plasmids (Table 3). The obtained expression plasmid was transformed into Saccharomyces cerevisiae BY4741 strain using the lithium acetate (LiAc) transformation method to obtain candidate UOX Saccharomyces cerevisiae expression strain (Table 4). This chassis strain was originally from the Jef D. Boeke team.

[0252] Example 2: Determination of the expression activity of candidate uricase in Saccharomyces cerevisiae

[0253] In this embodiment, the expression activity of the obtained Saccharomyces cerevisiae expression strain was measured to identify the total activity and specific activity of uricase expressed by candidate UOX in Saccharomyces cerevisiae.

[0254] First, a single clone of the candidate UOX Saccharomyces cerevisiae expression strain was inoculated into 500 μL of SC. -Ura The culture was incubated in liquid medium at 30°C with shaking at 200 rpm for 48 hours to allow for recovery until the plateau phase. The plateau-phase culture was then re-inoculated into 5 mL of SC medium. -Ura In liquid culture medium, to initiate OD 600 =0.02, cultured at 30℃ with shaking at 200 rpm for 16 hours. OD of the bacterial culture was measured using a microplate reader. 600 Values, based on bacterial OD 600Numerical calculations were used to determine the required bacterial culture volume for 1.8 OD units of cells. The bacterial culture containing 1.8 OD units of cells was transferred to a 2 mL centrifuge tube and centrifuged at 3000 rpm for 3 min using a benchtop centrifuge (Eppendorf, Centrifuge 5418R). The supernatant was discarded. 1.8 mL of Y-PER was added. TM Yeast protein extract (Thermo Scientific, Cat#78790) was thoroughly pipetted to homogenize the settled cells, and incubated for 20 min at 12°C and 180 rpm using a thermostatic mixer (Eppendorf, ThermoStat Plus). The obtained yeast cell lysate was centrifuged at 4°C and 13000 rpm for 10 min using a benchtop centrifuge (Eppendorf, Centrifuge 5418R), and 150 μL of supernatant was collected and stored at -80°C for protein concentration determination. Another 500 μM supernatant was transferred to a deep-well plate, and UA was added to a concentration of 600 μM. After thorough mixing, 50 μL of the sample was immediately added to a 96-well plate containing 150 μL of PBS solution. The plate was then vibrated for 30 s using a microplate reader (BioTek, SynergyH1), and the A value was read. 300 The reading was taken as the initial reading of the reaction. The remaining reaction solution was incubated at 37°C with shaking at 150 rpm, and 50 μL samples were taken every 30 minutes to determine A using the method described above. 300 Readings. Compare the UA standard curve with the same solution system and dilution conditions, and record the degradation process of UA. Figure 2 After the UOX activity assay, the total protein concentration in the cell lysate supernatant was determined using a BCA protein concentration kit (Abcam Cat#ab207003) compatible with reducing agents, and the specific activity of candidate UOX in *Saccharomyces cerevisiae* was calculated (Table 5). These tests revealed that candidate UOX from *Vibrio vulnificus*, *Peribacillus simplex*, *Rhodococcus erythropolis*, *Halobacillus sp.*, *Natrinema versiforme*, and *Haloferax* sp. exhibited high uric acid degradation activity in the *Saccharomyces cerevisiae* system. The *Vibrio vulnificus* UOX, which showed the best performance, was selected. Vvul The next step is to construct the uric acid-lowering functional pathway.

[0255] Table 5

[0256]

[0257]

[0258] Example 3: Construction of a candidate uric acid degradation regulation system

[0259] Selection of candidate UAT proteins from different sources (Schultz, AC, Nygaard, P. & Saxild, HHFunctional analysis of 14genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. J Bacteriol 183,3293–3302(2001). Leung, J., Karachaliou, M., Alves, C., Diallinas, G. & Byrne, B. Expression and purification of afunctional uric acid-xanthine transporter (UapA). Protein Expr Purif 72,139–146(2010). Goudela, S., Karatza, P., Koukaki, M., Frillingos, S. & Diallinas, G. Comparative substrate recognition by bacterial and fungal purinetransporters of the The coding sequence of the NAT / NCS2 family was optimized according to the method in Example 1. The optimized nucleic acid sequences are shown in Table 6. (Note: The original text also includes references to other articles, such as "NAT / NCS2 family. Mol Membr Biol 22, 263–275 (2005); Papakostas, K. & Frillingos, S. Substrate selectivity of YgfU, a uric acid transporter from Escherichia coli. J Biol Chem 287, 15684–15695 (2012)".)

[0260] Table 6

[0261] serial number protein Source genome Classification protein sequence Optimized nucleic acid sequence <![CDATA[UAT YgfU ]]> YgfU Escherichia coli Gram-negative SEQ ID NO: 11 SEQ ID NO: 37 <![CDATA[UAT PucJ ]]> PucJ Bacillus subtilis Gram-positive SEQ ID NO: 12 SEQ ID NO: 38 <![CDATA[UAT PucK ]]> PucK Bacillus subtilis Gram-positive SEQ ID NO: 13 SEQ ID NO: 39 <![CDATA[UAT Xut1 ]]> Xut1 Candida albicans fungi SEQ ID NO: 14 SEQ ID NO: 40 <![CDATA[UAT UapA ]]> UapA Aspergillus nidulans fungi SEQ ID NO: 15 SEQ ID NO: 41

[0262] UAT YgfU Protein sequences and optimized nucleic acid sequences:

[0263] MSAIDSQLPSSSGQDRPTDEVDRILSPGKLIILGLQHVLVMYAGAVAVPLMIGDRGLSKEAIAMLISSDL

[0264] FCCGIVTLLQCIGIGRFMGIRLPVIMSVTFAAVTPMIAIGMNPDIGLLGIFGATIAAGFITTLLAPLIGRLMP

[0265] LFPPLVTGVVITSIGLSIIQVGIDWAAGGKGNPQYGNPVYLGISFAVLIFILLITRYAKGFMSNVAVLLGIV

[0266] FGFLLSWMMNEVNLSGLHDASWFAIVTPMSFGMPIFDPVSILTMTAVLIIVFIESMGMFLALGEIVGRKL

[0267] SSHDIIRGLRVDGVGTMIGGTFNSFPHTSFSQNVGLVSVTRVHSRWVCISSGIILILFGMVPKMAVLVASI

[0268]

[0269] UAT PucJ Protein sequence and optimized nucleic acid sequence:

[0270] MKKRSFKVFTLSLQHVLAMYAGAILVPLLVGRALNVTTEQLSYLLAIDLLTCGVATLLQTLRGTYIGIGL

[0271] PVMLGSSFVAVTPMIAIGSNYGIHAIYGSIIAAGVFIFLFARFFGKLTVLFPPVVTGTVVTLIGLSLVPTGV

[0272] KNMAGGEKINGSANPEYGSLENLLLSVGVLVLILVLNRFLKGFARTLSVLIGIAAGTAAAAIMGKVSFSS

[0273] VTEAPFFQIPKPFYFGAPAFEIGPILTMLIVGIVIIVESTGVFYAIGKICGRPLTDKDLVKGYRAEGIAILIGG

[0274] LFNAFPYNTFAQNAGLLQLTKVKTRNIVVTAGCILVCLGLIPKIAALASAVPAAVLGGATVVMFGMVIA

[0275] SGVKMLSTADLKNQYHLLTIACSIALGIGASTAPGIFAEFPAPIRILVSDGTITGSLTAIFLNLFFSLRDKKELTAQQTELPVLEHTLALEKEV(SEQ ID NO: 12)

[0276] ATGAAAAAACGTAGCTTTAAAGTCTTCACTCTCTCTCTCCAACATGTACTTGCCATGTACGCAGGTG

[0277] CAATACTTGTCCCACTCCTCGTCGGACGGGCACTGAACGTTACCACAGAACAACTCTCTTATCTGCT

[0278] CGCAATTGATCTCCTTACGTGTGGTGTCGCTACGCTACTCCAGACCCTCAGAGGCACCTACATAGGA

[0279] ATAGGTCTTCCAGTTATGCTTGGTTCTTCCTTTGTTGCTGTCACACCTATGATTGCTATAGGTTCTAA

[0280] TTACGGAATTCATGCCATTTACGGAAGTATTATAGCAGCCGGCGTTTTCATTTTCCTTTTCGCACGG

[0281] TTTTTTGGTAAACTTACCGTCCTGTTCCCACCAGTCGTTACTGGCACAGTCGTAACTCTGATCGGTCT

[0282] GTCTCTCGTCCCCACTGGTGTCAAAAATATGGCAGGTGGTGAAAAAATTAACGGTAGCGCAAACCC

[0283] AGAGTACGGTAGTCTCGAAAATCTTCTCCTCTCCGTTGGAGTACTTGTACTTATTCTCGTACTGAAT

[0284] CGCTTCCTTAAGGGATTTGCAAGAACGCTAAGTGTCCTTATTGGTATAGCTGCTGGGACTGCTGCAG

[0285] CTGCTATTATGGGCAAAGTATCATTCTCATCTGTTACGGAAGCTCCATTCTTTCAGATCCCGAAACC

[0286] ATTCTACTTCGGCGCACCCGCTTTTGAGATTGGACCTATACTGACTATGCTCATTGTAGGCATAGTT

[0287] ATTATTGTTGAATCTACCGGTGTCTTTTACGCTATTGGTAAAATTTGTGGTAGACCACTGACCGATA

[0288] AGGATCTTGTAAAAGGGTACCGCGCCGAGGGCATCGCTATTCTTATAGGCGGACTCTTTAACGCAT

[0289] TCCCCTACAATACTTTTGCCCAAAATGCTGGGCTCCTGCAACTAACGAAAGTTAAAACGAGAAATA

[0290] TAGTAGTTACGGCCGGGTGCATACTGGTATGTCTCGGTCTTATCCCCAAAATCGCTGCACTTGCATC

[0291] AGCAGTACCCGCTGCAGTTCTAGGTGGAGCCACAGTAGTAATGTTTGGTATGGTTATAGCCAGCGG

[0292] TGTCAAGATGCTGTCTACTGCCGATCTCAAAAATCAATACCATCTTCTGACTATTGCGTGCTCCATT

[0293] GCACTGGGTATCGGTGCCTCAACTGCTCCAGGTATATTCGCGGAGTTCCCAGCACCCATTAGAATTC

[0294] TTGTCTCGGATGGAACTATAACAGGGAGTCTGACGGCTATTTTTCTGAATCTTTTCTTCTCTCTGCGC

[0295] GACAAAAAAGAGCTGACTGCACAGCAAACTGAGCTCCCAGTCCTCGAGCATACTCTGGCTCTAGAAAAGGAAGTT(SEQ ID NO:38)

[0296] UAT PucK Protein sequence and optimized nucleic acid sequence:

[0297] MKEQHNALQLMMLGLQHMLAMYAGAILVPLIVGAAIGLNAGQLTYLIAIDLFMCGAATLLQLWRNRY

[0298] FGIGLPVVLGCTFTAVGPMISIGSTYGVPAIYGAIIAAGLIVVLAAGFFGKLVRFFPPVVTGSVVMIIGISLI

[0299] PTAMNNLAGGEGSKEFGSLDNVLLGFGVTAFILLLFYFFKGFIRSIAILLGLIAGTAAAYFMGKVDFSEVL

[0300] EASWLHVPSLFYFGPPTFELPAVVTMLLVAIVSLVESTGVYFALADITNRRLSEKDLEKGYRAEGLAILL

[0301] GGLFNAFPYTAFSQNVGIVQLSKMKSVNVIAITGIILVAIGLVPKAAALTTVIPTPVLGGAMIVMFGMVIS

[0302] YGIKMLSSVDLDSQGNLLIIASSVSLGLGATTVPALFSSLSGAASVLAGSGIVIGSLTAIALHAFFQTKQPNSADIKT(SEQ ID NO:13)

[0303] ATGAAGGAACAACATAACGCACTGCAGCTTATGATGCTTGGACTTCAACATATGCTTGCAATGTAT

[0304] GCGGGCGCAATTCTGGTTCCACTCATTGTAGGTGCCGCGATAGGCCTCAACGCTGGTCAGCTCACTT

[0305] ACCTTATCGCAATTGATCTTTTTATGTGTGGAGCTGCGACCCTCCTTCAACTTTGGAGAAACAGATA

[0306] TTTTGGGATTGGACTTCCCGTCGTACTCGGTTGCACTTTCACGGCTGTTGGTCCTATGATCTCGATAG

[0307] GTAGCACGTATGGTGTTCCAGCCATTTACGGTGCTATTATTGCCGCAGGCCTCATAGTTGTTCTCGC

[0308] TGCTGGCTTCTTTGGCAAACTTGTCCGGTTCTTCCCGCCTGTAGTCACAGGCAGCGTCGTAATGATT

[0309] ATAGGCATTTCTCTCATCCCAACGGCGATGAATAATCTCGCGGGCGGTGAAGGTTCCAAAGAGTTT

[0310] GGGTCCCTAGATAACGTACTTCTCGGATTTGGCGTAACAGCATTCATTCTTCTGCTGTTCTATTTTTT

[0311] CAAGGGTTTCATTAGATCAATCGCCATCCTTCTGGGTCTTATTGCTGGGACTGCAGCCGCTTACTTT

[0312] ATGGGGAAAGTAGATTTCAGCGAAGTACTGGAAGCATCTTGGCTTCACGTCCCATCACTGTTTTATT

[0313] TTGGTCCCCCTACCTTTGAGCTGCCAGCAGTTGTTACCATGCTGCTGGTTGCTATTGTATCTCTTGTC

[0314] GAATCAACCGGTGTATATTTTGCGCTGGCTGACATCACTAATCGCCGCCTTTCTGAAAAAGATCTCG

[0315] AAAAAGGTTATCGTGCTGAAGGTCTTGCTATACTTCTTGGCGGACTTTTTAACGCCTTTCCTTACAC

[0316] TGCATTCAGCCAAAACGTCGGCATTGTACAGCTGTCCAAAATGAAGTCCGTAAACGTCATCGCTAT

[0317] AACCGGTATCATACTAGTCGCCATAGGTCTAGTTCCTAAAGCTGCAGCACTAACAACTGTTATTCCC

[0318] ACGCCAGTTCTTGGTGGGGCTATGATTGTAATGTTCGGTATGGTTATATCCTACGGGATTAAAATGC

[0319] TATCATCAGTAGATCTTGATTCGCAGGGTAACCTCCTAATAATTGCGTCTTCCGTTAGCCTGGGTCT

[0320] TGGGGCAACTACAGTTCCAGCACTGTTTTCCTCACTTAGCGGTGCTGCATCAGTACTGGCTGGTTCC

[0321] GGTATTGTTATCGGTAGCCTTACAGCCATCGCACTCCACGCCTTTTTTCAAACTAAACAACCTAATTCAGCCGATATTAAAACA(SEQ ID NO:39)

[0322] UAT Xut1 Protein sequence and optimized nucleic acid sequence:

[0323] MSAIQKTLKRMVRKWTTKDGLLGDYDYKYLFTPDIPFITKELKTQPFFGVDTDMPILLGAILGFQHALA

[0324] MLAGIVTVPIMVASTANLSVEIEQYLVSTSSIVSGVLSLIQITRFHIPKTPYYIGTGLLSVVGTSFATITIVTK

[0325] AFPMMYADGTCPMASDGVTKLPCPDGYGKILASACCCALLEILLSFMKPSMLQKIFPPLVTGPVVMLIG

[0326] VHLVESGFTDWVGGAGCQDSDTCTYGKFSAPWGDAKFIGLGFLVYVSIILAERFGAPIMKSCAVIFGLLI

[0327] GCIVAAAAGYFSSDNVDAAPAASFMWVHTFKLTVYGPVVLPFLAVYIVLMMECIGDVTATSDVSRLPV

[0328] SGEMYESRIQGGVLGDGICGILSTLMTMTPMSVFAQNNGVISITKCANRKVGYWCAFFLIVMGVFAKFA

[0329] GAITSIPKPVLGGMTSFLFCSVAISGIKIISTTEFTRRDRFVLTAAVSPGLGATMLPNWFEHVFTYQGGNK

[0330] SLKGFFNAIIVVVESGFCLSGVIAVILNLLIPQVEDDVEEINELVLDVVGSATESARQKFEEKEGVKLEAL

[0331] RSQLSNIKSNGGSRDAITVLHRNDGFPDLK(SEQ ID NO:14)

[0332] ATGTCGGCCATCCAAAAGACGCTCAAACGGATGGTTAGAAAGTGGACGACGAAAGACGGCCTTCT

[0333] TGGGGATTATGATTATAAGTATCTCTTCACCCCAGATATTCCATTCATCACTAAAGAACTAAAGACA

[0334] CAACCTTTTTTCGGCGTAGATACAGACATGCCAATTCTGCTCGGAGCAATACTAGGGTTTCAACATG

[0335] CACTAGCCATGCTAGCAGGTATAGTAACAGTTCCGATTATGGTCGCTAGCACAGCCAATCTTTCCGT

[0336] AGAGATTGAGCAGTACCTCGTCAGCACCTCTTCAATCGTCAGTGGAGTCCTATCTCTTATACAGATA

[0337] ACGAGATTCCATATTCCCAAGACTCCCTATTATATAGGGACAGGTCTGCTTTCCGTCGTTGGAACAT

[0338] CGTTTGCTACTATTACTATAGTAACGAAAGCATTTCCTATGATGTATGCTGATGGTACCTGTCCAAT

[0339] GGCAAGTGATGGTGTTACTAAGCTACCATGCCCGGATGGCTATGGAAAAATCCTTGCTAGTGCATG

[0340] TTGTTGTGCGCTTCTAGAAATACTTCTTAGCTTTATGAAGCCGTCTATGCTCCAAAAGATATTTCCA

[0341] CCCCTAGTCACGGGACCAGTTGTCATGCTTATTGGTGTACACCTAGTCGAAAGTGGCTTTACAGACT

[0342] GGGTCGGTGGGGCCGGTTGCCAAGATTCTGATACTTGTACATATGGCAAGTTTAGCGCCCCTTGGG

[0343] GTGACGCCAAATTTATAGGTCTTGGCTTTCTTGTTTATGTCTCCATCATACTCGCTGAGAGATTTGGC

[0344] GCTCCGATTATGAAATCATGTGCTGTCATATTCGGACTCCTAATCGGGTGCATCGTCGCTGCCGCTG

[0345] CCGGGTATTTTTCATCAGACAATGTAGACGCCGCTCCTGCTGCTTCCTTTATGTGGGTTCACACTTTC

[0346] AAGCTTACAGTCTACGGTCCAGTAGTTCTCCCTTTTCTCGCTGTCTACATCGTTCTAATGATGGAGT

[0347] GTATCGGGGATGTCACTGCAACCTCCGATGTATCCCGCCTCCCAGTTTCTGGTGAGATGTATGAGTC

[0348] TAGAATTCAAGGAGGTGTCCTAGGCGACGGCATATGTGGCATCCTTTCCACTCTAATGACCATGAC

[0349] ACCAATGTCTGTTTTTGCGCAGAATAATGGAGTTATATCTATCACAAAATGTGCTAACCGTAAAGTT

[0350] GGGTACTGGTGCGCATTCTTTCTCATTGTTATGGGCGTATTCGCAAAATTCGCTGGAGCCATCACTT

[0351] CCATACCTAAGCCGGTTCTTGGGGGCATGACGTCATTTCTTTTTTGTTCAGTCGCAATCTCAGGGAT

[0352] CAAAATCATCTCCACGACTGAGTTTACAAGAAGAGATCGCTTCGTACTTACAGCTGCAGTAAGTCC

[0353] AGGCCTTGGCGCTACTATGCTTCCTAATTGGTTTGAACATGTATTCACATACCAGGGGGGAAATAA

[0354] AAGTCTTAAGGGCTTTTTCAACGCCATCATAGTTGTCGTCGAGTCAGGCTTTTGTCTATCAGGGGTA

[0355] ATTGCCGTTATCCTCAATCTTCTGATACCACAGGTTGAAGACGACGTAGAGGAAATTAATGAACTC

[0356] GTACTGGATGTAGTCGGTTCAGCCACAGAATCTGCAAGACAGAAATTCGAGGAAAAAGAAGGAGT

[0357] AAAGCTCGAGGCTCTGCGGAGCCAACTAAGCAATATTAAATCAAATGGTGGTTCACGCGACGCCAT

[0358] TACGGTTCTCCATCGGAACGATGGATTTCCAGATCTGAAG(SEQ ID NO:40)

[0359] UAT UapA Protein sequence and optimized nucleic acid sequence:

[0360] MHDRAGLDHSPAPAVGPDPYSSDDSMDNSIHSTDGPDSVIPNSNPKKTVRQRVRLLARHLTTREGLIGD

[0361] YDYGFLFRPELPFMKKDPRAPPFFGLNEKIPVLLAFILGLQHALAMLAGVVTPPLIISSSLSLPSDLQQYLV

[0362] STSLIVCGLLSMVQITRFHIYKTPYYIGSGVLSVMGVSFSIISVASGAFNQMYSNGFCQLDEAGNRLPCPE

[0363] AYGALIGTSACCALVEILLAFVPPKVIQKIFPPIVTGPTVMLIGISLIGTGFKDWAGGSACMDDGMLCPSA

[0364] TAPRPLPWGSPEFIGLGFLVFVSIILCERFGAPIMKSCSVVIGLLVGCIVAAACGYFSHADIDAAPAASFIW

[0365] VKTFPLSVYGPMVLPIIAVFIICACECIGDVTATCDVSRLEVRGGTFESRIQGAVLADGINSVVAALATMT

[0366] PMTTFAQNNGVIALTRCANRWAGYCCCLILIVAGIFAKFAAAIVAIPNSVMGGMKTFLFASVVISGQAIV

[0367] AKAPFTRRNRFILTASMALGYGATLVPTWFGNVFPQTENRDLEGFENAIELVLETGFAVTAFVAMLLNAIMPAEVEEIGAVTPMPVSAHDNRDGEAEYQSKQA(SEQ ID NO:15)

[0368] ATGCACGATAGAGCTGGTCTTGATCACAGTCCTGCGCCTGCCGTTGGTCCCGATCCATATTCCTCTG

[0369] ACGATAGTATGGATAATTCAATCCACTCAACAGATGGCCCGGATAGTGTTATACCAAATAGTAACC

[0370] CAAAAAAAACGGTAAGACAAAGAGTACGTCTTCTTGCTAGACATCTTACCACTCGGGAAGGACTGA

[0371] TCGGAGACTATGATTACGGGTTTCTGTTTCGTCCAGAACTTCCGTTCATGAAAAAAGATCCACGCGC

[0372] ACCGCCATTTTTCGGCCTCAACGAAAAGATCCCAGTACTCCTCGCATTTATCCTTGGTCTTCAACAC

[0373] GCGCTCGCTATGCTTGCTGGTGTTGTCACTCCACCCCTTATAATATCTTCTTCTCTCTCCCTCCCATC

[0374] CGATCTACAACAGTACCTCGTATCAACTTCTCTCATCGTCTGTGGTCTTCTCTCTATGGTACAGATCA

[0375] CACGTTTTCATATATACAAAACCCCCTACTATATTGGGAGTGGAGTTCTCAGCGTAATGGGAGTTTC

[0376] CTTTTCAATCATTTCAGTCGCGAGCGGTGCATTCAATCAAATGTATTCGAATGGCTTTTGTCAACTG

[0377] GATGAAGCTGGAAATCGTCTTCCGTGTCCTGAAGCATACGGTGCCCTGATAGGAACTTCAGCGTGC

[0378] TGTGCCCTCGTAGAGATACTACTCGCTTTCGTCCCCCCCAAAGTTATTCAAAAAATCTTCCCGCCTA

[0379] TCGTCACCGGTCCAACCGTTATGCTCATAGGTATTTCGCTTATAGGTACTGGGTTTAAAGACTGGGC

[0380] CGGTGGCTCGGCCTGTATGGATGATGGTATGCTCTGTCCTAGCGCTACCGCCCCCCGCCCGCTACCT

[0381] TGGGGTAGCCCAGAATTTATTGGTCTGGGTTTCCTCGTCTTTGTTAGCATTATCCTGTGTGAAAGAT

[0382] TTGGCGCGCCAATTATGAAGAGCTGTTCGGTCGTTATAGGTCTGCTCGTCGGTTGTATCGTTGCTGC

[0383] CGCTTGTGGTTACTTTTCTCATGCAGATATTGATGCAGCTCCTGCAGCTTCATTCATTTGGGTAAAA

[0384] ACTTTCCCTCTCAGTGTTTACGGTCCGATGGTTCTCCCAATCATCGCCGTTTTCATTATTTGTGCCTG

[0385] TGAATGTATTGGTGACGTAACAGCTACTTGTGATGTCTCAAGACTTGAAGTAAGAGGCGGTACATT

[0386] CGAGTCGAGAATCCAAGGGGCCGTTCTCGCAGATGGCATCAACAGTGTTGTAGCTGCGCTCGCAAC

[0387] CATGACTCCAATGACAACCTTTGCGCAAAATAACGGTGTAATCGCACTCACGCGCTGTGCCAATAG

[0388] ATGGGCTGGATACTGCTGCTGTCTGATACTAATAGTTGCAGGTATTTTTGCTAAATTCGCGGCGGCC

[0389] ATTGTCGCCATCCCAAATTCGGTTATGGGGGGAATGAAGACTTTCCTCTTTGCCTCAGTTGTTATAA

[0390] GCGGTCAAGCTATTGTTGCAAAGGCACCATTTACAAGACGCAATAGATTCATCCTGACCGCCTCTA

[0391] TGGCACTTGGCTACGGCGCGACCCTCGTACCAACGTGGTTTGGCAATGTATTTCCACAGACCGAAA

[0392] ACAGAGATCTGGAAGGCTTTGAGAATGCGATTGAACTGGTCCTTGAAACCGGTTTTGCTGTAACAG

[0393] CATTTGTTGCTATGCTTCTAAACGCCATAATGCCTGCCGAAGTTGAAGAAATAGGTGCTGTTACACCGATGCCTGTATCGGCACATGATAATCGTGATGGCGAAGCCGAATATCAATCAAAGCAAGCT(SEQID NO:41)

[0394] The candidate UAT DNA coding sequences listed in Table 6 were artificially synthesized. Following the construction procedure in Example 1, these UAT sequences were ligated into the HcKan_O plasmid, ultimately obtaining a series of HcKan_UAT coding sequence element plasmids (Table 3). The primers used are shown in Table 2, SEQ ID NO: 62-71.

[0395] The constitutive promoter pEFB1 (SEQ ID NO: 17) and terminator tADH1 (SEQ ID NO: 25) were amplified by PCR using the same method (Table 3). Based on the different standardized sticky end sequences, they were assembled into HcKan_P and HcKan_T plasmids, respectively, to obtain HcKan_pEFB1 and HcKan_tADH1 plasmids (Table 3). Then, each of the obtained HcKan_UAT plasmids was combined with the HcKan_pEFB1 plus HcKan_tADH1 plasmid according to the rule of "promoter-UAT coding sequence-terminator," and ligated into the POT3 transcription unit vector to form a gene expression transcription unit. This unit was transformed into *E. coli* DH5α strain. Non-red transformants were selected, and colony PCR and Sanger sequencing were performed to confirm the sequence of the plasmids containing the transcription units within the transformants, ultimately obtaining a series of POT3_TU_UAT transcription unit plasmids (Table 3). The m416B pathway-carrying plasmid backbone was amplified by PCR, and a BsaI restriction endonuclease site with a standardized specific sticky end sequence was added. The PCR product was then purified, and the primers used are shown in Table 2 (SEQ ID NO: 92-93). Each obtained POT3_TU_UAT plasmid was then coupled with POT2_TU_UOX. Vvul The m416B backbone fragment was digested with BsaI restriction endonuclease and then ligated with T4 DNA ligase. Following the specific sticky end sequence "TU_UOX+TU_UAT", a uric acid transport and degradation pathway was formed. This pathway was then carried onto the m416B plasmid backbone and transformed into *E. coli* DH5α strain. Colony PCR and Sanger sequencing confirmed the sequence of the plasmid containing the transcription unit in the transformants, resulting in a series of m416B_UOX_UAT pathway expression plasmids (Table 3). These expression plasmids were then transformed into *Saccharomyces cerevisiae* BY4741 strain using the LiAc transformation method to obtain candidate uric acid-lowering *Saccharomyces cerevisiae* strains (Table 4).

[0396] Example 4: Determination of the expression activity of candidate uricase in Saccharomyces cerevisiae

[0397] The whole-cell uric acid-lowering activity of the candidate strains obtained in Example 3 was measured to identify the functional expression of candidate UAT in Saccharomyces cerevisiae and the unit cell uric acid degradation activity level. First, a single clone of the candidate strain was inoculated into 500 μL of SC. -Ura The culture was incubated in liquid medium at 30°C with shaking at 200 rpm for 48 hours to allow for recovery until the plateau phase. The plateau-phase culture was then re-inoculated into a solution containing 9 mL of SC. -Ura In 14 mL shake tubes of liquid culture medium, adjust the initial OD600 to 0.02 and incubate for 16 hours at 30°C and 200 rpm with shaking. Measure the OD600 value of the bacterial culture using a microplate reader. Centrifuge the bacterial culture at 3000 rpm for 5 min and discard the supernatant. Based on the OD600 value of the bacterial culture... 600 Numerical calculations were performed by resuspending the bacterial culture in PBS solution to a final volume of 7.5 OD units / mL (10⁻⁶). 8 (CFU / mL). Pipette 1 mL of the resuspended bacterial culture into a deep-well plate, add UA to the resuspended culture to a final concentration of 600 μM, and mix immediately. Add 50 μL of the supernatant to a 96-well plate containing 150 μL of PBS solution. After shaking for 30 seconds using a microplate reader (BioTek, Synergy H1), read the A300 value as the initial reading of the reaction. Incubate the remaining cell resuspended culture at 30°C and 200 rpm, and take 50 μL samples every 3 hours to measure the A300 value using the same method. Compare the A300 value with the UA standard curve under the same solution system and dilution conditions, and record the UA degradation process. The results are as follows: Figure 3 As shown.

[0398] Depend on Figure 3 The test results shown revealed UAT from Aspergillus nidulans. UapA It can be functionally expressed in Saccharomyces cerevisiae, and is associated with UOX from Vibrio vulnificus. Vvul After forming the functional pathway, its pathway activity expression plasmid m416B-pTDH3_UOX Vvul _tPGK1-pEFB1_UAT UapA _tADH1 can confer high levels of uric acid degradation activity on the whole cells of Saccharomyces cerevisiae, with a corresponding cell viability of 303.91±79.05 μM / hr / 10 9 CFU (Table 7).

[0399] Table 7

[0400]

[0401] Example 5: Optimization of the uric acid degradation regulation system in Saccharomyces cerevisiae.

[0402] The pathway activity plasmid m416B-pTDH3_UOX, which enables whole-cell uric acid degradation, was successfully expressed in Saccharomyces cerevisiae. Vvul _tPGK1-pEFB1_UAT UapA The transcriptional regulatory elements used in _tADH1 were further optimized through combination. First, constitutive promoters engineered from *Saccharomyces cerevisiae* (including pPGK1, pPDC1, pCCW12, pENO2, pFBA1, and pTEF2) and the terminator tSCW4, which enhances transcriptional expression, were selected, with sequences shown in SEQ ID NO: 18-23 and SEQ ID NO: 26, respectively. The above strong promoters were then used to replace UAT using the Gibson assembly method. UapA The promoter of pEFB1 (Gibson, DG, Young, L., Chuang, RY, Venter, JC, Hutchison, CA, 3rd, & Smith, HOEnzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods, 6 (5), 343–345 (2009).).

[0403] Specifically, the strong promoter was first amplified from the BY41741 genome by PCR, and homologous arms corresponding to the initial plasmid pEFB1 sequence were added to the 5' and 3' ends, respectively. The PCR product was then purified. The original plasmid backbone was amplified by reverse PCR starting upstream and downstream of pEFB1, and the PCR product was purified. The primers used are shown in Table 2, SEQ ID NO: 94-109. The obtained strong promoter fragments were mixed with the plasmid backbone at a fragment:backbone ratio of 3:1, added to a Gibson assembly mastermix (NEB, Cat#E2611), and incubated at 50°C for 1 hour using a thermal cycler. The mixture was then transformed into *E. coli* DH5α strain. Colony selection and Sanger sequencing confirmed the plasmids contained in the transformants, ultimately yielding UAT driven by strong promoters of different constitutive types. UapA The pathway activity plasmids encoding the sequences are shown in Table 3. The constructed pathway activity plasmid variants were transformed into strain BY4741 to obtain new whole-cell uric acid-lowering candidate strains. The whole-cell uric acid-lowering activity test in Example 4 was repeated using the above strains, and it was found that UAT was driven by five strong promoters (pPDC1, pPGK1, pCCW12, pFBA1, pTEF2). UapA Expression of the coding sequence can effectively improve the whole-cell uric acid degradation activity of the strain and eliminate the unstable m416B-pTDH3_UOX in the cell. Vvul_tPGK1-pPGK1_UAT UapA After combining _tADH1, m416B-pTDH3_UOX Vvul _tPGK1-pPDC1_UAT UapA The _tADH1 combination exhibited the highest level of activity, reaching 552.76±29.98 μM / hr / 10 9 CFU (Table 8). Based on these, using the Gibson method described above, UOX is... Vvul The transcription unit terminator was replaced with tSCW4, and the resulting strain was transformed into BY4741 to obtain a candidate strain for further modification. Using the new candidate strain, whole-cell uric acid degradation activity tests were repeated, revealing a significant increase in uric acid degradation activity in all modified expression systems, and the presence of m416B-pTDH3_UOX was also observed. Vvul _tSCW4-pPDC1_UAT UapA The _tADH1 plasmid combination conferred the highest level of uric acid degradation activity to the Saccharomyces cerevisiae strain, reaching 675.31±14.13 μM / hr / 10. 9 CFU (Table 8, Figure 3 ).

[0404] Table 8

[0405]

[0406]

[0407] pTDH3 nucleic acid sequence (SEQ ID NO: 16):

[0408] TCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTAT

[0409] TTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTA

[0410] CACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAAT

[0411] GGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTT

[0412] CACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGC

[0413] AAAAAACGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAA

[0414] TTGACCACGCATGTATCTATCTCATTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGA

[0415] AAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAATTTTCCCCTACTTGACTAATAAGTATA

[0416] TAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTTACTTTAT

[0417] AGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACATAAACAAACAAA(SEQ ID NO:16)

[0418] pEFB1 (SEQ ID NO:17):

[0419] ATTATCTGTTATTTACTTTGAATTTTTGTTTCTTGTAATACTTGATTACTTTTCTTTTGATGTGCTTATC

[0420] TTACAAATAGAAAATAAAACAACTTAAGTAAGAATTGGGAAACGAAACTACAACTCAATCCCT

[0421] TCTCGAAGATACATCAATCCACCCCTTATATAACCTTGAAGTCCTCGAAACGATCAGCTAATCTAAA

[0422] TGGCCCCCCTTCTTTTTGGGTTCTTTCTCCCTTTTGCCGCCGATGGAACGTTCTGGAAAAAGAAG

[0423] AATATTTAATTACTTTCTCAACTAAAATCTGGAGAAAAAACGCAAATGACAGCTTCTAAACGTTC

[0424] CGTGTGCTTTCTTTCTAGAATGTTCTGGAAAGTTTCAACAATCCACAAGAACGAAAATGCCGTTGA

[0425] CAATGATGAAACCATCATCCACACCGCGCACACGTGCTTTATTTCTTTTTCTGAATTTTTTTTTTC

[0426] CGCCATTTTCAACCAAGGAAATTTTTTTTCTTAGGGCTCAGAACCTGCAGGTGAAGAAGCGCTTTAG

[0427] AAATCAAAGCAACGTAACAATTTGTCGACAACCGAGCCTTTGAAAAAAATTTTTCACATTGT

[0428] CGCCTCTAAATAAATAGTTTAAGGTTATCTACCCACTATATTTAGTTGGTTCTTTTTTTTTTCCTTCTA

[0429] CTCTTTATCTTTTTACCTCATGCTTTCTACCTTTCAGCACTGAAGAGTCCAACCGAATATATACACACATA(SEQ ID NO:17)

[0430] pPDC1 (SEQ ID NO:18):

[0431] AGGGTAGCCTCCCCATAACATAAACTCAATAAAATATAGTCTTCAACTTGAAAAAGGAACAAGC

[0432] TCATGCAAAGAGGTGGTACCCGCACGCCGAAATGCATGCAAGTAACCTATTCAAAGTAATATCTCA

[0433] TACATGTTTCATGAGGGTAACAACATGCGACTGGGTGAGCATATGTTCCGCTGATGTGATGTGCAA

[0434] GATAAACAAGCAAGGCAGAAACTAACTTCTTCTTCATGTAATAAACACACCCCGCGTTTATTTACCT

[0435] ATCTCTAAACTTCAACACCTTATATCATAACTAATATTTCTTGAGATAAGCACACTGCACCCATACC

[0436] TTCCTTAAAAACGTAGCTTCCAGTTTTTGGTGGTTCCGGCTTCCTTCCCGATTCCGCCCGCTAAACGC

[0437] ATATTTTTGTTGCCTGGTGGCATTTGCAAAATGCATAACCTATGCATTTAAAAGATTATGTATGCTC

[0438] TTCTGACTTTTCGTGTGATGAGGCTCGTGGAAAAAATGAATAATTTATGAATTTGAGAACAATTTTG

[0439] TGTTGTTACGGTATTTTACTATGGAATAATCAATCAATTGAGGATTTTATGCAAATATCGTTTGAAT

[0440] ATTTTTCCGACCCTTTGAGTACTTTTCTTCATAATTGCATAATATTGTCCGCTGCCCCTTTTTCTGTTA

[0441] GACGGTGTCTTGATCTACTTGCTATCGTTCAACACCACCTTATTTTCTAACTATTTTTTTTTAGCTCA

[0442] TTTGAATCAGCTTATGGTGATGGCACATTTTTGCATAAACCTAGCTGTCCTCGTTGAACATAGGAAA

[0443] AAAAAATATATAAACAAGGCTCTTTCACTCTCCTTGCAATCAGATTTGGGTTTGTTCCCTTTATTTTC

[0444] ATATTTCTTGTCATATTCCTTTCTCAATTATTTCTACTCATAACCTCACGCAAAATAACAGTCAAATCAATCAAA(SEQ ID NO:18)

[0445] pCCW12 (SEQ ID NO:19):

[0446] AACCAGGGCAAAGCAAAATAAAGAAACTTAATACGTTATGCCGTAATGAAGGGCTACCAAAAAC

[0447] GATAATCTCAACTGTAAACAGGTACAATGCGGACCCTTTGCCACAAAACATACATCATTCATTCATGC

[0448] CGGAAAAAGAAAGAAGTGAAGACAGCAGTGCAGCCAGCCATGTTGCGCCAATCTAATTATAGATG

[0449] CTGGTGCCCTGAGGATGTATCTGGAGCCAGCCATGGCATCATGCGCTACCGCCGGATGTAAAATCC

[0450] GACACGCAAAAGAAAACCTTCGAGGTTGCGCACTTCGCCCACCCATGAACCACACGGTTAGTCCAA

[0451] AAGGGGCAGTTCAGATTCCAGATGCGGGAATTAGCTTGCTGCCACCCTCACCTCACTAACGCTGCG

[0452] GTGTGCGGATACTTCATGCTATTTATAGACGCGCGTGTCGGAATCAGCACGCGCAAGAACCAAAATG

[0453] GGAAAATCGGAATGGGTCCAGAACTGCTTTGAGTGCTGGCTATTGGCGTCTGATTTCCGTTTTGGGA

[0454] ATCCTTTGCCGCGCGCCCCTCTCAAAACTCCGCACAAGTCCCAGAAAGCGGGAAAGAAATAAAACG

[0455] CCACCAAAAAAAAAAATAAAAGCCAATCCTCGAAGCGTGGGTGGTAGGCCCTGGATTATCCCGTA

[0456] CAAGTATTTCTCAGGAGTAAAAAAACCGTTTGTTTTGGAATTCCCCATTTCGCGGCCACCTACGCCG

[0457] CTATCTTTGCAACAACTATCTGCGATAACTCAGCAAATTTTGCATATTCGTGTTGCAGTATTGCGAT

[0458] AATGGGAGTCTTACTTCCAACATAACGGCAGAAAGAAATGTGAGAAAATTTTGCATCCTTTGCCTC

[0459] CGTTCAAGTATATAAAGTCGGCATGCTTGATAATCTTTCTTTCCATCCTACATTGTTCTAATTATTCT

[0460] TATTCTCCTTTATTCTTTCCTAACATACCAAGAAATTAATCTTCTGTCATTCGCTTAAACACTATATCAATA(SEQ ID NO:19)

[0461] pENO2 nucleic acid sequence (SEQ ID NO:20):

[0462] ACGCGGCGTTATGTCACTAACGACGTGCACCAACTTGCGGAAAGTGGAATCCCGTTCCAAAACTGG

[0463] CATCCACTAATTGATACATCTACACACCGCACGCCTTTTTTCTGAAGCCCACTTTCGTGGACTTTGC

[0464] CATATGCAAAATTCATGAAGTGTGATACCAAGTCAGCATACACCTCACTAGGGTAGTTTCTTTGGTT

[0465] GTATTGATCATTTGGTTCATCGTGGTTCATTAATTTTTTTTCTCCATTGCTTTCTGGCTTTGATCTTAC

[0466] TATCATTTGGATTTTTGTCGAAGGTTGTAGAATTGTATGTGACAAGTGGCACCAAGCATATAAAAA

[0467] AAAAAAAGCATTATCTTCCTACCAGAGTTGATTGTAAAAACGTATTTATAGCAAACGCAATTGTA

[0468] ATTAATTCTTATTTTGTATCTTTTCTTCCCTTGTCTCAATCTTTTATTTTTATTTTTTCTTTTCTTAGTTTCTTTCATAACACCAAGCAACTAATACTATAACATACAATAATA(SEQ ID NO:20)

[0469] pFBA1(SEQ ID NO:21):

[0470] ATAACAATACTGACAGTACTAAATAATTGCCTACTTGGCTTCACATACGTTGCATACGTCGATATAG

[0471] ATAATAATGATAATGACAGCAGGATTATCGTAATACGTAATAGTTGAAAATCTCAAAATGTGTGG

[0472] GTCATTACGTAAATAATGATAGGAATGGGATTCTTCTATTTTTCCTTTTTCCATTCTAGCAGCCGTCG

[0473] GGAAAACGTGGCATCCTCTCTTTCGGGCTCAATTGGAGTCACGCTGCCGTGAGCATCCCTCTTTCC

[0474] ATATCTAACAACTGAGCACCGTAACCAATGGAAAAGCATGAGCTTAGCGTTGCTCCAAAAAAGTATT

[0475] GGATGGTTAATACCATTTGTCTGTTCTCTTCTGACTTTGACTCCTCAAAAAAAAAAATCTACAATC

[0476] AACAGATCGCTTCAATTACGCCCTCACAAAAACTTTTTTCCTTCTTCTTCGCCCACGTTAAATTTTAT

[0477] CCCTCATGTTGTCTAACGGATTTCTGCACTTGATTTATTATAAAAAGACAAAGACATAATACTTCTC

[0478] TATCAATTTCAGTTATTGTTCTTCCTTGCGTTATTCTTCTGTTCTTCTTTTTCTTTTGTCATATATAACCATAACCAAGTAATACATATTCAAA(SEQ ID NO: 21)

[0479] Nucleic acid sequence of pTEF2 (SEQ ID NO: 22):

[0480] ACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTG

[0481] TATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTA

[0482] GATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCG

[0483] CCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACA

[0484] AATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCA

[0485] AAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACT

[0486] ATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTT

[0487] CTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCT

[0488] CTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCT

[0489] TCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAAC(SEQ ID NO:22)

[0490] pPGK1 nucleic acid sequence (SEQ ID NO:23):

[0491] ACTGTAATTGCTTTTAGTTGTGTATTTTTAGTGTGCAAGTTTCTGTAAATCGATTAATTTTTTTTTCTT

[0492] TCCTCTTTTTATTAACCTTAATTTTTATTTTAGATTCCTGACTTCAACTCAAGACGCACAGATATTAT

[0493] AACATCTGCATAATAGGCATTTGCAAGAATTACTCGTGAGTAAGGAAAGAGTGAGGAACTATCGCA

[0494] TACCTGCATTTAAAGATGCCGATTTGGGCGCGAATCCTTTATTTTGGCTTCACCCTCATACTATTATC

[0495] AGGGCCAGAAAAAGGAAGTGTTTCCCTCCTTCTTGAATTGATGTTACCCTCATAAAGCACGTGGCC

[0496] TCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACC

[0497] CAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCT

[0498] AGCGACGGCTCACAGGTTTTGTAACAAGCAATCGAAGGTTCTGGAATGGCGGGAAAGGGTTTAGTA

[0499] CCACATGCTATGATGCCCACTGTGATCTCCAGAGCAAAGTTCGTTCGATCGTACTGTTACTCTCTCT

[0500] CTTTCAAACAGAATTGTCCGAATCGTGTGACAACAACAGCCTGTTCTCACACACTCTTTTCTTCTAA

[0501] CCAAGGGGGTGGTTTAGTTTAGTAGAACCTCGTGAAACTTACATTTACATATATATAAACTTGCATA

[0502] AATTGGTCAATGCAAGAAATACATATTTGGTCTTTTCTAATTCGTAGTTTTTCAAGTTCTTAGATGCT

[0503] TTCTTTTTCTCTTTTTTACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATATAAAACA(SEQ ID NO:23)

[0504] tPGK1 nucleic acid sequence (SEQ ID NO:24):

[0505] ATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAAT

[0506] AATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTA

[0507] ATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTT

[0508] CCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTAATAACTCGAAAATTCTGCGTT(SEQ ID NO:24)

[0509] tADH1 nucleic acid sequence (SEQ ID NO:25):

[0510] GCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAAT

[0511] TTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGG(SEQ ID NO:25) tSCW4 nucleic acid sequence (SEQ ID NO:26):

[0512] GTCTACTCAATTGTAAAAAAAAACAAAAAAAATCTTGATTTAAAAAACGATACCCAATATTTTTAA

[0513] CTTTAAATTAAGGCTTTGATAATCTTCTTTTTCTCTGTTATATCAAAGGACTGCTTTCTGGTTTGCCT

[0514] TCGCTAATCTTTTTTTTTTTTTTTCATTTTCCTATTTTAGTTCGTTTATGATCTCATTTGTATTTCTGTA

[0515] TATTTGTCTTAATACGAAATACGAGTTTTTATTTTATATTCTACCTTTCTTTTTTTTTTGGCTAAAAGC

[0516] GTGTACGGTTCTATGTAAATATGTACGATTACATGTGTAATGTTTGTTTGGCCGGGCTCTTCATCTATGCCGTTGCCTTCTTGCAGGCGGAGTCCTGGATCTGTTACGATGGTTTCTCGATCCTCTTGG(SEQ ID NO:26)

[0517] Example 6: Transplantation of the function of reducing uric acid

[0518] After establishing the optimized uric acid degradation pathway, the optimal combination of regulatory elements from Example 5 was transplanted into the probiotic *Saccharomyces boulardii* MYA-796 strain. This strain was purchased from the American Type Culture Collection (ATCC). To achieve the goal of pathway activity transplantation, the MYA-796 strain needed to be preliminarily modified to free up selectable tags for transformation. First, the reverse complementary sequence of the URA3 gene at positions 229-248 of strain MYA-796 was selected as the CRISPR / Cas9 knockout target sequence. The genomic template in the product was removed by reverse PCR using KLD enzyme mix (NEB, Cat#M0554) and then enzymatically circularized. This 20bp target sequence was inserted into the 5′ end of the gRNA scaffold sequence carried by the pCAS plasmid (Addgene, Plasmid#60847) and transformed into E. coli DH5α strain. The plasmid contained in the transformants was confirmed by colony selection and Sanger sequencing to obtain the pCAS-sgRNA-SbURA3 plasmid. Then, using OE-PCR, the 1-261bp and 224-520bp URA3 coding sequences were amplified from the MYA-796 genome and spliced ​​together. Simultaneously, the TCC in the 241-243bp sequence was replaced with TAA, forming a 520bp URA3 double-strand break repair template SbURA3-TAA with an early stop codon. The primers used are listed in Table 2, SEQ ID NO: 110-115. The pCAS-sgRNA-SbURA3 plasmid and the repair template SbURA3-TAA were simultaneously transformed into the MYA-796 strain. Transformants resistant to G418 antibiotics were randomly selected and simultaneously transformed into SC... -URA Colony agar plates were prepared using SC solid culture medium. Corresponding colony spots were selected from SC plates. -URA Colonies that failed to grow on plates were confirmed by colony PCR and Sanger sequencing to have a TAA mutation in the URA3 gene at 241-243 bp in strain MYA-796. Finally, a mutant strain, MYA-796ΔURA3 (Table 4), which failed to grow under Uracyl conditions, was obtained. This strain can be used for plasmid transformation screening with the URA3 gene tag. The pathway activity plasmid m416B-pTDH3_UOX, expressing the optimal uric acid degradation activity, was then transformed. Vvul _tSCW4-pPDC1_UAT UapA_tADH1 was transformed into the MYA-796ΔURA3 strain and, together with the Saccharomyces cerevisiae strain carrying the same pathway activity plasmid in Example 4, the whole-cell uric acid-lowering activity test was repeated at 37°C (close to human body temperature). The results are shown in Table 9 and Figure 4 As shown.

[0519] Tests revealed that, under human body temperature conditions, plasmids carrying the uric acid degradation pathway could still achieve high levels of uric acid degradation activity in Saccharomyces cerevisiae, reaching 676.43±19.08 μM / hr / 10. 9 CFU levels (Table 9). Whole-cell uric acid-lowering agents were successfully transplanted into the probiotic *Saccharomyces boulardii* strain, achieving an activity of 372.22 ± 23.98 μM / hr / 10 under human body temperature conditions. 9 The CFU levels (Table 9) all showed a highly significant uric acid degradation effect compared to their unmodified chassis bacteria, indicating that the uric acid degradation regulation system proposed in this application can be effectively used in eukaryotic intestinal bacteria such as yeast and effectively degrade uric acid.

[0520] Table 9

[0521]

[0522] Table 2 Primer sequences involved in the embodiments of this application

[0523]

[0524]

[0525]

[0526] Table 3. Plasmid templates and expression vectors constructed based on them involved in the embodiments of this application.

[0527]

[0528]

[0529]

[0530]

[0531]

[0532] Table 4. Starting bacteria and recombinant bacteria based thereon involved in the embodiments of this application.

[0533]

[0534]

[0535] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0536] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A regulatory system for degrading uric acid to lower uric acid levels, characterized in that, The control system includes: A first functional gene, the first functional gene encoding a first functional protein capable of degrading uric acid, the first functional protein comprising one or more sequences as shown in or having at least 85% identity with SEQ ID NO: 1-10; A second functional gene, which encodes a second functional protein capable of transporting uric acid, the second functional protein comprising one or more sequences as shown in or having at least 85% identity with SEQ ID NO: 11-15; A first promoter, wherein the first promoter is used to regulate the expression of the first functional gene and / or the second functional gene; and A first terminator is used to terminate the expression of the first functional gene and / or the second functional gene.

2. The control system according to claim 1, characterized in that, The control system also includes: A second promoter, used to regulate the expression of the first functional gene and / or the second functional gene; and A second terminator, used to terminate the expression of the first functional gene and / or the second functional gene. Optionally, the second promoter and the first promoter are constitutive promoters.

3. The control system according to claim 1 or 2, characterized in that, The control system specifically includes: The first promoter is used to regulate the expression of the first functional gene; The first terminator is used to terminate the expression of the first functional gene; The second promoter, which regulates the expression of the second functional gene; and The second terminator is used to terminate the expression of the second functional gene. The first promoter and the second promoter respectively comprise any sequence selected from SEQ ID NO: 16-23 or any sequence having at least 85% identity with it. The first promoter preferably comprises the sequence shown in SEQ ID NO: 16 or any sequence having at least 85% identity with it. The second promoter preferably comprises any sequence shown in SEQ ID NO: 17-23 or any sequence having at least 85% identity with it. The first terminator and the second terminator each comprise any sequence selected from SEQ ID NO: 24-26. The first terminator preferably comprises the sequence shown in SEQ ID NO: 24 or SEQ ID NO: 26, more preferably the sequence shown in SEQ ID NO:

26. The second terminator preferably comprises the sequence shown in SEQ ID NO:

25.

4. The control system according to claim 3, characterized in that, The control system includes: The first functional gene encoding the first functional protein, wherein the first functional protein comprises any one of the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 6-10, preferably comprising the sequence shown in SEQ ID NO: 2; The second functional gene encoding the second functional protein, the second functional protein comprising the sequence shown in SEQ ID NO: 15; The first promoter comprises the sequence shown in SEQ ID NO: 16; The first terminator comprises the sequence shown in SEQ ID NO: 24 or SEQ ID NO: 26, preferably the sequence shown in SEQ ID NO: 26; The second promoter comprises any of the sequences shown in SEQ ID NO: 18-23, preferably the sequences shown in SEQ ID NO: 18 or SEQ ID NO: 23; and The second terminator comprises the sequence shown in SEQ ID NO:

25.

5. The control system according to claim 4, characterized in that, The first functional protein comprises the sequence shown in SEQ ID NO: 2; The first terminator comprises the sequence shown in SEQ ID NO: 26; and The second promoter contains the sequence shown in SEQ ID NO: 18 or SEQ ID NO:

23.

6. An expression vector comprising a regulatory system as described in any one of claims 1 to 5 for degrading uric acid to lower uric acid levels. Optionally, the expression vector is a eukaryotic expression vector.

7. A recombinant microorganism comprising a regulatory system for degrading uric acid to lower uric acid levels as described in any one of claims 1 to 5, or an expression vector as described in claim 6. Optionally, the microorganism is a yeast (Saccharomyces sp.), preferably Saccharomyces cerevisiae, and more preferably Saccharomyces cerevisiae var. boulardii.

8. A composition for degrading uric acid to lower uric acid levels, comprising the recombinant microorganisms as described in claim 7 and / or their metabolites and pharmaceutically or food-grade excipients. Optionally, the microorganisms in the composition remain active. Alternatively, the composition may be administered orally.

9. The use of the recombinant microorganism according to claim 7 or the composition according to claim 8 in the preparation of food, health products, food additives or pharmaceuticals for degrading uric acid to lower uric acid levels.

10. A pharmaceutical product or health supplement for degrading uric acid to lower uric acid levels, comprising the recombinant microorganisms according to claim 7 and / or their metabolites, or the composition according to claim 8. Optionally, the medicine or health product may be in the form of tablets, capsules, granules, pills, powders, gels, or solutions.

11. A pharmaceutical remedy for treating hyperuricemia-related diseases, comprising the recombinant microorganism of claim 7 and / or its metabolites or the composition of claim 8.

12. The use of the recombinant microorganism according to claim 7 or the composition according to claim 8 in the preparation of a medicament for treating hyperuricemia-related diseases. Optionally, the hyperuricemia-related diseases include: Hyperuricemia, gout, uric acid stones, acute and chronic uric acid nephropathy.

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