Recombinant enterobacter and use thereof in degradation of tyrosine and phenylalanine

Abstract

The present invention relates to the technical field of biology. Disclosed are recombinant enterobacter and a use thereof in degradation of tyrosine and phenylalanine. According to the present invention, enzyme molecules in nature that have both phenylalanine and tyrosine degradation activities are collected by means of gene search, and the phenylalanine and tyrosine degradation activities of the enzyme molecules are tested in vitro; enzyme molecules having excellent activities are obtained by screening; the selected enzyme molecules are transferred into enterobacter, engineered bacteria highly expressing the enzyme molecules and having the ability to efficiently degrade phenylalanine and tyrosine are further selected, and then protein tags are introduced to improve the degradation activities. Finally, an optimal structure is selected by means of gastrointestinal fluid tolerance experiments, an engineered bacterium having degradation activities far higher than the existing degradation activities is obtained, and the engineered bacterium is expected to be used for preparing a drug for treating phenylketonuria and tyrosinase.

Description

A recombinant enterobacterium and its application in degrading tyrosine and phenylalanine Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant enterobacterium and application thereof in degrading tyrosine and phenylalanine. Background Art

[0002] Phenylketonuria (PKU) is the most common inborn error in amino acid metabolism. It is an autosomal recessive genetic disorder caused by mutations in the phenylalanine hydroxylase gene, which reduces enzyme activity and leads to the accumulation of phenylalanine and its metabolites in the body. Symptoms typically begin 3-6 months after birth, with clinical manifestations including intellectual and motor developmental delays, decreased skin and hair pigmentation, and foul-smelling urine. If left untreated, high levels of phenylalanine accumulation can lead to serious medical problems, such as epileptic seizures and intellectual disability.

[0003] Currently, most common treatments for phenylketonuria (PKU) are chemical drugs, which have drawbacks such as high toxicity and side effects and the development of drug resistance. Among biological drugs, the current effective treatment for PKU is injection of PEG-modified recombinant PAL protein (PEG-PAL, polyethylene glycol-conjugated phenylalanine ammonia lyase), which can reduce phenylalanine concentrations in human plasma to a safer level. However, PEG-PAL can cause a severe immune response. Unlike invasive PEG-PAL formulations and treatments, oral PAL formulations have also been studied both domestically and internationally. While oral delivery offers the advantage of avoiding immune rejection, it also carries significant drawbacks: oral PAL is degraded by proteases such as trypsin, pepsin, and chymotrypsin in the gastrointestinal tract. Whether using invasive injections or oral protein formulations, the primary goal is to improve the catalytic activity and stability of the PAL protein while reducing drug dosage. Therefore, the targeted modification of native PAL is particularly important.

[0004] Tyrosinemia is caused by enzyme defects in the tyrosine metabolism pathway, leading to elevated plasma tyrosine concentrations. Enzyme defects at different steps can lead to a variety of clinically diverse diseases, including damage to multiple organs such as the brain, liver, kidneys, and bones, with poor prognosis and high mortality and disability rates. Tyrosine is derived from both dietary intake and endogenous synthesis. The substrate for endogenous synthesis is the essential amino acid tyrosine, which is derived from food.

[0005] Tyrosinemia type I, also known as hepatorenal tyrosinemia (HT-1), is caused by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH) and is characterized by liver, kidney, and peripheral neuropathy. HT-1 affects males and females equally, with an overall incidence of 1 / 2,000 to 1 / 100,000. The mutation carrier frequency in the US population is 1 / 150 to 1 / 100. Due to a founder effect, the incidence of HT-1 in live births is approximately 1 / 74,000 in Scandinavia and approximately 1 / 60,000 in Finland and Norway. Furthermore, in Quebec, Canada, the incidence is approximately 1 / 16,000 live births, with a carrier frequency of approximately 1 / 66. The estimated prevalence in the Saguenay-Lac Saint-Jean region of Quebec is 1 / 1,850. Based on the age of onset of HT-1 patients, the disease is clinically divided into acute (onset age <2 months), subacute (onset age 2-6 months), and chronic (onset age >6 months). Acute patients often develop the disease within 2 months of birth, with rapid deterioration of their condition, and often die from liver failure between 3 and 9 months of age. The subacute form is similar to the acute form, but symptoms appear between 2 and 6 months of age. The chronic form usually develops after 6 months of age, with clinical manifestations primarily characterized by progressive liver and kidney damage. As the disease progresses, it can eventually lead to cirrhosis, Fanconi syndrome, and even rickets. FAH enzyme deficiency leads to the accumulation of toxic metabolites, including fumarylacetoacetate (FAA) and maleylacetoacetate. Elevated levels of the upstream metabolites tyrosine and 4-hydroxyphenylpyruvate lead to further accumulation of the intermediate metabolite maleylacetoacetate, stimulating a secondary metabolic pathway that results in increased levels of the secondary metabolites succinylacetoacetate and succinylacetone (SA). These secondary metabolites can bind to sulfhydryl groups on proteins and are the primary cause of liver and kidney damage. Without treatment, most patients with HT-1 die from acute, severe liver and kidney failure in early infancy. Tyrosinemia type II, caused by a deficiency in tyrosine aminotransferase (TAT), is characterized by corneal thickening, palmoplantar keratosis, and developmental delay. Tyrosinemia type III, an extremely rare condition caused by a deficiency in 4-hydroxyphenylpyruvate dioxygenase (HPPD), presents primarily with neuropsychiatric symptoms.

[0006] Currently, the only marketed drug for tyrosinemia is nitisinone (NTBC). NTBC is a competitive inhibitor of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), inhibiting the normal catabolism of tyrosine in HT-1 patients, preventing the accumulation of toxic metabolites, thereby preventing liver and kidney damage and improving survival. During nitisinone treatment, patients must restrict their dietary intake of tyrosine and tyrosine. Common adverse reactions reported in studies include thrombocytopenia, leukopenia, and visual system symptoms, including conjunctivitis, corneal opacity, keratitis, and photophobia. However, because NTBC is a 4-HPPD inhibitor, it inhibits the metabolism of tyrosine to 4-HPPD, leading to elevated blood tyrosine levels. If blood tyrosine levels exceed 500 μmol / L, protein intake should be controlled and a tyrosine-free diet or a tyrosine-enriched nutritional powder should be administered. Otherwise, elevated blood tyrosine levels can lead to corneal damage.

[0007] Other treatments for HT-1 include liver transplantation and gene therapy. Liver transplantation has been used for over 20 years to treat HT-1, significantly improving patients' liver, kidney, and neurological symptoms. In recent years, the number of patients requiring liver transplants has gradually decreased with the application of NTBC. However, after liver transplantation, children still have an approximately 5%-10% mortality rate and require lifelong immunosuppressive therapy. This approach is limited by surgical conditions and the limited availability of donors, making clinical application difficult. Therefore, liver transplantation is considered an option only for children with severe liver failure who are refractory to nitisinone or who cannot receive nitisinone for other reasons, and who have evidence of liver malignancy. Gene therapy is still in the animal research stage.

[0008] The present invention aims to treat the above diseases from the perspective of food-borne metabolism by developing living microbial drugs. There are currently no related reports.

[0009] Summary of the Invention

[0010] To address these issues, the present invention provides a recombinant Enterobacterium that efficiently expresses the phenylalanine and tyrosine ammonia lyases PcXAL from Phanerochaete chrysosporium, maintaining the degradation activity of the entire bacterium. Furthermore, gastrointestinal fluid tolerance experiments were conducted on engineered strains with different protein tags, identifying constructs with optimal phenylalanine and tyrosine degradation, laying the foundation for the development of therapeutics for phenylketonuria and tyrosinemia.

[0011] The first object of the present invention is to provide a use of a recombinant Enterobacter in the preparation of a tyrosine and / or phenylalanine degradation product, wherein the recombinant Enterobacter heterologously expresses the amino acid sequence shown in SEQ ID NO.1 and expresses a protein tag fused to the N-terminus of the amino acid sequence.

[0012] Furthermore, the protein tag is selected from SUMO tag, GST tag or GroE tag.

[0013] Furthermore, the nucleotide sequence of the SUMO tag is shown in SEQ ID NO.7; the nucleotide sequence of the GST tag is shown in SEQ ID NO.8; and the nucleotide sequence of the GroE tag is shown in SEQ ID NO.9.

[0014] Furthermore, the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO.2.

[0015] Furthermore, Escherichia coli was used as the starting strain.

[0016] Furthermore, pGEX-4T-2 was used as a vector.

[0017] Furthermore, the degraded tyrosine and / or phenylalanine product is a drug for treating or preventing phenylketonuria and / or tyrosinemia.

[0018] Furthermore, the degraded tyrosine and / or phenylalanine product is an oral preparation.

[0019] A second object of the present invention is to provide a recombinant Enterobacter, which heterologously expresses the amino acid sequence shown in SEQ ID NO.1 and expresses a protein tag fused to the N-terminus of the amino acid sequence, wherein the protein tag is selected from a SUMO tag, a GST tag or a GroE tag.

[0020] Furthermore, Enterobacteriaceae include Escherichia coli.

[0021] The third object of the present invention is to provide a method for constructing the above-mentioned recombinant Enterobacter, comprising the following steps:

[0022] S1. Fusing the protein tag to the N-terminus of the amino acid sequence shown in SEQ ID NO. 1 and ligating it to the vector pGEX-4T-2 to obtain a recombinant plasmid;

[0023] S2. Introduce the recombinant plasmid obtained in S1 into an Enterobacter host to obtain the above-mentioned recombinant Enterobacter.

[0024] The fourth object of the present invention is to provide a microbial agent for degrading phenylalanine and / or tyrosine in vivo or in vitro, comprising the above-mentioned recombinant Enterobacter.

[0025] Furthermore, the microbial agent is a liquid agent or a solid agent.

[0026] Beneficial effects of the present invention:

[0027] (1) The present invention obtains a recombinant Enterobacter with high whole-cell activity through macroscreening technology, and further improves the activity by fusion expression of protein tags. It is found that it not only exhibits significantly higher phenylalanine or tyrosine degradation activity than other phenylalanine ammonia lyases and tyrosine ammonia lyases, but also has no significant decrease in activity under simulated intestinal fluid and simulated gastric fluid conditions, thereby effectively degrading phenylalanine and tyrosine to prevent their accumulation in the body.

[0028] (2) The present invention utilizes a biotherapy with minimal toxic side effects, low resistance to drug resistance, and stable and long-lasting properties. Enterobacteriaceae are selected as the expression host, into which vectors containing phenylalanine and tyrosine ammonia lyase are transferred, and the activity of phenylalanine and tyrosine ammonia lyase is enhanced by adding protein tags. In vivo experiments have demonstrated that the engineered bacteria possess strong resistance and tolerance to the gastrointestinal digestive system. The enzymes can degrade food-borne phenylalanine and tyrosine ingested in the gastrointestinal tract. The bacteria can be prepared into a bacterial agent or, after combining with other excipients, applied to degrade phenylalanine and tyrosine in the body, achieving the purpose of treating phenylketonuria and tyrosinemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows the activity test results of the purified protein.

[0030] Figure 2 shows the results of whole-cell activity detection after phenylalanine ammonia lyase and tyrosine ammonia lyase from different sources were introduced into Escherichia coli.

[0031] Figure 3 shows the effect of fusion expression of different protein tags on the degradation activity of the whole bacteria.

[0032] Figure 4 shows the results of the intestinal fluid tolerance experiment.

[0033] Figure 5 shows the results of the gastric juice tolerance experiment.

[0034] FIG6 shows the basal body weight, food intake and blood Phe levels of mice.

[0035] FIG7 shows the changes in body weight and blood Phe levels of mice after the phenylalanine-deficient feed was replaced with a normal feed.

[0036] FIG8 shows the grouping of mice after being fed with a phenylalanine-deficient diet.

[0037] FIG9 shows the changes in blood Phe levels in PKU mice after a single oral administration of Phe.

[0038] FIG10 shows the effect of a single administration of PcXAL engineered bacteria on the blood Phe level in PKU mice after oral administration of Phe.

[0039] FIG11 shows the effect of continuous administration of PcXAL engineered bacteria on the blood Phe level in PKU mice after oral administration of Phe. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0041] The detection methods involved in the following embodiments are:

[0042] (1) Tyrosine detection method: Tyrosine was used as the reaction substrate. By detecting the reduction of tyrosine in the reaction system, a liquid chromatography detection method was established and optimized. The detection was performed using a Shimadzu high-performance liquid chromatography LC-2050C. The detection conditions and parameters were as follows:

[0043] Chromatographic column: SHIMADZU C18 column (250 mm × 4.6 mm, 5 μm);

[0044] Mobile phase: 1% acetic acid and acetonitrile;

[0045] Column temperature: 40°C;

[0046] Flow rate: 0.8 mL / min

[0047] Gradient elution:

[0048] 0-8 min, 1.5% acetic acid-acetonitrile (95:5) gradually changed to 1.5% acetic acid-acetonitrile (0:100);

[0049] 8-13 minutes, maintain 1.5% acetic acid-acetonitrile (0:100)

[0050] 13-14 minutes, 1.5% acetic acid-acetonitrile (0:100) gradually changed to 1.5% acetic acid-acetonitrile (95:5);

[0051] 14-23 minutes, maintain 1.5% acetic acid-acetonitrile (95:5)

[0052] Detection: UV, wavelength 280nm

[0053] Injection volume: 10 μL

[0054] Phenylalanine detection method: Phenylalanine was used as the reaction substrate. By detecting the reduction of phenylalanine in the reaction system, a liquid chromatography detection method was established and optimized. This method can achieve the simultaneous detection of phenylalanine and cinnamic acid. The detection was performed using a Shimadzu LC-2050C high-performance liquid chromatography. The detection conditions and parameters are as follows:

[0055] Chromatographic column: Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm);

[0056] Mobile phase: 1% acetic acid and acetonitrile;

[0057] Column temperature: 40°C;

[0058] Flow rate: 0.8 mL / min;

[0059] Gradient elution:

[0060] 0-8 min, 1% acetic acid-acetonitrile (95:5) gradually changed to 1.5% acetic acid-acetonitrile (0:100);

[0061] 8-13 minutes, maintain 1% acetic acid-acetonitrile (0:100);

[0062] 13-14 minutes, 1% acetic acid-acetonitrile (0:100) gradually changed to 1.5% acetic acid-acetonitrile (95:5);

[0063] 14-23 minutes, maintain 1% acetic acid-acetonitrile (95:5).

[0064] Detection: UV, wavelength 260nm;

[0065] Injection volume: 10 μL.

[0066] (2) The conditions for the detection of the degradation activity of phenylalanine and tyrosine ammonia lyase in whole cells were as follows: reaction time 3 h, 37°C water bath, substrate 1 mM Phe / Tyr, cell volume 1*10 10 CFU.

[0067] The materials involved in the following examples are as follows:

[0068] SOC medium: 2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM D-glucose, adjusted to pH 7.5.

[0069] Simulated gastric fluid and intestinal fluid were purchased from Nanjing Yuanzhixin Biotechnology Co., Ltd. and stored in a -20℃ refrigerator.

[0070] Plasmid pGEX-4T-2 and Escherichia coli EcN1917 were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0071] The sequences involved in the following examples are as follows:

[0072] The amino acid sequence encoding phenylalanine and tyrosine ammonia lyase PcXAL is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2;

[0073] The amino acid sequence encoding tyrosine ammonia lyase SeSam8 is shown in SEQ ID NO.3;

[0074] The amino acid sequence encoding tyrosine ammonia lyase TcPAL is shown in SEQ ID NO.4;

[0075] The amino acid sequence encoding phenylalanine ammonia lyase AVPAL is shown in SEQ ID NO.5;

[0076] The amino acid sequence encoding phenylalanine ammonia lyase ZmPAL is shown in SEQ ID NO.6;

[0077] The nucleotide sequence encoding the SUMO tag is shown in SEQ ID NO.7;

[0078] The nucleotide sequence encoding the GST tag is shown in SEQ ID NO.8;

[0079] The nucleotide sequence encoding the GroE tag is shown in SEQ ID NO.9.

[0080] Example 1 Screening of phenylalanine and tyrosine ammonia lyases and study of their tyrosine degradation abilities

[0081] Using database-based macroscreening technology, we conducted virtual screening and functional prediction of phenylalanine and tyrosine ammonia lyases from various databases, including NCBI, PDB, and Uniprot. Phenylalanine and tyrosine ammonia lyases with high scores in whole-gene synthesis were expressed in vitro and their activity verified. Enzymes with high activity after purification were selected (Figure 1). The five selected enzymes were recombinantly expressed using plasmid pGEX-4T-2 as the expression vector, and primers designed based on its nucleotide sequence were amplified. The target genes were amplified by PCR and then ligated to the expression vector using enzyme digestion and ligation. After sequence verification, fermentation testing was performed.

[0082] The details are as follows: After the plasmid pGEX-4T-2 was digested, it was purified and recovered according to the instructions of the Norwegian gel recovery kit (DC301-01). Using primers PcXAL-F and PcXAL-R and pUC57-PcXAL as templates, PCR amplification was used to obtain the PcXAL fragment, which was then recovered and purified by gel (DC301-01); using primers SeSam8-F and SeSam8-R and pUC57-SeSam8 as templates, PCR amplification was used to obtain the SeSam8 fragment, which was then recovered and purified by gel (DC301-01); using primers TcPAL-F and TcPAL-R and pUC57-TcPAL as templates, PCR amplification was used to obtain the TcPAL fragment, which was then recovered and purified by gel (DC301-01); using primers AVPAL-F and AVPAL-R and pUC57-AVPAL as templates, PCR amplification was used to obtain the AVPAL fragment, which was then recovered and purified by gel The ZmPAL fragment was amplified by PCR using primers ZmPAL-F and ZmPAL-R and pUC57-ZmPAL as templates. The fragment was recovered and purified on gel (DC301-01). The purified fragments were ligated using T4 DNA ligase and electroporated into Escherichia coli EcN1917. The following positive clones were obtained after sequencing verification: pGEX-4T-2-PcXAL, pGEX-4T-2-SeSam8, pGEX-4T-2-TcPAL, pGEX-4T-2-AVPAL, and pGEX-4T-2-ZmPAL. Single positive clones were selected to prepare seed solution, which was fermented, induced, and the activities of phenylalanine and tyrosine ammonia lyase were determined. The results are shown in Figure 2.

[0083] As shown in Figures 1-2, the purified PcXAL, TcPAL, and SeSam8 proteins all have high Tyr degradation activity, PcXAL, AVPAL, and ZmPAL have high phenylalanine degradation activity, and PcXAL has both phenylalanine and tyrosine degradation activity. Possibly due to differences in protein properties, there are large differences in their expression levels, solubility, and activity within the bacteria, so there are also large differences in whole-cell activity under the same CFU conditions. Figure 2 shows that by performing whole-cell activity tests on the five screened phenylalanine and tyrosine ammonia lyases, the enzyme obtained by expressing the Phanerochaete chrysosporium encoding gene (named PcXAL, amino acid sequence shown in SEQ ID NO.1, nucleotide sequence shown in SEQ ID NO.2) in Enterobacteriaceae has high activity and can efficiently degrade phenylalanine and tyrosine simultaneously.

[0084] Table 1 Amplification primers

[0085] Example 2 Expression element optimization

[0086] Based on the results of Example 1, PcXAL was the primary research target. To further enhance the activity of the engineered strains, different protein tags were fused to the N-terminus of the PcXAL fragment using a (G4S)*3 ligation. The engineered strains expressing SUMO, GST, and GroE tags were named PcXAL-1, PcXAL-2, and PcXAL-3, respectively. The results are shown in Figure 3.

[0087] Among them, the tyrosine degradation rate of the strain PcXAL without adding protein tags was 26.5%; after adding the SUMO tag, the tyrosine degradation rate of PcXAL-1 was 78%, an increase of 190% compared with PcXAL; after adding the GST tag, the tyrosine degradation rate of PcXAL-2 was 52%, an increase of 96% compared with PcXAL; after adding the GroE tag, the tyrosine degradation rate of PcXAL-3 was 68.5%, an increase of 158% compared with PcXAL.

[0088] The phenylalanine degradation rate of the strain PcXAL without protein tag was 62%; after adding the SUMO tag, the phenylalanine degradation rate of PcXAL-1 was 93.5%, an increase of 51% compared with PcXAL; after adding the GST tag, the phenylalanine degradation rate of PcXAL-2 was 76.5%, an increase of 23% compared with PcXAL; after adding the GroE tag, the phenylalanine degradation rate of PcXAL-3 was 82%, an increase of 32% compared with PcXAL.

[0089] In summary, PcXAL-1, PcXAL-2, and PcXAL-3 have excellent phenylalanine and tyrosine degradation activities and can be used as candidate strains for further study.

[0090] Example 3 Artificial gastrointestinal fluid resistance test of strains

[0091] 1. Material preparation:

[0092] (1) Simulated gastric juice and intestinal juice: Thaw the simulated gastric juice and intestinal juice in a refrigerator at 4°C.

[0093] (2) Preparation of bacterial solution:

[0094] The strains PcXAL, PcXAL-1, PcXAL-2, and PcXAL-3 of Example 2 were inoculated into SOC liquid culture medium. When the cells grew to OD = 0.6, isopropyl-β-D-thiogalactoside was added to induce protein expression overnight. The next day, the cells were collected by centrifugation, washed three times with phosphate-buffered saline, and resuspended with a protective agent for use in gastrointestinal fluid tolerance experiments.

[0095] 2. Gastrointestinal fluid tolerance test operation

[0096] Artificial gastric juice / artificial intestinal juice was inoculated into the heavy suspension of bacteria containing a protective agent. After reacting for 30, 60, 90, 120, 150, and 180 minutes, the degradation rates of phenylalanine and tyrosine were determined by high performance liquid chromatography.

[0097] The results are shown in Figures 4-5, where Figure 4 shows 1*10 10 Figure 5 shows the degradation rates of phenylalanine and tyrosine after the CFU bacteria (containing protective agent) were mixed with intestinal fluid (pH = 6.8) at a ratio of 1:1. 10 Phenylalanine and tyrosine degradation rates after a 1:1 mixture of CFU bacteria (containing a protective agent) and gastric fluid (pH = 2). It can be seen that the engineered bacterium PcXAL-1 performed better in the tolerance test of artificial gastrointestinal fluid, with almost no significant decrease in degradation rate compared to Example 2 under these conditions, indicating that it is more suitable for degrading phenylalanine and tyrosine in vivo, thereby achieving the purpose of treating or alleviating phenylketonuria and tyrosinemia.

[0098] Example 4: Drug Efficacy Study in Disease Animal Models

[0099] PcXAL-1 in Example 3 was selected as a candidate strain and named PcXAL engineered bacteria for the next step of drug efficacy study in disease animal models.

[0100] Experimental Animals: All experimental animals were purchased from Southern Model Organisms. Ten male PKU mice aged 8-16 weeks were selected and fed a special diet (phenylalanine-deficient diet). They were divided into two groups of five mice based on body weight and blood phenylalanine levels. One group received a blank control bacterial solution, while the other group received a PcXAL engineered bacterial solution. The activity of the PcXAL engineered bacterial solution was evaluated in the animal model using a single daily and bi-daily dose.

[0101] Experimental Design: PKU mice were gavaged once (0.4 mL) with the engineered bacteria solution at 4:00 PM the day before. The following day, after baseline blood sampling, each group was simultaneously gavaged with a mixture of phenylalanine (approximately 12.5 mg / kg, with an actual gavage volume of 0.1 mL, containing 0.25 mg of phenylalanine) and either the engineered bacteria solution sample or a blank control solution sample (0.3 mL) (a total gavage volume of 0.4 mL, the maximum gavage volume for mice of this weight). Tail tip blood samples were collected 2 and 4 hours thereafter, and blood phenylalanine levels were measured by fluorescence spectrophotometry.

[0102] 1. General Condition of PKU Mice and Monitoring of Phenylalanine Levels in Blood

[0103] The normal control group consisted of 8-week-old normal male C57 mice fed a normal diet, while the disease model group consisted of 8-16-week-old male PKU mice fed a phenylalanine-deficient diet (special diet). After 2 weeks of feeding, changes in body weight, food intake, water intake, and random blood phenylalanine levels were monitored. The results are shown in Figure 6A. Compared with normal mice (C57), PKU mice had lower body weight and slower weight gain; as shown in Figure 6B, compared with C57 mice, PKU mice had relatively less daily food and water intake; the random blood phenylalanine level of PKU mice fed a special diet was around 100 μM, while the blood phenylalanine level of normal C57 mice was around 150 μM (Figure 6C).

[0104] After monitoring the basal blood phenylalanine level, the PKU mice were fed a normal diet to induce the formation of a disease model. After 8 days of feeding, the phenylalanine-deficient diet was replaced with a normal diet, and the changes in the body weight and blood phenylalanine levels of the PKU mice were monitored. As shown in Figure 7A, compared with C57 mice, the PKU mice that were fed a normal diet showed significant weight gain within 8 days (an average weight gain of about 4g); random blood samples were taken to detect the phenylalanine level in the blood. As shown in Figure 7B, due to mutations in the metabolic enzyme gene, the PKU mice had significantly increased phenylalanine levels in their blood, reaching about 2800μM. Compared with when they were fed a diet deficient in diet, the phenylalanine in the blood increased by nearly 30 times. This result shows that feeding with a normal diet can cause PKU mice to develop more severe hyperphenylalaninemia.

[0105] 2. PKU mice were fed with a phenylalanine-deficient diet and then screened and grouped

[0106] Based on previous monitoring of the general condition of PKU mice and the effects of feed change on blood phenylalanine levels, a single oral gavage of phenylalanine was performed to simulate the elevated blood phenylalanine levels induced by feed feeding. The effect of the engineered bacteria samples on reducing blood phenylalanine levels in PKU mice after oral administration of phenylalanine was investigated. First, PKU mice fed a normal diet were switched to a special phenylalanine-deficient diet. This resulted in a modest weight loss, with a mean of approximately 20 g (Figure 8A), and blood phenylalanine levels returned to normal, approximately 130-150 μM (Figure 8B). Subsequently, 10 PKU mice with a broad range of weights were divided into two groups (Group 1 and Group 2) based on their weight and blood phenylalanine levels for subsequent experimental studies.

[0107] 3. Study on the changes of phenylalanine levels in the blood of PKU mice after a single oral administration of phenylalanine

[0108] The two groups of PKU mice described above were gavaged with 25 mg / kg and 50 mg / kg phenylalanine solutions, respectively. Changes in blood phenylalanine levels were monitored 1 and 2 hours after oral administration of phenylalanine. The results, shown in Figure 9, show that after a single oral administration of 25 mg / kg and 50 mg / kg phenylalanine to PKU mice, blood phenylalanine levels increased significantly by 3-5 times within 1 and 2 hours. This result demonstrates that a single oral administration of phenylalanine can elevate blood phenylalanine levels in PKU mice, with the increase occurring slowly over 1-2 hours. Therefore, a single oral administration of phenylalanine, combined with co-administration of the engineered bacterial solution or prior administration, can be used to study the inhibitory effect of engineered bacteria on the absorption of oral phenylalanine into the blood of PKU mice.

[0109] 4. Effect of a single administration of PcXAL engineered bacteria on blood phenylalanine levels in PKU mice after oral administration of phenylalanine

[0110] As shown in Figure 10, compared with the blank control group (model group, given an equal volume of control bacterial solution 1917), a single oral administration of 0.3 mL of the PcXAL engineered bacteria solution significantly reduced blood phenylalanine levels in PKU mice two hours after oral administration of 0.25 mg of phenylalanine (P<0.05), with a reduction percentage of 35.5%. This reduction effect was maintained four hours later, with a reduction percentage of 19.7%. These results demonstrate that a single administration of the engineered bacteria PcXAL significantly reduced the elevated blood phenylalanine levels in PKU mice following oral administration of 0.25 mg of phenylalanine.

[0111] 5. Effect of continuous administration of PcXAL engineered bacteria on blood phenylalanine levels in PKU mice after oral administration of phenylalanine

[0112] As shown in Figure 11, compared with the blank control group (model group, administered an equal volume of control bacterial solution 1917), the PcXAL engineered bacteria significantly reduced blood phenylalanine levels in PKU mice 2 hours after oral administration of 0.25 mg of phenylalanine (P = 0.06), with a reduction percentage of 47.8%. This significant reduction was maintained 4 hours later (P < 0.05), with a reduction percentage of 61.3%. Compared with a single dose, a 2-day administration of PcXAL significantly inhibited the absorption of oral phenylalanine. These results demonstrate that increasing the oral dose of the engineered bacteria PcXAL significantly increases its inhibitory effect on PKU mice's absorption of oral phenylalanine, achieving a more pronounced effect than a single dose.

[0113] The results above demonstrate that, in a PKU mouse model, PcXAL engineered bacteria inhibited phenylalanine absorption after a single oral dose, both when administered once and over two days, reducing blood phenylalanine levels. Compared to the blank control group, the inhibition rate reached over 30% two hours after oral administration, and the effect persisted for at least four hours. These results demonstrate that oral administration of PcXAL engineered bacteria in the PKU mouse model can inhibit phenylalanine absorption through the digestive tract, thereby reducing the elevated blood phenylalanine levels following oral administration, thereby achieving the goal of treating phenylketonuria.

[0114] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a recombinant Enterobacter in preparing a product that degrades tyrosine and / or phenylalanine, characterized in that: The recombinant Enterobacter heterologously expresses the amino acid sequence shown in SEQ ID NO.1, and a protein tag is fused and expressed at the N-terminus of the amino acid sequence.

2. The use according to claim 1, characterized in that: The protein tag is selected from SUMO tag, GST tag or GroE tag.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the SUMO tag is shown in SEQ ID NO.7; the nucleotide sequence of the GST tag is shown in SEQ ID NO.8; and the nucleotide sequence of the GroE tag is shown in SEQ ID NO.

9.

4. The use according to claim 1, characterized in that: The nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO.

2.

5. The use according to claim 1, characterized in that: Escherichia coli was used as the starting strain.

6. The use according to claim 1, characterized in that: The degraded tyrosine and / or phenylalanine products are drugs for treating or preventing phenylketonuria and / or tyrosinemia.

7. The use according to claim 1, characterized in that: The degraded tyrosine and / or phenylalanine product is an oral preparation.

8. A recombinant Enterobacter, characterized in that: The recombinant Enterobacter heterologously expresses the amino acid sequence shown in SEQ ID NO.1, and expresses a protein tag shown in any one of SEQ ID NOs.7-9 at the N-terminus of the amino acid sequence.

9. The method for constructing a recombinant Enterobacter according to claim 8, characterized in that: The following steps are involved: S1. Fusion of the protein tag to the N-terminus of the amino acid sequence shown in SEQ ID NO.1 and ligation to the expression vector to obtain a recombinant plasmid; S2. Introducing the recombinant plasmid obtained in S1 into an Enterobacter host to obtain the recombinant Enterobacter.

10. A microbial agent for degrading phenylalanine and / or tyrosine, characterized in that: Contains the recombinant Enterobacter as claimed in claim 8.

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