CN116790468A - Recombinant enterobacteria and application thereof in degradation of tyrosine and phenylalanine - Google Patents

Abstract

The invention discloses recombinant enterobacteria and application thereof in degrading tyrosine and phenylalanine, belonging to the technical field of biology. According to the invention, through a gene searching means, enzyme molecules with phenylalanine and tyrosine degradation activities in nature are collected, in-vitro activity detection is carried out on the phenylalanine and tyrosine degradation activities, enzyme molecules with excellent activities are obtained through screening, the screened enzyme molecules are transferred into enterobacteria, engineering bacteria with high expression of the enzyme molecules and high efficiency of degrading phenylalanine and tyrosine are further selected, and then protein tags are introduced for improving the degradation activities. Finally, the optimal structure is obtained through screening by a gastric juice tolerance experiment, and an engineering bacterium with far higher degradation activity than the prior art is obtained, which is expected to be used for preparing the phenylketonuria and tyrosinemia treatment medicine.

Description

Recombinant enterobacteria and application thereof in degradation of tyrosine and phenylalanine

Technical Field

The invention belongs to the technical field of biology, and particularly relates to recombinant enterobacteria and application thereof in degrading tyrosine and phenylalanine.

Background

Phenylketonuria (PKU), which is the most common disease among congenital amino acid metabolic disorders, is also an autosomal recessive genetic disease, and is a disease caused by the accumulation of phenylalanine and its metabolites in the body due to the decrease of enzyme activity caused by the mutation of phenylalanine hydroxylase gene. Symptoms generally appear after 3-6 months after birth, and clinical manifestations include mental retardation, movement and development retardation, skin hair pigment reduction, rat urine odor and the like. If not treated in time, high amounts of phenylalanine accumulation can cause more serious medical problems such as seizures, intellectual disabilities, and the like.

At present, most of common medicaments for treating phenylketonuria are chemical medicaments, have the defects of higher toxic and side effects, easiness in generating drug resistance and the like, and in biological medicaments, the specific medicaments for treating PKU at present are recombinant PAL proteins (PEG-PAL, polyethylene glycol conjugated phenylalanine ammonia lyase) modified by PEG, so that the concentration of phenylalanine in human blood plasma can be reduced to a safer concentration level, but the PEG-PAL can cause a more serious immune response; unlike PEG-PAL invasive preparation and treatment method, oral PAL preparation has been studied at home and abroad, and oral delivery has the advantage of no immunological rejection reaction, but has obvious defect that oral PAL can be decomposed by trypsin, pepsin, chymotrypsin and the like in intestines and stomach. Whether the PAL protein is an invasive injection or an oral protein, the primary solution is to improve the catalytic activity and the stability of the PAL protein on the premise of reducing the dosage of medicaments, so that the directional transformation of the natural PAL is particularly important.

Tyrosinemia (tyrosinemia) is a disease in which the concentration of tyrosine in blood plasma is increased due to enzyme deficiency in the metabolic pathway of tyrosine, and the enzyme deficiency in different steps can cause various clinical manifestations, such as damage to multiple organs of brain, liver, kidney, bones, etc., bad prognosis, mortality and disability rate are high. Tyrosine is a source of food-borne essential amino acid tyrosine in human body, and comprises two parts of dietary intake and endogenous synthesis.

Type i tyrosinase, also known as liver and kidney tyrosinase (hepatorenal tyrosinemia, HT-1), is a defect in fumarylacetoacetic acid hydrolase (fumarylacetoacetate hydrolase, FAH) and is characterized by liver, kidney and peripheral neuropathy. HT-1 affects men and women equally in number, with an overall incidence of 1/2000-1/100000. The mutation carrying frequency of the US population is 1/150 to 1/100, and the incidence of live neonates of the type HT-1 of the scandinavia peninsula is about 1/74 000 due to the founder effect, finland and Norway is about 1/60 000. In addition, in Quebec, canada, the incidence of live birth newborns is about 1/16 000, with a frequency of about 1/66. Neonatal prevalence in the Saguenay-Lac Saint-Jean region of Quebec was estimated to be 1/1850. The disease is clinically classified into an acute type (age of onset <2 months), a subacute type (age of onset 2-6 months) and a chronic type (age of onset >6 months) according to the age of onset of HT-1 patients. Acute infants usually develop acute illness within 2 months after birth, and rapidly worsen, usually die from liver failure at 3-9 months after birth. Subacute forms are similar to acute forms, but symptoms occur between 2-6 months after birth. The chronic onset time is usually 6 months after birth, clinical manifestations are mainly progressive liver and kidney damage, and with the development of the disease, cirrhosis and fanconi syndrome can finally result, even rickets appear. Deficiency of the FAH enzyme results in accumulation of toxic metabolites, including fumarylacetoacetic acid (FAA) and maleylacetoacetic acid (maleylacetoacetate). As the upstream metabolite tyrosine (tyrosine), 4-hydroxyphenylpyruvate (4-hydroxyphenylpyruvate) content is increased, the intermediate metabolite maleylacetoacetate is further accumulated, thereby stimulating the generation of a side metabolic pathway, resulting in the increase of the metabolites Succinylacetoacetate (SA) and Succinylacetone (SA). These two paracbolites can bind to thiol groups of proteins and are the main cause of liver and kidney damage. When untreated, most HT-1 patients die in early infancy from acute severe liver and kidney failure. Type ii tyrosinemia, a defect in tyrosine aminotransferase (tyrosine aminotransferase, TAT), is characterized by corneal thickening, palmoplantar keratosis and developmental lag. Type III tyrosinemia, which is very rare, is caused by defects of 4-hydroxyphenylpyruvate dioxygenase (hydroxyphenylpyruvic acid dioxygenase, HPPD) and is mainly manifested by neuropsychiatric symptoms.

Currently, there is only one marketed drug against tyrosinase, nitenpyram (NTBC). NTBC is a competitive inhibitor of 4-hydroxyphenylpyruvate dioxygenase, and can inhibit the conventional catabolism of tyrosine in HT-1 patients, prevent toxic metabolites from accumulating in the body, thereby avoiding the damage of liver and kidney functions and improving the survival rate of the patients. Patients must limit the dietary tyrosine and tyrosine intake during the treatment of nitenpyram. Common adverse reactions reported in the study were thrombocytopenia, leukopenia and vision system disorders including conjunctivitis, corneal haze, keratitis and photophobia. However, since NTBC is a 4-hydroxyphenylpyruvate dioxygenase inhibitor, it inhibits the metabolism of tyrosine into 4-hydroxyphenylpyruvate, so that the blood tyrosine is increased, if the blood tyrosine level exceeds 500 mu mol/L, it is necessary to control protein intake, and a dietary therapy is given to a formula nutritional powder without tyrosine, otherwise, cornea damage is caused by the excessive blood tyrosine level.

Other methods of treatment for HT-1 include liver transplantation and gene therapy. Liver transplantation has been used for over 20 years to treat HT-1, and can significantly improve the symptoms of the liver, kidney and nervous system of patients. Liver transplants have been increasingly required in recent years with the use of NTBC. In addition, the death rate of the infant after the liver transplantation is still about 5% -10%, the immunosuppression treatment is still needed after the operation, and the method is limited by the operation condition and the small number of donors, and has difficulty in clinical application, so the liver transplantation treatment is selected only when the liver function of the infant is seriously exhausted, the treatment of the Nitixiong is ineffective or the basis of the treatment of the Nitixiong and the occurrence of malignant changes of liver tissues is not available for other reasons. Gene therapy is still currently in the animal research phase.

The invention aims at treating the diseases from the food metabolism perspective by developing living microbial medicaments, and no related report exists at present.

Disclosure of Invention

In order to solve the problems, the invention provides a method for efficiently expressing Phanerochaete chrysosporiumPhanerochaete chrysosporium) The recombinant enterobacteria of phenylalanine ammonia acid and tyrosine ammonia lyase PcXAL realize the maintenance of the degradation activity of the whole bacteria. Meanwhile, the engineering strains added with different protein tags are subjected to gastrointestinal fluid tolerance experiments, and the structure with optimal phenylalanine ammonia acid and tyrosine degradation effect is obtained through screening, so that a foundation is laid for preparing phenylketonuria and tyrosinemia treatment drugs.

The first object of the invention is to provide an application of recombinant enterobacteria in preparing products for degrading tyrosine and/or phenylalanine, wherein the recombinant enterobacteria heterologously express an amino acid sequence shown as SEQ ID NO.1, and fusion express a protein tag at the N end of the amino acid sequence.

Further, the protein tag is selected from the group consisting of a SUMO tag, a GST tag, or a GroE tag.

Further, the nucleotide sequence of the SUMO tag is shown as SEQ ID NO. 7; the nucleotide sequence of the GST tag is shown as SEQ ID NO. 8; the nucleotide sequence of GroE tag is shown as SEQ ID NO. 9.

Further, the nucleotide sequence for encoding the amino acid sequence is shown as SEQ ID NO. 2.

Further, escherichia coli is used as an initial strain.

Further, pGEX-4T-2 was used as a vector.

Further, the tyrosine and/or phenylalanine degrading product is a medicament for treating or preventing phenylketonuria and/or tyrosinemia.

Further, the tyrosine and/or phenylalanine degrading product is an oral preparation.

The second object of the present invention is to provide a recombinant enterobacterium which heterologously expresses an amino acid sequence as shown in SEQ ID NO.1 and fusion-expresses a protein tag at the N-terminus of the amino acid sequence, wherein the protein tag is selected from SUMO tag, GST tag or GroE tag.

Further, the E.coli includes E.coli.

The third object of the present invention is to provide a construction method of the recombinant enterobacteria, comprising the steps of:

s1, fusing a protein tag to the N end of an amino acid sequence shown in SEQ ID NO.1, and connecting to a vector pGEX-4T-2 to obtain a recombinant plasmid;

s2, introducing the recombinant plasmid obtained in the S1 into an enterobacter host to obtain the recombinant enterobacter.

The fourth object of the present invention is to provide a microbial agent for in vivo or in vitro degradation of phenylalanine ammonia acid and/or tyrosine, comprising the recombinant enterobacteria described above.

Further, the microbial agent is a liquid microbial agent or a solid microbial agent.

The invention has the beneficial effects that:

the recombinant enterobacteria with high whole-cell activity is obtained by a macro-screening technology, and the activity is further improved by fusing and expressing protein tags, so that the recombinant enterobacteria not only shows the phenylalanine or tyrosine degradation activity remarkably higher than that of other phenylalanine ammonia lyase and tyrosine ammonia lyase, but also has no obvious reduction in the activity under the conditions of simulating intestinal juice and gastric juice, thereby effectively degrading phenylalanine and tyrosine and avoiding accumulation in a human body.

The invention adopts a biological therapy which has small toxic and side effects, is not easy to generate drug resistance, is stable and durable, selects enterobacteria as an expression host, transfers the enterobacteria into a carrier with phenylalanine and tyrosine ammonia lyase, and improves the activity of the phenylalanine and tyrosine ammonia lyase by adding a protein tag. In vivo experiments prove that the engineering bacteria have stronger resistance and tolerance to gastrointestinal digestive systems, and can degrade food-borne phenylalanine and tyrosine ingested in the stomach and the intestines through the enzyme, and can be prepared into a microbial inoculum or be applied to degradation of phenylalanine and tyrosine in vivo after being matched with other auxiliary materials, thereby achieving the aim of treating phenylketonuria and tyrosinemia.

Drawings

FIG. 1 shows the results of the protein activity assay after purification.

FIG. 2 shows the results of detection of the activity of whole cells of E.coli after introduction of phenylalanine ammonia lyase and tyrosine ammonia lyase of different sources.

FIG. 3 shows the effect on whole cell degradation activity after fusion expression of different protein tags.

Fig. 4 shows the results of intestinal juice tolerance test.

FIG. 5 shows the results of gastric juice tolerance experiments.

FIG. 6 shows the basal body weight, feeding and blood Phe levels of mice.

FIG. 7 shows changes in body weight and blood Phe levels of mice after replacement of phenylalanine deficiency diet with normal diet.

FIG. 8 is a grouping of mice fed with phenylalanine deficiency feed.

FIG. 9 shows changes in blood Phe levels after single Phe administration in PKU mice.

FIG. 10 is the effect of single administration of PcXAL engineering bacteria on blood Phe levels after oral administration of Phe to PKU mice.

FIG. 11 shows the effect of continuous administration of PcXAL engineering bacteria on blood Phe levels after oral administration of Phe to PKU mice.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The following examples relate to detection methods:

(1) The tyrosine detection method comprises the following steps: tyrosine is used as a reaction substrate, a liquid chromatography detection method is established and optimized by detecting the reduction condition of tyrosine in a reaction system, and the detection adopts Shimadzu high performance liquid chromatography LC-2050C, and the detection condition parameters are as follows:

chromatographic column: SHIMADZU C18 column (250 mm ×4.6 mm,5 μm);

mobile phase: 1% acetic acid and acetonitrile;

column temperature: 40. the temperature is lower than the temperature;

flow rate: 0.8 mL/min

Gradient elution:

0-8 minutes, 1.5% acetic acid-acetonitrile (95:5) is gradually changed into 1.5% acetic acid-acetonitrile (0:100);

8-13 minutes, 1.5% acetic acid-acetonitrile (0:100)

13-14 minutes, 1.5% acetic acid-acetonitrile (0:100) is gradually changed into 1.5% acetic acid-acetonitrile (95:5);

14-23 minutes, 1.5% acetic acid-acetonitrile (95:5)

And (3) detection: ultraviolet, wavelength 280 nm

Sample injection amount: 10 mu L

Phenylalanine detection method: the method can realize synchronous detection of the phenylalanine and the cinnamic acid by detecting the reduction condition of the phenylalanine in a reaction system, and the detection condition parameters are as follows:

chromatographic column: eclipse XDB-C18 column (250 mm X4.6 mm,5 μm);

mobile phase: 1% acetic acid and acetonitrile;

column temperature: 40. the temperature is lower than the temperature;

flow rate: 0.8 mL/min;

gradient elution:

0-8 minutes, 1% acetic acid-acetonitrile (95:5) is gradually changed into 1.5% acetic acid-acetonitrile (0:100);

8-13 minutes, keeping 1% acetic acid-acetonitrile (0: 100);

13-14 minutes, 1% acetic acid-acetonitrile (0:100) is gradually changed into 1.5% acetic acid-acetonitrile (95:5);

14-23 minutes, 1% acetic acid-acetonitrile (95:5) was maintained.

And (3) detection: ultraviolet, wavelength 260 nm;

sample injection amount: 10 muL.

(2) The degradation activity detection conditions of the whole-cell phenylalanine and tyrosine ammonia lyase are as follows: the reaction time was 3 hours, water bath at 37 ℃, substrate 1 mM Phe/Tyr, cell mass 1X 10 ¹⁰ CFU。

The following examples relate to the following materials:

SOC medium: 2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl ₂ 、10mM MgSO ₄ 20 mM D-glucose, adjusted to pH7.5.

Gastric juice and intestinal juice were simulated: were purchased from Xin-Biotechnology Co., ltd. From Nanjing source and stored in a-20℃refrigerator.

Plasmid pGEX-4T-2, E.coli EcN1917: all purchased from wuhan vast, bio-technology limited.

The sequences involved in the examples below are as follows:

the amino acid sequences of the coded phenylalanine and tyrosine ammonia lyase PcXAL are shown in SEQ ID NO.1, and the nucleotide sequences are shown in SEQ ID NO. 2;

the amino acid sequence of the coded tyrosine ammonia lyase SeSam8 is shown as SEQ ID NO. 3;

the amino acid sequence of the coded tyrosine ammonia lyase TcPAL is shown as SEQ ID NO. 4;

the amino acid sequence of the coded phenylalanine ammonia lyase AVPAL is shown as SEQ ID NO. 5;

the amino acid sequence of the coded phenylalanine ammonia lyase ZmPAL is shown as SEQ ID NO. 6;

the nucleotide sequence for encoding the SUMO tag is shown as SEQ ID NO. 7;

the nucleotide sequence of the coding GST tag is shown as SEQ ID NO. 8;

the nucleotide sequence for encoding GroE tag is shown as SEQ ID NO. 9.

Example 1 screening of phenylalanine and tyrosine Ammonia lyase and investigation of its ability to degrade tyrosine

The macro screening technology based on the database is utilized to carry out virtual screening and function prediction on phenylalanine and tyrosine ammonia lyase with different sources in the databases such as NCBI, PDB, uniprot, the phenylalanine and tyrosine ammonia lyase with higher total gene synthesis score is subjected to in vitro expression and activity verification, and the enzyme with higher enzyme activity after purification is selected, and the result is shown in figure 1. Recombinant expression was performed on the 5 enzymes screened: plasmid pGEX-4T-2 is used as an expression vector, a primer is designed by a nucleotide sequence of the plasmid pGEX-4T-2 for amplification, a target gene is connected to the expression vector through an enzyme digestion connection method after PCR amplification, and fermentation detection is carried out after the sequence is verified to be correct.

The method comprises the following steps: plasmid pGEX-4T-2 was digested and purified according to the instructions of the Novain recovery box (DC 301-01). PCR amplification using primers PcXAL-F and PcXAL-R and pUC57-PcXAL as templates to obtain PcXAL fragments, and gel recovery purification (DC 301-01); PCR amplification using primers SeSam8-F and SeSam8-R and pUC57-SeSam8 as templates to obtain SeSam8 fragment, gel recovery and purification (DC 301-01); PCR amplification to obtain TcPAL fragment and gel recovery purification (DC 301-01) using the primers TcPAL-F and TcPAL-R and pUC57-TcPAL as templates; using the primers AVPAL-F and AVPAL-R and pUC57-AVPAL as templates, PCR amplifying to obtain AVPAL fragments, and recovering and purifying (DC 301-01); PCR amplification using primers ZmPAL-F and ZmPAL-R and pUC57-ZmPAL as templates, obtaining ZmPAL fragments, gel recovery purification (DC 301-01), ligation of the purified fragments using T4 DNA ligase, electrotransformation of E.coli EcN1917, sequencing and verification to obtain the correct positive clone: pGEX-4T-2-PcXAL, pGEX-4T-2-SeSam8, pGEX-4T-2-TcPAL, pGEX-4T-2-AVPAL, pGEX-4T-2-ZmPAL; the positive gram Long Shan clone is selected to prepare seed liquid, and the activity of phenylalanine and tyrosine ammonia lyase is fermented, induced and measured, and the result is shown in figure 2.

As can be seen from FIGS. 1-2, the purified PcXAL, tcPAL, seSam proteins have high Tyr degradation activity, pcXAL, AVPAL,ZmPAL has high phenylalanine degrading activity, and PcXAL has phenylalanine and tyrosine degrading activity. The protein may have a large difference in the expression level, solubility and activity in the cell due to the difference in the properties of the protein, and thus the activity of the whole cell may also have a large difference under the same Cfu condition. FIG. 2 shows the detection of the activity of whole cells of Phanerochaete chrysosporium by screening 5 kinds of phenylalanine and tyrosine ammonia lyasePhanerochaete chrysosporium) The activity of the enzyme obtained by expressing the coding gene (named PcXAL, the amino acid sequence is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO. 2) in enterobacteria is higher, and phenylalanine and tyrosine can be simultaneously and efficiently degraded.

TABLE 1 amplification primers

Example 2 expression element optimization

According to the results of example 1, pcXAL was the main subject. To further increase the activity of the engineered strain, different protein tags were fused to the N-terminus of the PcXAL fragment, each by (G ₄ S) 3, and respectively named as PcXAL-1, pcXAL-2 and PcXAL-3 by constructing fusion expression SUMO label, GST label and GroE label. The results are shown in FIG. 3.

Wherein, the tyrosine degradation rate of the bacterial strain PcXAL without the protein tag is 26.5%; after SUMO label is added, the degradation rate of PcXAL-1 tyrosine is 78%, which is improved by 190% compared with PcXAL; after GST label is added, the degradation rate of PcXAL-2 tyrosine is 52%, which is improved by 96% compared with PcXAL; after GroE label is added, the degradation rate of PcXAL-3 tyrosine is 68.5%, and is 158% higher than that of PcXAL.

The phenylalanine degradation rate of the strain PcXAL without the protein tag is 62%; after SUMO label is added, the degradation rate of PcXAL-1 phenylalanine is 93.5%, which is improved by 51% compared with PcXAL; after GST label is added, the degradation rate of PcXAL-2 phenylalanine is 76.5%, which is improved by 23% compared with PcXAL; after GroE tag is added, the degradation rate of PcXAL-3 phenylalanine is 82%, which is improved by 32% compared with PcXAL.

In conclusion, pcXAL-1, pcXAL-2 and PcXAL-3 have excellent phenylalanine and tyrosine degradation activity, and can be used as candidate strains for further research.

Example 3 Artificial gastrointestinal fluid resistance experiment of Strain

1. Material preparation:

(1) Gastric juice and intestinal juice were simulated: simulated gastric and intestinal fluids were thawed in a refrigerator at 4 ℃.

(2) Preparing bacterial liquid:

strains PcXAL, pcXAL-1, pcXAL-2, and PcXAL-3 of example 2 were inoculated into SOC liquid medium, isopropyl- β -D-thiogalactoside was added when the cells grew to od=0.6, and protein expression was induced overnight; the following day, the cells were collected by centrifugation, washed 3 times with phosphate buffered saline, and resuspended in protectant for gastrointestinal fluid tolerance experiments.

2. Gastrointestinal fluid tolerance test procedure

Absorbing artificial gastric juice/artificial intestinal juice, inoculating into bacterial body weight suspension added with protective agent, reacting for 30, 60, 90, 120, 150, 180 minutes, and measuring phenylalanine and tyrosine degradation rate by high performance liquid chromatography.

The results are shown in FIGS. 4-5, wherein FIG. 4 shows 1X 10 ¹⁰ Phenylalanine and tyrosine degradation rates after CFU cells (containing protectant) were mixed 1:1 with intestinal fluid (ph=6.8), and fig. 5 shows 1×10 ¹⁰ Phenylalanine and tyrosine degradation rate after CFU cells (containing protectant) were mixed with gastric juice (ph=2) 1:1. The engineering bacterium PcXAL-1 is more excellent in tolerance experiments of artificial gastrointestinal fluids, and the degradation rate under the condition is almost not obviously reduced compared with that of the example 2, which indicates that the engineering bacterium PcXAL-1 is more suitable for degrading phenylalanine and tyrosine in vivo, thereby achieving the purpose of treating or relieving phenylketonuria and tyrosinemia.

EXAMPLE 4 research on efficacy of animal models of diseases

PcXAL-1 in example 3 was selected as a candidate strain and designated as PcXAL engineering bacterium for the next disease animal model efficacy study.

Experimental animals: experimental animals were purchased from southern model organisms. Male PKU mice of 8-16 weeks of age were selected, 10 were fed with special feed (phenylalanine-deficient feed), and equally divided into 2 groups of 5 according to body weight and phenylalanine level in blood. The model group was given a blank bacterial solution, the other group was given a PcXAL engineering bacterial solution, and activity evaluation studies were conducted on animal models using the PcXAL engineering bacterial solution once a day and once a day.

Experiment design: on the next day, 4 pm, the test engineering bacteria solution was perfused 1 time (0.4 mL) in PKU mice. After the next day, basal blood samples were collected, phenylalanine was administered to each group by simultaneous gavage (dose: about 12.5. 12.5 mg/kg, actual gavage volume: 0.1mL, phenylalanine content: 0.25 mg) and a mixture of test engineering bacteria liquid sample or blank bacteria liquid sample (0.3. 0.3 mL) (total gavage liquid: 0.4. 0.4 mL, which is the maximum gavage volume of the mice in this weight state), tail blood samples were collected 2 hours and 4 hours later, respectively, and the levels of phenylalanine in the blood were measured by fluorescence spectrophotometry.

Monitoring of phenylalanine levels in the general status and blood of PKU mice

Normal control groups selected 8 week old normal male C57 mice fed normal diet, disease model groups selected 8-16 week old male PKU mice fed phenylalanine deficient diet (special diet). After 2 weeks of feeding, changes in body weight, food intake and water intake and random blood levels of phenylalanine were monitored. The results are shown in fig. 6 a, where PKU mice had lower body weight and a slower body weight gain than normal mice (C57); as shown in fig. 6B, PKU mice consumed relatively less daily and consumed water than C57 mice; the phenylalanine level in the random blood of PKU mice fed special feeds was around 100. Mu.M, and the phenylalanine level in the blood of normal C57 mice was around 150. Mu.M (C in FIG. 6).

After monitoring phenylalanine levels in basal blood, replacement with normal feed feeding prompted PKU mice to develop a disease model. After 8 days of feeding with phenylalanine-deficient feed changed to normal feed, PKU mice were monitored for changes in body weight and blood phenylalanine levels. As shown in fig. 7 a, PKU mice that were replaced with normal diet showed significant weight gain (average weight gain of about 4 g) over 8 days as compared to C57 mice; random blood samples were taken to detect the phenylalanine level in the blood, and as shown in fig. 7B, PKU mice had significantly elevated phenylalanine levels in the blood due to metabolic enzyme gene mutations, which could reach around 2800 μm, approximately 30-fold increase in phenylalanine in the blood compared to when fed in the absence of the feed. The results show that: feeding with normal feed can lead to severe hyperphenylalanine in PKU mice.

PKU mice were fed with phenylalanine-deficient feed and screened for groups

Based on the influence of the early period on the general state monitoring of PKU mice and the replacement of feeds on the phenylalanine level in blood, the method of single gastric lavage of phenylalanine is adopted to simulate the increase of phenylalanine in blood induced by feed feeding, and the reduction effect of a test engineering bacteria sample on the phenylalanine level in blood of PKU mice after oral administration of phenylalanine is examined. Firstly, PKU mice fed with normal feed were again replaced with special feed for phenylalanine deficiency, the body weight thereof was reduced to some extent, the average value was about 20 g (FIG. 8A), and the phenylalanine level in the blood was recovered to the normal level, about 130 to 150. Mu.M (FIG. 8B). Thereafter, 10 PKU mice with more concentrated body weight ranges were divided into two groups (group 1 and group 2) based on body weight and phenylalanine levels in the blood for subsequent experimental study.

Studies of changes in phenylalanine levels in blood after single oral administration of phenylalanine in PKU mice

Using these two groups of PKU mice, the phenylalanine solutions of 25 mg/kg and 50 mg/kg were administered orally, and the changes in the phenylalanine levels in the blood after 1 hour and 2 hours after oral administration of phenylalanine were monitored, and as a result, as shown in FIG. 9, when the PKU mice were given phenylalanine of 25 mg/kg and 50 mg/kg orally at a single time, the phenylalanine levels in the blood could be significantly increased by 3-5 times in 1 hour and 2 hours. The results show that: a single oral administration of phenylalanine causes an increase in phenylalanine levels in the blood of PKU mice, and slowly increases over 1-2 hours. Therefore, the method of singly taking phenylalanine orally can be combined with the methods of taking the engineering bacteria liquid to be tested together or taking the engineering bacteria liquid in advance, and the like, so that the inhibition effect of the engineering bacteria on the absorption and blood of PKU mice by taking phenylalanine orally can be studied.

Effect of single administration of PcXAL engineering bacteria on phenylalanine levels in blood after oral administration of phenylalanine to PKU mice

As shown in fig. 10, compared with the blank control group (model group, the same volume of control bacterial liquid 1917 was administered), the PcXAL engineering bacterial liquid was infused with stomach 0.3 and mL once, and the phenylalanine level in the blood 2 hours after oral administration of 0.25 and mg phenylalanine by PKU mice was significantly reduced (P < 0.05), the reduction percentage was 35.5%, the reduction effect was still maintained after 4 hours, and the reduction percentage was 19.7%. The results show that: the increase of phenylalanine level in blood of PKU mice after oral administration of 0.25 mg phenylalanine can be significantly reduced by single administration of the test engineering bacteria PcXAL.

Effect of continuous administration of PcXAL engineering bacteria on phenylalanine levels in blood after oral administration of phenylalanine in PKU mice

As shown in fig. 11, compared with the blank control group (model group, the same volume of control bacterial liquid 1917 is given), pcXAL engineering bacteria can significantly reduce phenylalanine level (p=0.06) in blood 2 hours after oral administration of phenylalanine of 0.25 mg to PKU mice, the reduction percentage can reach 47.8%, obvious reduction effect (P < 0.05) can be maintained after 4 hours, and the reduction percentage can reach 61.3%; the absorption inhibition effect of PcXAL on oral phenylalanine was more pronounced for 2 days compared to single administration. The results show that: by increasing the oral dose of the test engineering bacteria PcXAL, the absorption inhibition effect of PKU mice on oral phenylalanine can be obviously increased, and the effect is more obvious than that of single administration.

Taken together, the results show that in a phenylketonuria mouse model (PKU mouse), the PcXAL engineering bacteria can inhibit the absorption of PKU mice after single oral administration of phenylalanine and reduce the phenylalanine level in blood after single administration and 2 days of administration. Compared with the blank control group, the inhibition rate of 2 hours after phenylalanine oral administration can reach more than 30%, and the effect can last for at least 4 hours. The result proves that in a PKU mouse model, the PcXAL engineering bacteria can inhibit the effect of phenylalanine absorbed into blood through the alimentary canal by oral administration, thereby reducing the rise of the phenylalanine level in blood after oral administration of phenylalanine, and further achieving the purpose of treating phenylketonuria.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. An application of recombinant enterobacteria in preparing products for degrading tyrosine and/or phenylalanine is characterized in that: the recombinant enterobacteria heterologously express an amino acid sequence shown as SEQ ID NO.1, and fusion expresses a protein tag at the N end of the amino acid sequence.

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

3. The use according to claim 2, characterized in that: the nucleotide sequence of the SUMO tag is shown as SEQ ID NO. 7; the nucleotide sequence of the GST tag is shown as SEQ ID NO. 8; the nucleotide sequence of the GroE tag is shown as SEQ ID NO. 9.

4. The use according to claim 1, characterized in that: the nucleotide sequence of the coding amino acid sequence is shown as SEQ ID NO. 2.

5. The use according to claim 1, characterized in that: coli is used as an initial strain.

6. The use according to claim 1, characterized in that: the tyrosine and/or phenylalanine degradation product is a medicament for treating or preventing phenylketonuria and/or tyrosinemia.

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

8. A recombinant enterobacteria, characterized in that: the recombinant enterobacteria heterologously express an amino acid sequence shown as SEQ ID NO.1, and fusion expresses a protein tag shown as any one of SEQ ID NO.7-9 at the N end of the amino acid sequence.

9. The method for constructing recombinant enterobacteria of claim 8, comprising the steps of:

s1, fusing a protein tag to the N end of an amino acid sequence shown in SEQ ID NO.1, and connecting the protein tag to an expression vector to obtain a recombinant plasmid;

s2, introducing the recombinant plasmid obtained in the S1 into an enterobacter host to obtain the recombinant enterobacter.

10. A microbial agent for degrading phenylalanine and/or tyrosine, characterized in that: comprising the recombinant enterobacteria of claim 8.