CN116731945A - A recombinant Escherichia coli expressing tyrosine ammonia lyase SeSam8 and its application - Google Patents

Abstract

The invention discloses recombinant escherichia coli for expressing tyrosine ammonia lyase SeSam8 and application thereof, and belongs to the technical field of biology. According to the invention, through a means of gene searching, enzyme molecules with tyrosine degradation activity in nature are collected, in-vitro activity detection is carried out on the tyrosine degradation activity of the enzyme molecules, the enzyme molecules with excellent activity are obtained through screening, the screened enzyme molecules are transferred into escherichia coli to further select engineering bacteria which express tyrosine ammonia lyase molecules and have high-efficiency tyrosine degradation capability, then protein tags are introduced for improving the degradation activity, and one engineering bacteria is found to be good in the tolerance experiment of intestinal gastric juice, so that the engineering bacteria are expected to be used for preparing the tyrosine blood treatment medicine.

Description

A recombinant Escherichia coli expressing tyrosine ammonia lyase SeSam8 and its application

Technical field

The invention relates to a recombinant Escherichia coli expressing tyrosine ammonia lyase SeSam8 and its application, and belongs to the field of biotechnology.

Background technique

Tyrosinemia is due to enzyme defects in the tyrosine metabolism pathway, which causes an increase in tyrosine concentration in the plasma. Enzyme defects in different steps can lead to a variety of diseases with different clinical manifestations, such as brain, The liver, kidneys, bones and other organs are damaged, the prognosis is poor, and the death and disability rates are high. The sources of tyrosine in the human body include dietary intake and endogenous synthesis. The substrate source of endogenous production is the food-derived essential amino acid tyrosine. There are 3 types of tyrosinemia.

Tyrosinemia type I, also known as hepatorenal tyrosinemia (HT-1), is caused by fumarylacetoacetate hydrolase (FAH) deficiency and affects the liver, kidneys and Characterized by peripheral neuropathy. HT-1 affects men and women equally, with an overall incidence of 1/2,000 to 1/100,000. The frequency of mutation carriers in the American population is 1/150 to 1/100. Due to the founder effect, the incidence of HT-1 type live births in Scandinavia is about 1/74,000, and that in Finland and Norway is about 1/74,000. /60,000. In addition, in the Canadian province of Quebec, the incidence rate is approximately 1/16,000 live births, and the carrier frequency is approximately 1/66. The estimated prevalence in the Saguenay-Lac Saint-Jean region of Quebec is 1/1850 births. According to the age of onset of HT-1 patients, the disease is clinically divided into acute type (age of onset < 2 months), subacute type (age of onset 2-6 months) and chronic type (age of onset > 6 months). Children with acute disease usually develop acute disease within 2 months after birth, and their condition worsens rapidly. They often die of liver failure between 3 and 9 months after birth. The subacute form is similar to the acute form, but symptoms appear between 2 and 6 months after birth. The onset of the chronic type usually occurs after 6 months of life, and the clinical manifestations are mainly progressive liver and kidney damage. As the disease progresses, it will eventually lead to cirrhosis, Fanconi syndrome, and even rickets. FAH enzyme deficiency leads to the accumulation of toxic metabolites, including fumaryl acetoacetate (FAA) and maleylacetoacetate. Due to the increased content of its upstream metabolites tyrosine and 4-hydroxyphenylpyruvate, the intermediate metabolite maleylacetoacetate further accumulates, thereby stimulating the production of parametabolic pathways and leading to the production of its metabolites Increased levels of succinylacetoacetate and succinylacetone (SA). These two parametabolites can combine with the sulfhydryl groups of proteins and are the main causes of liver and kidney damage. When left untreated, most patients with HT-1 die in early infancy from acute severe liver and kidney failure. Tyrosinemia type II is caused by tyrosine aminotransferase (TAT) deficiency and is characterized by corneal thickening, palmoplantar keratosis and developmental retardation. Tyrosinemia type III is extremely rare and is caused by 4-hydroxyphenylpyruvic acid dioxygenase (HPPD) deficiency, with neuropsychiatric symptoms as the main manifestation.

Currently, there is only one marketed drug for tyrosinemia - nitisinone (NTBC). NTBC is a competitive inhibitor of 4-hydroxyphenylpyruvate dioxygenase. It can inhibit the routine catabolism of tyrosine in HT-1 patients, prevent the accumulation of toxic metabolites in the body, thereby avoiding damage to liver and kidney function and improving the patient's health. Survival rate. Patients must limit dietary tyrosine and tyrosine intake during nitisinone treatment. Common adverse reactions reported in studies were thrombocytopenia, leukopenia, and visual system disorders, including conjunctivitis, corneal opacification, keratitis, and photophobia. However, because NTBC is a 4-hydroxyphenylpyruvate dioxygenase inhibitor, which inhibits the metabolism of tyrosine to 4-hydroxyphenylpyruvate, blood tyrosine increases. If the blood tyrosine level exceeds 500 μmol/L, It is necessary to control the protein intake and provide dietary treatment without tyrosine or tyrosine formula nutritional powder, otherwise corneal damage will occur due to excessive blood tyrosine levels.

Other treatments for HT-1 include liver transplantation and gene therapy. Liver transplantation has been used to treat HT-1 for more than 20 years and can significantly improve patients' liver, kidney and neurological symptoms. In recent years, with the application of NTBC, the number of liver transplant recipients has gradually decreased. In addition, children after liver transplantation still have a mortality rate of about 5%-10%, and they still need lifelong immunosuppressive treatment after surgery. This method is limited by surgical conditions and the small number of donors, making it difficult to apply clinically. , so only when the child has severe liver failure and is ineffective on nitisinone treatment or cannot receive nitisinone treatment due to other reasons and there is evidence of malignant transformation of the liver tissue, liver transplantation should be selected. As for gene therapy, it is still in the animal research stage.

The present invention aims to treat this disease from the perspective of food-borne metabolism by developing living microbial drugs. There are currently no relevant reports.

Contents of the invention

In order to solve the above problems, the present invention provides a recombinant Escherichia coli, which maintains the degradation activity of the whole cell by introducing the tyrosine ammonia lyase SeSam8 derived from Saccharothrix espanaensis, and further fuses the expression protein tag. , the structure with the best tyrosine degradation effect was screened. At the same time, gastrointestinal fluid tolerance experiments were conducted on different strains, and it was found that one of the engineered bacteria performed best in the tolerance experiment, laying the foundation for the preparation of drugs for treating tyrosinemia.

The first object of the present invention is to provide a recombinant Escherichia coli expressing tyrosine ammonia lyase, which heterologously expresses the tyrosine ammonia lyase SeSam8 with the amino acid sequence shown in SEQ ID NO.1, and in the tyrosine ammonia lyase SeSam8 The N-terminal fusion of the amino acid ammonia lyase SeSam8 expresses the protein tag.

Further, the protein tag is selected from SUMO tag, GST tag or GroE tag.

Further, 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; the nucleotide sequence of the GroE tag is shown in SEQ ID NO.9 .

Further, the nucleotide sequence of tyrosine ammonia lyase SeSam8 is shown in SEQ ID NO. 6.

Further, Escherichia coli Nissle1917 (EcN1917) was used as the starting strain.

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

The second object of the present invention is to provide a method for constructing the above-mentioned recombinant E. coli, which includes the following steps:

S1. Fusion the protein tag to the N-terminus of tyrosine ammonia lyase SeSam8 and connect it to the vector pGEX-4T-2 to obtain the recombinant plasmid;

S2. Introduce the recombinant plasmid obtained in S1 into the E. coli host to obtain the above recombinant E. coli.

The third object of the present invention is to provide the application of the above-mentioned recombinant Escherichia coli in degrading tyrosine, such as preparing a tyrosine-degrading product for degrading tyrosine in vitro or in vivo.

The fourth object of the present invention is to provide the use of the above-mentioned recombinant Escherichia coli in preparing drugs for preventing or treating tyrosinemia.

The fifth object of the present invention is to provide a microbial agent capable of degrading tyrosine, containing the above-mentioned recombinant Escherichia coli.

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

Beneficial effects of the present invention:

(1) The present invention provides a recombinant Escherichia coli strain with high enzyme activity in vitro and in vivo. This engineering strain fuses and expresses a protein tag and tyrosine ammonia lyase SeSam8, which can not only express tyrosine ammonia lyase with high efficiency, but also It also exhibits significantly higher tyrosine degradation activity than other tyrosine ammonia lyases, thereby effectively degrading tyrosine and avoiding its accumulation in the body.

(2) Currently, most of the common drugs used to treat tyrosinemia are chemical drugs, which have high side effects and are prone to drug resistance. PEG-modified drugs and oral protein preparations also suffer from immune rejection and instability. And other disadvantages. The present invention adopts a biological therapy with low toxic and side effects, is less likely to produce drug resistance, and is stable and long-lasting. Enterobacteriaceae are selected as the expression host, and a vector with tyrosine ammonia lyase is transferred into it, so that the constructed engineering bacteria can High expression of tyrosine ammonia lyase, which can degrade food-borne tyrosine ingested in the gastrointestinal tract. In order to further meet the purpose of effectively treating tyrosinemia, the activity of tyrosine ammonia lyase is increased by adding protein tags. And it has been verified that the engineered bacteria of the present invention have strong resistance and tolerance to the gastrointestinal digestive system, and can be prepared into bacterial agents or used in combination with other auxiliary materials to degrade tyrosine in the body to achieve the treatment of tyrosine. Purpose of Acidemia.

Description of drawings

Figure 1 shows the purified protein activity detection results;

Figure 2 shows the whole cell activity test results after tyrosine ammonia lyase from different sources was introduced into E. coli;

Figure 3 is a schematic diagram of the pGEX-4T-2 plasmid structure after optimization of expression elements;

Figure 4 shows the effect of fusion expression of different protein tags on the degradation activity of the whole cell;

Figure 5 shows the results of intestinal fluid tolerance experiment;

Figure 6 shows the results of the gastric juice tolerance test.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.

Detection methods involved in the following examples:

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

Tyr liquid phase detection method

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

Mobile phase: 1% acetic acid and acetonitrile;

Column temperature: 40℃;

Flow rate: 0.8mL/min

Gradient elution:

0-8min, 1.5% acetic acid-acetonitrile (95:5) gradually changes to 1.5% acetic acid-acetonitrile (0:100);

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

13-14min, gradually change from 1.5% acetic acid to acetonitrile (0:100) to 1.5% acetic acid to acetonitrile (95:5);

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

Detection: UV, wavelength 280nm

Injection volume: 10μL

(2) The conditions for testing the degradation activity of whole-cell tyrosine ammonia-lyase are: reaction time 3h, 37°C water bath, substrate 1mMTyr, and bacterial volume 1*10 ¹⁰ .

The materials involved in the following examples are as follows:

SOC liquid medium: 2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5mM KCl, 10mMgCl ₂ , 10mM MgSO ₄ , 20mM D-glucose, adjust pH to 7.5.

Simulated gastric juice and intestinal juice: were purchased from Nanjing Yuanzhixin Biotechnology Co., Ltd. and stored in a -20°C refrigerator.

The sequences involved in the following examples are as follows:

The amino acid sequence encoding tyrosine ammonia lyase SeSam8 is shown in SEQ ID NO.1;

The amino acid sequence encoding tyrosine ammonia lyase TcPAL is shown in SEQ ID NO.2;

The amino acid sequence encoding tyrosine ammonia lyase PcXAL is shown in SEQ ID NO.3;

The amino acid sequence encoding tyrosine ammonia lyase RtXAL is shown in SEQ ID NO.4;

The amino acid sequence encoding tyrosine ammonia lyase RmXAL is shown in SEQ ID NO.5;

The nucleotide sequence encoding tyrosine ammonia lyase SeSam8 is shown in SEQ ID NO. 6;

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

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

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

Example 1 Screening of tyrosine ammonia lyase and study on its ability to degrade tyrosine

Database-based macro screening technology is used to conduct virtual screening and functional prediction of tyrosine ammonia lyases from different sources in NCBI, PDB, Uniprot and other databases, and the tyrosine ammonia lyases with higher scores for full gene synthesis are expressed in vitro And activity verification, select the enzyme with higher enzyme activity after purification. The results are shown in Figure 1. Recombinant expression of the five selected enzymes: plasmid pGEX-4T-2 is used as the expression vector, and primers are designed based on its nucleotide sequence for amplification. The target gene is amplified by PCR and then connected to the expression vector through enzyme digestion and ligation. , conduct fermentation testing after verifying that the sequence is correct.

The details are as follows: After the plasmid pGEX-4T-2 is digested, purify and recover it according to the instructions of the Novozin recovery box (DC301-01). Using primers SeSam8-F and SeSam8-R and pUC57-SeSam8 as templates, PCR amplified the SeSam8 fragment, which was recovered and purified by gel (DC301-01); using primers PcXAL-F, PcXAL-R and pUC57-PcXAL as templates, PCR amplified The PcXAL fragment was amplified and purified by gel (DC301-01); using primers TcPAL-F and TcPAL-R and pUC57-TcPAL as templates, the TcPAL fragment was obtained by PCR amplification and purified by gel (DC301-01); using primer RtXAL- F and RtXAL-R and pUC57-RtXAL were used as templates, and the RtXAL fragment was obtained by PCR amplification, which was recovered and purified by gel (DC301-01); using primers RmXAL-F, RmXAL-R and pUC57-RmXAL as templates, the RmXAL fragment was obtained by PCR amplification , gel recovery and purification (DC301-01); use T4 DNA ligase to ligate the above purified fragments, electrotransform E. coli EcN1917, and obtain the correct positive clones after sequencing verification: pGEX-4T-2-SeSam8, pGEX- 4T-2-TcPAL, pGEX-4T-2-PcXAL, pGEX-4T-2-RtXAL, pGEX-4T-2-RmXAL; pick positive clones to prepare seed liquid, ferment, induce, and measure tyrosine hydrolysis. Ammonia enzyme activity, the results are shown in Figure 2.

According to Figure 1-2, it can be seen that the purified proteins all have high Tyr degradation activity. However, due to differences in protein properties, there are large differences in expression, solubility, and activity in the bacteria. Therefore, under the same Cfu conditions, the whole bacteria There are also large differences in activity. Figure 2 shows that the Saccharothrix espanaensis encoding gene (named SeSam8, SEQ ID NO. 6) was expressed in Enterobacteriaceae through whole-cell activity detection of the five selected tyrosine ammonia lyases. The resulting enzyme has high activity and can efficiently degrade tyrosine.

Table 1 Amplification primers

name sequence Restriction sites SeSam-F CGGAATTCATGACCCAGGTGGTTGAACG EcoRI SeSam-R CC CTCGAG ACCGAAATCTTTTACCGTCAGCT ikB TcPAL-F CG GAATTC ATGTTCATTGAAAACCAACGTGGCT EcoRI TcPAL-R CC CTCGAG ACGACCGTCACGCATCG ikB PcXAL-F CGGAATTCATGCCGAGCCGTATCGACTA EcoRI PcXAL-R CC CTCGAG CGCTTTGATGGATTTAACCAGC ikB RmXAL-F CGGAATTCATGGCACCGAGCGTTGATAG EcoRI RmXAL-R CC CTCGAG CGCCATCATTTTAACCAGCACC ikB RtXAL-F CGGAATTCATGGCTCCGAGCCTGGATTC EcoRI RtXAL-R CC CTCGAG CGCCAGCATTTTCAGCAGAA ikB

Example 2 Optimization of expression elements

According to the results of Example 1, SeSam8 was taken as the main research object. In order to further improve the activity of the engineered strain, different protein tags were fused to the N-terminus of the SeSam8 fragment (see Figure 3 for a schematic diagram of the construction), and were connected through (G ₄ S)*3 linker respectively to construct the fusion expression SUMO tag, GST tag, and GroE tag. The engineering strains were named SeSam8-2, SeSam8-3, and SeSam8-4 respectively. The results are shown in Figure 4. Among them, the degradation rate of SeSam8 without protein tag was 22.5%; after adding SUMO tag, the degradation rate of SeSam8-2 was 61.5%, which was 173.3% higher than SeSam8; after adding GST tag, the degradation rate of SeSam8-3 was 36.5%, which was higher than SeSam8. SeSam8 increased by 62.2%; after adding the GroE tag, the degradation rate of SeSam8-4 was 44%, which was 95.6% higher than that of SeSam8. In summary, SeSam8-2, SeSam8-3, and SeSam8-4 have excellent tyrosine degradation activity and can be used as candidate strains for further research.

Example 3 Artificial gastrointestinal fluid resistance test of bacterial strains

1. Material preparation:

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

(2) Bacterial liquid preparation:

The strains SeSam8, SeSam8-2, SeSam8-3, and SeSam8-4 of Example 2 were inoculated into SOC liquid culture medium. When the bacterial cells grew to OD = 0.6, IPTG was added to induce protein expression overnight; the next day, centrifugation was performed to collect The cells were washed three times with PBS and resuspended with protective agent for gastrointestinal fluid tolerance test.

2. Gastrointestinal fluid tolerance experimental operation

Absorb the artificial gastric juice/artificial intestinal juice and inoculate it into the bacterial resuspension with protective agent. After reacting for 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes, use HPLC to measure the tyrosine degradation rate.

The results are shown in Figure 5-6. Figure 5 shows the tyrosine degradation rate after mixing 1*10 ¹⁰ CFU bacteria (including protective agent) and intestinal fluid (pH = 6.8) at a ratio of 1:1. Figure 6 shows the tyrosine degradation rate of 1*10 ¹⁰ Tyrosine degradation rate after mixing CFU bacteria (containing protective agent) and gastric juice (pH=2) at a ratio of 1:1. It can be seen that the engineered bacterium SeSam8-2 performed better in the tolerance test of artificial gastrointestinal fluid, indicating that it is more suitable for tyrosine degradation in the body, thereby treating or alleviating tyrosinemia.

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or modifications may be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (10)

1. A recombinant escherichia coli expressing a tyrosine ammonia-lyase selam 8, characterized by: the recombinant escherichia coli heterologously expresses tyrosine ammonia-lyase SeSam8 with an amino acid sequence shown as SEQ ID NO.1, and a protein tag is fused and expressed at the N end of the tyrosine ammonia-lyase SeSam 8.

2. The recombinant escherichia coli of claim 1, wherein: the protein tag is selected from the group consisting of a SUMO tag, a GST tag, or a GroE tag.

3. The recombinant escherichia coli of claim 2, wherein the recombinant escherichia coli is: 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 recombinant escherichia coli of claim 1, wherein: the nucleotide sequence of the coding gene of the tyrosine ammonia lyase SeSam8 is shown as SEQ ID NO. 6.

5. The recombinant escherichia coli of claim 1, wherein: escherichia coliNissle1917 was used as starting strain.

6. The recombinant escherichia coli of claim 1, wherein: pGEX-4T-2 was used as the expression vector.

7. The method for constructing recombinant E.coli according to any one of claims 1 to 6, comprising the steps of:

s1, fusing a protein tag to the N end of tyrosine ammonia lyase SeSam8, 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 escherichia coli host to obtain the recombinant escherichia coli.

8. Use of the recombinant escherichia coli of any one of claims 1-6 for degrading tyrosine.

9. Use of the recombinant escherichia coli of any one of claims 1-6 for preparing a medicament for preventing or treating tyrosinase.

10. A microbial agent capable of degrading tyrosine is characterized in that: comprising the recombinant E.coli of any one of claims 1-6.