CN116410942B - Prephenate dehydrogenase SaPD and its encoding gene and application - Google Patents

Abstract

The invention discloses prephenate dehydrogenase SaPD, and a coding gene and application thereof. The protein provided by the invention is derived from helicobacter salis (Salinispirillum sp.) and is named SaPD protein, and is composed of an amino acid sequence shown as a sequence 1 in a sequence table. The invention also protects the application of SaPD protein as prephenate dehydrogenase.

Description

Prephenate dehydrogenase SaPD and its encoding gene and application

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to prephenate dehydrogenase SaPD and a coding gene and application thereof.

Background technique

L-Tyrosine is an aromatic amino acid and an important conditionally essential amino acid for organisms. It plays an important role in the growth, development and metabolism of humans and animals. It is an important exogenous nutritional supplement for infants and children and patients with phenylketonuria. It is widely used in the food and feed industries. In the pharmaceutical field, L-Tyrosine can improve memory, resist depression and anxiety, and is an important precursor of the Parkinson's disease drug 3,4-dihydroxy-L-phenylalanine (L-DOPA). It is also a precursor for the synthesis of high-value-added drugs such as L-DOPA, thyroxine, adrenaline, and tyrosine sulfite. In the chemical industry, L-Tyrosine is a raw material for the preparation of products such as p-hydroxycinnamic acid and p-hydroxystyrene.

Traditional L-tyrosine production mainly includes chemical method, protein hydrolysis method, enzyme method and fermentation method. Protein hydrolysis method has the disadvantages of limited material sources, complex process and products, long cycle, etc., and has been eliminated. The chemical method has complicated process, low product conversion rate, heavy environmental pollution, especially it can only synthesize D and L-type racemates, which is seriously restricted.

Microbial fermentation is currently the main method for producing L-tyrosine. In the metabolic pathway for synthesizing L-tyrosine in microorganisms, chorismate serves as a branching point for the synthesis of L-tyrosine, L-tryptophan, and L-phenylalanine, and the three aromatic amino acids compete for substrates. In order to improve the production efficiency of L-tyrosine, it is often necessary to modify and optimize the microbial metabolic network and expression regulation network. Therefore, production strains often have to undergo mutagenesis or metabolic engineering. The process control of producing L-tyrosine by fermentation is also relatively complex, and needs to meet the dual requirements of microbial growth and product accumulation.

Biological enzyme catalysis has strong specificity, mild reaction conditions, and easy control of conditions, which provides an important idea for the cheap and efficient production of L-tyrosine and has received more and more attention. Prephenate dehydrogenase can catalyze the oxidative decarboxylation of prephenate to produce p-hydroxyphenylpyruvate, and then synthesize L-tyrosine through transamination. It is a key enzyme for microbial de novo and L-tyrosine production, and is also a feasible way to produce L-tyrosine in vitro. However, most of the prephenate dehydrogenases discovered so far are subject to feedback inhibition by the end product L-tyrosine during the catalytic synthesis of L-tyrosine, which greatly limits the production of L-tyrosine. It is necessary to remove the feedback inhibition of tyrosine on prephenate dehydrogenase by means of tyrosine, or to breed new prephenate dehydrogenases with high activity and low feedback inhibition, which is crucial to the production of L-tyrosine by enzymatic and microbial methods.

Microbial fermentation to produce L-tyrosine uses prephenate dehydrogenase from Escherichia coli, Brevibacterium, Corynebacterium, or genetically engineered strains, and biological enzymes from extreme microbial strains are rare. Extreme enzymes often have better pH, temperature, salinity adaptability and tolerance to heavy metals, organic solvents, denaturants, or features that biological enzymes from other sources do not have. The present invention provides a prephenate dehydrogenase SaPD derived from a halophilic alkali microbial strain and a coding gene thereof, which is tolerant to L-tyrosine and can be used in a wide range of production conditions and can be used for the production of L-tyrosine.

Summary of the invention

The purpose of the present invention is to provide prephenate dehydrogenase SaPD and its encoding gene and application.

The protein provided by the present invention is derived from Salinispirillum sp. NM, is a prephenate dehydrogenase, named SaPD protein, and the amino acid sequence is shown in Sequence 1 in the sequence table. Due to the particularity of the amino acid sequence, any fragment or mutant of the peptide protein containing the amino acid sequence shown in Sequence 1 in the sequence table, as long as it has more than 99% homology with the aforementioned amino acid sequence and has the function of prephenate dehydrogenase, belongs to the protection scope of the present invention. Specifically, the change includes substitution and/or deletion and/or addition and/or replacement of one or more amino acid residues in the amino acid sequence.

The gene encoding the SaPD protein also falls within the protection scope of the present invention.

Preferably, the base sequence of the gene is shown in Sequence 2 in the sequence table, and the gene sequence is derived from Salinispirillum sp. and consists of 900 bases. Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in Sequence 2 in the sequence table, as long as it has more than 90% homology with the polynucleotide, belongs to the scope of protection of the present invention. The variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes. The variant of this polynucleotide includes substitution variants, deletion variants and insertion variants.

The recombinant expression vector, expression cassette or recombinant microorganism containing the gene all fall within the protection scope of the present invention.

The present invention also protects the use of SaPD protein as prephenate dehydrogenase. When SaPD protein is used as prephenate dehydrogenase, the temperature used is 25-70°C and the pH used is 3-11. When SaPD protein is used as prephenate dehydrogenase, the temperature used is 40°C and the pH used is 8.

The prephenate dehydrogenase provided by the invention has high enzyme activity and thermal stability, high tolerance to the product L-tyrosine, and a wide reaction temperature and a wide reaction pH.

Salinispirillum sp. NM was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China; Postal Code: 430072) on April 26, 2021, with the deposit number CCTCC NO: M 2021460.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a scanning electron micrograph of strain NM.

Figure 2 is a phylogenetic tree of strain NM.

FIG3 is the SDS-APGE electrophoresis diagram of SaPD protein.

FIG4 shows the relative enzyme activity results at the optimal pH.

FIG5 shows the relative enzyme activity results when detecting the optimal reaction temperature.

FIG. 6 shows the effect of L-tyrosine on the enzyme activity of SaPD protein.

Detailed ways

The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

Example 1. Isolation, identification and preservation of Halospira NM

1. Separation

The sample was taken from an alkaline lake in Ordos, Inner Mongolia. 50 μL of the alkaline lake sample was added to 450 μL of the alkaline lake filtrate, and the sample was mixed by blowing to obtain a 10-1 concentration sample, which was then diluted to obtain samples with concentrations of ^10-1 , ^10-2 , ^10-3 , ^10-4 , and ^10-5 . 20 μL, 40 μL of the original solution of the alkaline lake water sample and the diluted sample were spread on LBH solid culture medium, and the growth was observed after culturing in a 35°C constant temperature incubator for 3-4 days.

2. Identification

The purified strain was inoculated into LBH solid medium and cultured at 30°C for 3 days. The colony color, protrusion, edge regularity, size and transparency of the strain were recorded. The morphology, size and extracellular appendages of the strain were observed using a transmission electron microscope and an inverted fluorescence microscope. The strain was treated with Gram staining and the Gram staining of the bacteria was observed using an inverted fluorescence microscope. In addition, the strain was inoculated into a semi-solid LBH medium using the puncture inoculation method to observe the motility and aerobicity of the bacteria.

Using LBH liquid medium as the basic medium, 0-10.0% concentration (in increments of 1%, w/v) of NaCl was added respectively, the strain was inoculated into the above medium, and cultured at 30°C for 7 days. The blank control group was LBH medium without inoculation of the strain, and the bacterial density was detected at a wavelength of 600nm using a spectrophotometer to determine the optimal salinity for the growth of the strain; the pH value of the LBH liquid medium was adjusted within the range of pH 3.0-11.0 (in intervals of 1.0 unit), and the strain was inoculated into the above liquid medium. The blank control group was The growth density of the bacteria was detected at a wavelength of 600nm using a spectrophotometer in the LBH medium without inoculation of the strain, so as to determine the optimal pH for the growth of the strain; the strain was inoculated into LBH liquid culture medium and cultured at 4℃, 10℃, 15℃, 20℃, 25℃, 28℃, 30℃, 37℃, 40℃, 45℃ and 50℃ for 7d, respectively; the blank control group was LBH medium without inoculation of the strain, and the growth density of the bacteria was detected at a wavelength of 600nm using a spectrophotometer, so as to determine the optimal temperature and temperature growth range of the strain; the utilization of different carbon sources by the strain was detected using the Biolog GEN III identification plate.

The strains were identified using API 20NE and API 32GN kits, such as nitrate reduction test, nitrite reduction test, enzyme activity characteristics and carbon source utilization test. The results of strain NM and reference strain Salinispirillum marinumGCWy1 ^T are shown in Table 1.

Table 1

The genomic DNA of the strain was extracted using a genome extraction kit, and the 16SrDNA sequence of the strain was amplified using PCR technology. The PCR system used in this experiment was: 4μL template DNA, 1μL primer 27F, 1μL primer 1492R, 5μL 10×TaqBuffer, 4μL dNTP, 0.8μL Taq DNA polymerase, and 34.2μL sterilized ultrapure water. The PCR amplification product was verified by agarose gel electrophoresis. If the band size was consistent with the expected experimental results, the PCR amplification product was sent to Qingdao Qingke Biological Co., Ltd. for sequencing. If the sequencing results were correct, the PCR amplification product was excised and recovered, and the target band was cloned using the pClone007Simple Vector Kit cloning kit, and the cloned target gene was connected to the plasmid pMD18-T. Finally, the recombinant plasmid was transferred into E. coli DH5α. The transformation process was strictly carried out in accordance with the instructions for use of the competent cell E. coli DH5α. The measured 16S rDNA sequence results were uploaded to https://www.ezbiocloud.net/ for sequence comparison analysis, to determine the species of the isolated bacteria, and to download the 16S rDNA sequences of bacteria with a high affinity to the strains. By selecting a suitable outgroup and based on the 16SrDNA gene sequences of the isolated strains and their related strains, the phylogenetic tree of the strains was constructed using MEGA 7.0 software. The phylogenetic analysis of bacteria was performed using the neighbor joining method (NJ). Based on 1000 repetitions, the topological structure of the phylogenetic tree was determined using Bootstrap analysis. The phylogenetic tree is shown in Figure 2.

The above identification results showed that strain NM belonged to the family Saccharospirillaceae and the genus Salinispirillum.

3. Preservation

Salinispirillum sp. NM was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China; Postal Code: 430072) on April 26, 2021, with the deposit number CCTCC NO: M 2021460.

Example 2. Preparation of prephenate dehydrogenase (SaPD protein)

After a large number of sequence analyses, alignments and functional verifications, a new protein was found from Halospirillum NM, which was named SaPD protein, as shown in Sequence 1 of the sequence listing. The gene encoding SaPD protein in Halospirillum NM was named SaPD gene, and its coding frame was shown in Sequence 2 of the sequence listing.

1. Construction of recombinant plasmid

1. Using the genomic DNA of Halspirillum NM as a template, PCR amplification was performed using a primer pair consisting of PD-F and PD-R, and the PCR amplification product was recovered.

PD-F: 5'- CCGG AATTCATGATCAATCAAGCG-3';

PD-R: 5’-CCCAAGCTTTTACCTGGAACGTTCT-3’.

2. Take the PCR amplification product obtained in step 1 and connect it with the pET-28a vector to obtain the recombinant plasmid pET-28a-SaPD.

pET-28a Vector: Anolun (Beijing) Biotechnology Co., Ltd., product catalog number 69864-3.

After sequencing, the recombinant plasmid pET-28a-SaPD contained the DNA molecule shown in Sequence 2 of the sequence listing.

2. Preparation of recombinant bacteria

The recombinant plasmid pET-28a-SaPD was introduced into Escherichia coli BL21 (DE3) to obtain recombinant bacteria A.

The pET-28a vector was introduced into Escherichia coli BL21 (DE3) to obtain recombinant bacteria B.

3. Protein Expression

1. Inoculate the recombinant bacteria into liquid LB medium containing 50 μg/mL kanamycin, and culture at 37°C and 150 rpm for 12 hours to obtain seed solution.

2. Inoculate 1 volume of seed liquid into 99 volumes of liquid LB medium containing 50 μg/mL kanamycin, and culture at 37°C and 200 rpm with shaking until the OD _600nm value is about 0.6. At this time, add IPTG inducer and make its concentration in the system 0.5 mmol/L, then culture at 25°C and 200 rpm with shaking for 6 h (induced expression), then centrifuge at 4°C and 8000×g for 10 min, and collect the bacterial precipitate.

3. Take the precipitate obtained in step 2, wash it with Tris-HCl buffer (0.05M, pH 7.4), then suspend it in Tris-HCl buffer (0.05M, pH 7.2) and perform ultrasonic disruption (ultrasonic disruption parameters: power 200W, 4s of ultrasound and 6s of pause, total time 40min), then centrifuge at 4°C, 10000×g for 20min, and collect the supernatant.

The supernatant obtained by carrying out the above steps on the recombinant bacteria A is named crude enzyme solution A.

The supernatant obtained by carrying out the above steps with the recombinant bacteria B is named crude enzyme solution B.

4. Protein Purification

Take the crude enzyme solution A obtained in step three, filter it with a microfiltration membrane with a pore size of 0.22μm, and collect the filtrate. Take the filtrate and use a Superdex ^TM 200 10/60 gel chromatography column for separation and purification. Connect the Superdex ^TM 20010/60 gel chromatography column to a fast protein liquid phase system, use 1×PBS buffer (pH 8.0) as the mobile phase, and the flow rate is 0.5mL/min. Collect the post-column solution with a retention volume of 18-20mL corresponding to the elution peak, which is the SaPD protein solution. The electrophoresis diagram of the SaPD protein solution is shown in Figure 3, with only one protein band, and it is consistent with the estimated molecular weight (about 33.2KD).

Example 3. Enzymatic properties of prephenate dehydrogenase (SaPD protein)

Tris-HCl buffer (100 mM, pH 8.0): Weigh 1.211 g Tris, dissolve it in deionized water, adjust the pH to 8.0 with HCl, and make up to 1 L.

5 mM prephenic acid solution: weigh 0.018 g of barium prephenate salt, dissolve it in deionized water, and make up to 10 mL.

100mM EDTA: Weigh 2.92g EDTA, dissolve in deionized water, and make up to 100mL.

100mM DTT: Weigh 1.54g DTT, dissolve in deionized water, and make up to 100mL.

100mM NAD ⁺ : Weigh 0.663g NAD ⁺ , dissolve it in deionized water, and make up to 10mL.

The volume of the enzymatic reaction system is 1 mL, containing 50 mM Tris (pH 8.0), 1 mmol/L EDTA, 1 mmol/L DTT, 2 mmol/L NAD ⁺ , and 0.5 mM prephenic acid. Preheat in a water bath or metal bath set to a certain temperature for 5 minutes.

Add 20 μL of crude enzyme solution A, mix well to start the reaction, and time for 20 minutes.

The enzymatic reaction was terminated by adding 500 μL of 1 mol/L NaOH solution, and the mixture was centrifuged at 10000 g for 10 min to remove the precipitate. The OD _{340 nm} value was measured using a 96-well plate in an ELISA reader.

1. Effect of pH on the activity of prephenate dehydrogenase

1. Optimum pH

The SaPD protein solution prepared in Example 2 was diluted to 2 times its volume with a buffer solution, and the dilution was used as the test solution.

The following buffers were used respectively: pH 3.0 citrate buffer, pH 4.0 citrate buffer, pH 5.0 citrate buffer, pH 6.0 citrate buffer, pH 6.0 phosphate buffer, pH 7.0 phosphate buffer, pH 8.0 phosphate buffer, pH 8.0 carbonate buffer, pH 9.0 carbonate buffer, pH 10.0 carbonate buffer, pH 11.0 carbonate buffer. The formula of the citrate buffer is shown in Table 2. The formula of the phosphate buffer is shown in Table 3. The formulas of various salt buffers are shown in Table 4.

Table 2

table 3

Table 4

2. The optimum pH of SaPD protein is 8. The OD _340nm value when using the buffer corresponding to the optimum pH is taken as 100%, and the relative values when using various buffers are calculated as the relative enzyme activity. The results are shown in Figure 4.

2. Effect of temperature on the activity of prephenate dehydrogenase

1. Optimum reaction temperature

The SaPD protein solution prepared in Example 2 was diluted to twice its volume with Tris-HCl buffer (100 mM, pH 8.0) and then used as the test solution.

Detection method: The total reaction volume is 1 mL, containing 50 mM Tris (pH 8.0), 1 mmol/L EDTA, 1 mmol/L DTT, 2 mmol/L NAD+, and 0.5 mM prephenic acid, and preheated at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 5 min. Add 20 μL of the test solution, react for 20 min, and then add 100 μL of 1 mol/L NaOH to terminate the reaction. Centrifuge at 10000 g for 10 min, take 200 μL of the supernatant, and measure the OD _340nm value in a 96-well plate in an ELISA reader.

2. The optimum reaction temperature is 40°C. The OD _340nm value at the optimum reaction temperature is taken as 100%, and the relative values at various reaction temperatures are calculated as the relative enzyme activity. The results are shown in Figure 5.

3. Determination of enzyme activity

Enzyme activity (IU) is defined as: at 30°C and pH 7.0, the amount of enzyme that catalyzes the production of 1 mol of NADH is one enzyme activity unit U.

Detection method: Add 20 μL of test solution, 50 mM Tris (pH 8.0), 1 mmol/L EDTA, 1 mmol/L DTT, 2 mmol/L NAD+, 0.5 mM prephenic acid, react at 30 °C for 10 min, and continuously record the OD _{340 nm} value.

The crude enzyme solution A prepared in Example 2 was used as the test solution, and the enzyme activity was 527 U/mL.

The crude enzyme solution B prepared in Example 2 was used as the test solution, and the enzyme activity was 0 U/mL.

The SaPD protein solution prepared in Example 2 was used as the test solution to detect the enzyme activity per unit volume of the test solution. The protein concentration in the SaPD protein solution prepared in Example 2 was detected. The enzyme activity per unit volume of the test solution was divided by the protein content per unit volume of the test solution to obtain the protein specific activity, which was 757 U/mg.

Example 4: Tolerance of SaPD to L-Tyrosine

The SaPD protein solution prepared in Example 2 was diluted to 2 times the volume with Tris-HCl buffer (100 mM, pH 8.0) as the test solution;

The total reaction volume was 1 mL, containing 50 mM Tris (pH 8.0), 1 mmol/L EDTA, 1 mmol/L DTT, 2 mmol/L NAD+, and 0.5 mM prephenic acid. L-tyrosine was added at concentrations of 0.01 mM, 0.1 mM, 1 mM, 5 mM, and 10 mM, respectively.

Preheat in a water bath or metal bath at 40°C for 5 min.

Add 20 μL of test solution, mix well to start the reaction, and time for 20 minutes.

The enzymatic reaction was terminated by adding 500 μL of 1 mol/L NaOH solution, and the mixture was centrifuged at 10000 g for 10 min to remove the precipitate. The OD _{340 nm} value was measured using a 96-well plate in an ELISA reader.

The OD _340nm value without L-tyrosine was taken as 100%, and the relative values when different concentrations of L-tyrosine were added were calculated as the relative enzyme activity. The results are shown in FIG6 .

Example 5: Use of SaPD protein to prepare L-tyrosine

1. Experimental process

(1) The SaPD protein solution prepared in Example 2 was diluted to 500 U/mL with PBS buffer (100 mM, pH 8.0) as the test solution.

(2) The total volume of the reaction was 100 mL, containing 50 mM Tris (pH 8.0), 1 mmol/L EDTA, 1 mmol/L DTT, 2 mmol/L NAD+, and 0.5 mM prephenic acid. The reaction temperature was 40°C, the stirring speed was 150 rpm, and the reaction time was 12 h.

(3) Aspartate aminotransferase and aspartic acid were added to the reaction mixture, the reaction pH was 8.0, the reaction temperature was 40°C, the stirring speed was 150 rpm, and the reaction time was 24 h. After the reaction was completed, acetic acid was added for recrystallization to obtain L-tyrosine crystals.

(4) L-tyrosine was analyzed by reverse phase chromatography using a Zorbax SB-C18 (4.6×150 mm) column, a column temperature of 20° C., a flow rate of 0.5 mL/min, a mobile phase of 8.0 mM sodium acetate solution (pH 4.0): methanol = 4:1, and a detection wavelength of 230 nm.

2. Prephenate dehydrogenase (SaPD protein) is used to produce L-tyrosine

After the reaction, HPLC analysis showed that the conversion rate of L-tyrosine could reach 90%, indicating that the enzyme has great application potential in the preparation of L-tyrosine.

Claims (5)

1. The protein coded by the prephenate dehydrogenase gene is characterized in that the amino acid sequence is shown as a sequence 1 in a sequence table.

2. A gene encoding the protein of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is shown in sequence table 2.

4. A recombinant expression vector, expression cassette or recombinant microorganism comprising the gene of claim 2 or 3.

5. Use of the protein of claim 1 as prephenate dehydrogenase.