A recombinant strain and its construction method and application

Abstract

The present invention belongs to the field of biochemical engineering technology, and discloses a recombinant vector, a vector containing a DNA molecule encoding a purine base; wherein the 5' end of the DNA molecule encoding the purine base is connected to an additional start codon. The recombinant vector is used to transform a host strain to obtain a recombinant strain. The present invention can effectively improve the expression level of a gene by introducing a double start codon into a DNA molecule encoding a purine base, and can obtain a high-activity nucleoside hydrolase by heterologously expressing a nucleoside hydrolase gene in the recombinant vector of the present invention through a pET system, and can obtain a nucleoside hydrolase with higher activity under the same preparation conditions, thereby improving the efficiency of hydrolyzing adenosine and guanosine, and obtaining more purine bases and D-ribose.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical engineering technology, and specifically relates to a recombinant vector, a recombinant strain, a construction method and an application thereof, and in particular to a recombinant vector, a recombinant strain, a construction method and an application thereof for improving the in vitro catalytic activity of expressed nucleoside hydrolase. Background Art

[0002] Purine base is a base compound. It is an important component of nucleic acids (DNA, RNA) and some small molecules in organisms. Common ones in biology are: guanine (G) and adenine (A).

[0003] Adenine, also known as 6-aminopurine, is a component of nucleic acid and participates in the synthesis of genetic material. The molecular formula of adenine is C5H5N5, the molecular weight is 135.1, and its properties are white needle-shaped crystals. It is soluble in acid and alkali, slightly soluble in alcohol, and its aqueous solution is neutral. Adenine can promote leukocyte proliferation and increase the number of leukocytes. It is used to prevent and treat leukopenia caused by various reasons, especially for leukopenia caused by radiotherapy, benzene poisoning and anti-tumor, and is also used for acute granulocytopenia, medicine and biochemical research. Adenine is an important pharmaceutical raw material, mainly used in the production of anti-hepatitis B drug adefovir dipivoxil and anti-AIDS drug tenofovir dipivoxil, as well as plant growth hormone 6-benzyladenine and other pharmaceutical products. Guanine (2-amino-6-hydroxypurine, 2-aminohypoxanthine, C5H5N5O) is an important intermediate for a series of highly effective and low-toxic anti-herpes drugs such as acyclovir and famciclovir.

[0004] There are three methods for producing adenine: chemical synthesis, acid hydrolysis, and enzyme catalysis. The chemical synthesis method is to obtain 4,6-diamino-5-nitropyrimidine by ammoniation of 4,6-dichloro-5-nitropyrimidine with ammonia water, and then cyclize it with formic acid, formamide, and sodium thiosulfate. The chemical synthesis method has long reaction steps, low yield, harsh reaction conditions, high equipment requirements, and is not environmentally friendly. The acid hydrolysis method uses adenosine as a raw material, hydrolyzes it with hydrochloric acid and neutralizes it with ammonia water to obtain adenine. The acid hydrolysis method is the current mainstream method with high yield, but the mother liquor is difficult to handle. The enzyme catalysis method uses adenosine as a substrate, adds nucleoside hydrolase to catalyze the reaction, and can obtain adenine and D-ribose at the same time. The enzyme catalysis method is efficient, fast, safe, pollution-free, and has a low production cost, which is very competitive.

[0005] Guanine can also be prepared by chemical and enzyme-catalyzed methods. The chemical method can use 2,4,5-triamino-6-hydroxypyrimidine sulfate, formic acid and formamide to prepare guanine, but its disadvantages are large loss of raw materials, difficulty in refining guanine, cumbersome operation, complex process, high requirements for equipment, and environmental unfriendly. Enzymatic hydrolysis is that under certain conditions, nucleoside hydrolase can hydrolyze N-glycosyl bonds to hydrolyze guanosine into guanine and D-ribose. Using this technology, guanine and D-ribose can be prepared efficiently, quickly, safely and pollution-free.

[0006] Nucleoside hydrolases can decompose nucleosides to generate nitrogenous bases and pentoses. Nucleoside hydrolases are key substances for the enzymatic preparation of purine bases. Currently, nucleoside hydrolases are mainly produced by microbial fermentation. Excellent production strains are the key to nucleoside hydrolase fermentation. However, the fermentation performance of current nucleoside hydrolase strains is still poor, and the in vitro catalytic activity of the produced nucleoside hydrolases is low, which cannot meet the needs of large-scale industrial production. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a recombinant vector, a recombinant strain and a construction method thereof for expressing nucleoside hydrolase to improve the in vitro catalytic activity of the expressed nucleoside hydrolase in order to address the defects of the prior art.

[0008] To achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0009] A recombinant vector contains a DNA molecule encoding a purine base; wherein the 5' end of the DNA molecule encoding a purine base is connected to an additional start codon.

[0010] In some embodiments, the start codon in the recombinant vector is ATG.

[0011] In some embodiments, the DNA molecule encoding the purine base in the recombinant vector is the rihC gene sequence of Escherichia coli BL21 (DE3) or the iagnh gene sequence of Trypanosoma brucei.

[0012] In some embodiments, the vector in the recombinant vector is a pET24a vector.

[0013] The present invention also provides a method for constructing the above-mentioned recombinant vector, using Phusion high-fidelity polymerase to introduce an additional ATG start codon into the DNA molecule encoding the purine base through PCR amplification, and then connecting it with the vector to obtain the recombinant vector.

[0014] The present invention also provides a recombinant bacterial strain containing the recombinant vector.

[0015] In some embodiments, the strain in the recombinant strain is Escherichia coli BL21 (DE3).

[0016] The present invention also provides the use of the recombinant strain in the fermentation production of nucleoside hydrolase.

[0017] The present invention also provides a method for producing nucleoside hydrolase, which comprises fermenting and culturing the above recombinant strain, collecting the bacterial bodies, and crushing and collecting the supernatant nucleoside hydrolase crude enzyme liquid.

[0018] It can be seen from the above technical scheme that the present invention provides a recombinant vector, a vector containing a DNA molecule encoding a purine base; wherein the 5' end of the DNA molecule encoding the purine base is connected to an additional start codon. The recombinant vector is used to transform a host strain to obtain a recombinant strain. The present invention can effectively improve the expression level of the gene by introducing a double start codon into the DNA molecule encoding the purine base, and can obtain a high-activity nucleoside hydrolase by heterologously expressing the nucleoside hydrolase gene in the recombinant vector of the present invention through the pET system. A higher-activity nucleoside hydrolase is obtained under the same preparation conditions, thereby improving the efficiency of hydrolyzing adenosine and guanosine, and obtaining more purine bases and D-ribose. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0020] Figure 1 shows the nucleoside hydrolase expression vector of rihC gene of Escherichia coli BL21(DE3);

[0021] Figure 2 Shown is the nucleoside hydrolase expression vector of the iagnh gene of Trypanosoma brucei. DETAILED DESCRIPTION

[0022] The present invention discloses a recombinant vector, a recombinant strain, a construction method and an application thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the above. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and products of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0023] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels. Among them, the host strain of this experiment is Escherichia coli BL21 (DE3). The LB liquid culture medium formula is 5g / L yeast powder, 5g / L NaCl, 10g / L peptone solution, and the LB solid culture medium formula is 5g / L yeast powder, 5g / L NaCl, 10g / L peptone solution and 1.8% agar powder.

[0025] Example 1: Recombinant plasmid pET24a-rihC eh , pET24a-iagnh eh Construction

[0026] The ORF nucleotide sequence of the rihC gene of Escherichia coli BL21 (DE3) was obtained from the NCBI GenBank database, and two primers were designed to synthesize the positions on both sides of the gene (as shown in Table 1). The full-length fragment of the rihC gene was amplified using Phusion super-fidelity polymerase (New England BioLabs) with A1 / A2 as primers and the Escherichia coli BL21 (DE3) genome as a template. The full-length fragment of the rihC gene with an additional ATG start codon introduced was amplified using Phusion super-fidelity polymerase (New England BioLabs) with B1 / A2 as primers and the Escherichia coli BL21 (DE3) genome as a template.

[0027] The PCR program was: denaturation at 98°C for 10s, renaturation at 50°C for 20s, extension at 72°C for 20s, and cycled 30 times. The obtained fragment was purified by an agarose gel recovery kit (Tiangen), digested with NdeI / HindIII, and pET24a was digested with NdeI / HindIII, and the fragment was ligated to the vector pET24a with T4 DNA ligase (TransGen Biotech). Trans1T1 competent cells (TransGenBiotech) were transformed, kanamycin-resistant clones were picked, and positive clones with rihC gene fragments inserted into pET24a were identified by NdeI / HindIII digestion. The plasmid was extracted, the fragment was amplified with T1 / T2, and sequencing (Invitrogen) confirmed that the inserted fragment was indeed the rihC gene fragment, that is, the pET24a-rihC recombinant plasmid. A similar method was used to construct pET24a-rihC eh Recombinant plasmid (containing an extra ATG start codon).

[0028] The construction of pET24a-iagnh plasmid was similar to that described above. The C1 / C2 and D1 / C2 primer pairs were used to amplify the iagnh gene using the DAL972 genome of Trypanosoma brucei as a template, and two expression plasmids pET24a-iagnh recombinant plasmid and pET24a-iagnh were constructed. eh Recombinant plasmid (containing an extra ATG start codon).

[0029] Table 1 Primer sequences

[0030]

[0031]

[0032] Example 2: Preparation of nucleoside hydrolase expression strain

[0033] The four recombinant plasmids pET24a-rihC, pET24a-rihC eh , pET24a-iagnh, pET24a-iagnh eh The pET24a empty plasmid was co-transformed into competent E. coli BL21 (DE3) to obtain four recombinant strains: pET24a-rihC / BL21 (DE3), pET24a-rihC eh / BL21(DE3),pET24a-iagnh / BL21(DE3),pET24a-iagnh eh / BL21(DE3) and one control strain (pET24a / BL21(DE3)).

[0034] Example 3: Preparation of nucleoside hydrolase

[0035] The four recombinant strains described in Example 2 were cultured to express the nucleoside hydrolase, specifically: a single colony of the recombinant strain was inoculated into a kanamycin-resistant LB medium, cultured overnight at 37°C, the culture after shaking culture was transferred to the same medium at an inoculum of 1%, and cultured at 37°C until the OD600 value reached 0.4-0.6, IPTG was added to make the final concentration of IPTG 0.5mM / L, and then placed at 25°C for overnight shaking culture. After the shaking culture, the cells were collected by centrifugation, and a phosphate buffer of pH 7.0 was added to the collected cells to resuspend the cells, and the cells were placed in an ice bath for ultrasonic cell disruption, the disrupted liquid was centrifuged, and the supernatant was collected to obtain the crude enzyme solution of the nucleoside hydrolase.

[0036] Example 4: Nucleoside hydrolase hydrolysis of adenosine and enzyme activity test

[0037] Prepare a 6 g / L adenosine solution, adjust the solution pH to 7.0, add the crude nucleoside hydrolase solution prepared in Example 3, and react at 37° C. with stirring for 10 min.

[0038] Enzyme activity calculation formula:

[0039] Enzyme activity

[0040] Table 2 Adenosine hydrolysis and enzyme activity determination

[0041]

[0042]

[0043] As shown in Table 2, under the same expression conditions, the activity of the nucleoside hydrolase derived from Escherichia coli in hydrolyzing adenosine is better than that of the nucleoside hydrolase derived from Trypanosoma brucei.

[0044] Double start codon expression can effectively improve translation efficiency, which is manifested in that a crude enzyme solution with higher enzyme activity units can be obtained under the same preparation conditions.

[0045] Example 5: Hydrolysis of guanosine by nucleoside hydrolase and enzyme activity test

[0046] Prepare a 6 g / L guanosine solution, adjust the solution pH to 7.0, add the crude nucleoside hydrolase solution prepared in Example 3, and react at 37° C. with stirring for 10 min.

[0047] Enzyme activity calculation formula:

[0048] Enzyme activity

[0049] Table 3 Guanosine hydrolysis and enzyme activity determination

[0050]

[0051] As shown in Table 2, under the same expression conditions, the activity of the nucleoside hydrolase derived from Trypanosoma brucei in hydrolyzing guanosine is better than that of the nucleoside hydrolase derived from Escherichia coli.

[0052] Double start codon expression can effectively improve translation efficiency, which is manifested in that a crude enzyme solution with higher enzyme activity units can be obtained under the same preparation conditions.

[0053] In summary, by heterologously expressing nucleoside hydrolase genes through the pET system, high-activity nucleoside hydrolase can be obtained, and adenosine and guanosine can be effectively hydrolyzed to obtain purine bases and D-ribose. By introducing double start codons, the expression level of the gene can be effectively improved, and nucleoside hydrolase with higher activity can be obtained under the same preparation conditions.

Claims

1. A recombinant vector comprising a DNA molecule encoding a purine nucleoside hydrolase; wherein the 5' end of the DNA molecule encoding the purine nucleoside hydrolase is connected to an additional start codon; The start codon is ATG; The DNA molecule encoding purine nucleoside hydrolase is the rihC gene sequence of Escherichia coli BL21 (DE3) or the iagnh gene sequence of Trypanosoma brucei; and the vector is a pET system heterologous expression vector.

2. The recombinant vector according to claim 1, wherein the vector is a pET24a vector.

3. The method for constructing the recombinant vector according to claim 1 or 2, using Phusion high-fidelity polymerase to introduce an additional ATG start codon into the DNA molecule encoding the purine base through PCR amplification, and then connecting it with the vector to obtain the recombinant vector.

4. A recombinant strain containing the recombinant vector according to any one of claims 1 to 2, wherein the strain is Escherichia coli BL21 (DE3).

5. Use of the recombinant strain according to claim 4 in the fermentation production of nucleoside hydrolases.

6. A method for producing a nucleoside hydrolase, characterized in that: The recombinant strain according to claim 4 is fermented and cultured, and the bacterial cells are collected, and the supernatant nucleoside hydrolase crude enzyme liquid is collected by crushing.

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