CN108753808B

CN108753808B - Recombinant expression vector, recombinant expression host and method for synthesizing adenosine triphosphate by using recombinant expression vector

Info

Publication number: CN108753808B
Application number: CN201810540239.6A
Authority: CN
Inventors: 杨勇; 江林林; 吴磊; 许海霞; 黄坚丽; 张永进; 徐期
Original assignee: Zhejiang Hisun Pharmaceutical Co Ltd
Current assignee: Zhejiang Hisun Pharmaceutical Co Ltd
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2021-02-23
Anticipated expiration: 2038-05-30
Also published as: CN108753808A

Abstract

The invention belongs to the field of bioengineering, and relates to preparation of adenosine triphosphate. The present invention provides a recombinant expression vector comprising polynucleotides encoding adenosine kinase, adenylate kinase and acetate kinase; the invention also provides a method for synthesizing adenosine triphosphate by using the recombinant expression host. The invention uses the recombinant expression host to catalyze and synthesize the adenosine triphosphate through whole cells, improves the adenosine conversion rate, the ATP yield and the reaction efficiency, and simultaneously has simple reaction system components and low cost.

Description

Recombinant expression vector, recombinant expression host and method for synthesizing adenosine triphosphate by using recombinant expression vector

Technical Field

The invention belongs to the field of bioengineering, and relates to preparation of adenosine triphosphate.

Background

Adenosine Triphosphate (ATP) is an important metabolite in organisms, has a molecular weight of 507 and a molecular formula of C₁₀H₁₆N₅O₁₃P₃The compounds which are used as metabolic intermediates, coenzymes and energy donors participate in various biochemical reactions in organisms and are high-energy compounds necessary for life activities and biochemical reactions; in vitro, ATP is often used as a co-substrate for enzymatic reactions in the production of industrial products such as glutathione. Clinically, ATP is also commonly used for the adjuvant treatment of diseases such as progressive muscular atrophy, myocardial infarction, myocarditis, and the like.

The ATP synthesis method mainly includes a chemical synthesis method, a biological enzyme catalysis method, a microbial enzyme system fermentation method and the like. The existing method for industrial production mainly uses adenosine or Adenosine Monophosphate (AMP) as a substrate and utilizes an enzyme system in a glycolysis pathway of yeast to synthesize ATP, but the method has the problems of complex reaction system, difficult separation and purification, difficult control of reaction process, large quality difference among product batches and the like.

Compared with fermentation, the enzyme catalysis method has the advantages of high efficiency, stability, simple reaction system, easy control of reaction process and the like. For example, Akihiko MARUYAMA et al (Akihiko MARUYAMA, Tatsuro FUJIO. ATP Production from Adein by a Self-linking enzyme Process: High-level Accumulation under Ammonia-limited Conditions, Biosci, Biotechnol, biochem.,65(3):644 and 650) report that ATP is synthesized under catalysis of Corynebacterium ammoniagenes (Corynebacterium ammoniagenes) with adenine as a substrate, the amount of ATP accumulated after 28 hours of reaction reaches 117mmol/L, and the substrate conversion rate reaches 82%. Patent application CN 106191170a discloses the construction of recombinant strains expressing adenosine kinase, adenylate kinase and polyphosphate-adenylate phosphotransferase, respectively, and after extraction or purification, the three enzymes convert adenosine as free enzymes or immobilized enzymes to produce ATP.

However, in the ATP synthesis field, there is still a need for a method with high substrate conversion rate, high ATP yield, high reaction efficiency, simple reaction system components, and low cost.

Disclosure of Invention

The invention aims to improve the substrate conversion rate and ATP yield of ATP synthesis, improve reaction efficiency, simplify reaction system components, facilitate operation and subsequent separation and purification and save cost. To achieve the above objects, the present invention provides an expression vector comprising a polynucleotide encoding adenosine kinase (AK, EC 2.7.1.20), adenylate kinase (ADK, EC 2.7.4.3) and acetate kinase (ACK, EC 2.7.2.1) and a related host; and a method for synthesizing adenosine triphosphate by using the host cell whole cell catalysis.

The technical problem to be solved by the invention is realized by the following technical scheme:

in a first aspect, the present invention provides a recombinant expression vector comprising polynucleotides encoding adenosine kinase, adenylate kinase, and acetate kinase.

Where the polynucleotides encoding adenosine kinase, adenylate kinase and acetate kinase may be present in the expression vector in any suitable manner, e.g. in different operons, or in the same operon. In a particular embodiment, the polynucleotides encoding adenosine kinase, adenylate kinase, and acetate kinase, respectively, are arranged sequentially in the same operon. The sequence arrangement means that the polynucleotides of the three enzymes are arranged in sequence in the same operon after a promoter, and the sequence of the three enzymes is not unique and can be determined according to actual requirements or operation.

The adenosine kinase, adenylate kinase and acetate kinase may be derived from any organism or may be artificially modified to have the same catalytic function. In a particular embodiment, the adenylate kinase and/or acetate kinase is of bacterial origin, such as e.coli (e.coli), e.g. e.coli BL21(DE 3). In a particular embodiment, the adenosine kinase is derived from a yeast, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae). In a more specific embodiment, the sequence of the polynucleotide encoding the adenosine kinase is shown in SEQ ID NO. 1, the sequence of the polynucleotide encoding the adenylate kinase is shown in SEQ ID NO. 2, and the sequence of the polynucleotide encoding the acetate kinase is shown in SEQ ID NO. 3.

The expression vector may be any vector suitable for protein expression. Preferably, the expression vector is a plasmid; further, the expression vector is pET24 a.

In another aspect, the present invention provides a recombinant expression host comprising a recombinant expression vector comprising polynucleotides encoding adenosine kinase, adenylate kinase, and acetate kinase.

The host may be any host cell capable of stably expressing the protein in the expression vector. Preferably, the host is escherichia coli, more preferably, escherichia coli BL21(DE 3).

In another aspect, the present invention provides the use of a recombinant expression vector or a recombinant expression host as described above, or an expression product thereof, or a bacterial suspension thereof, or a disrupted product thereof, for the synthesis of adenosine triphosphate.

In another aspect, the present invention provides a method for synthesizing adenosine triphosphate, comprising the steps of:

(1) constructing a recombinant expression host as described above, and inducing the expression of adenosine kinase, adenylate kinase and acetate kinase;

(2) adding adenosine into the reaction system of the step (1), and simultaneously adding acetyl phosphate (ACP) and/or salt thereof according to a proportion to perform catalytic synthesis, wherein the reaction system further comprises magnesium ions, and anions and cations which play a buffering role.

In a particular embodiment, the buffering cation is one or more of potassium ion, sodium ion; the anion playing a buffering role is one or more of phosphate radical ion and tetraborate radical ion.

Wherein the recombinant expression host expressing adenosine kinase, adenylate kinase and acetate kinase functions as a catalytic enzyme source. The substrate, enzyme and other components added in the invention can be added into the reaction system at one time or added in batches according to the process flow.

In a specific embodiment, before the constructing step (1)), the method may further comprise: extracting genomes from the source organisms of the adenosine kinase, the adenylate kinase and the acetate kinase, amplifying to obtain polynucleotides encoding corresponding enzymes, and further constructing a recombinant expression vector containing the polynucleotides encoding the three enzymes. In this case, the step (1) further comprises transforming the recombinant expression vector into an expression host, amplifying the recombinant expression host cell, and collecting the bacterial cells.

In the step of inducing expression, the inducer used may be any suitable inducer. In a particular embodiment, the expression vector is pET24a and the inducer used is lactose.

In a preferred embodiment, the reaction temperature of the catalytic synthesis step (2)) is from 20 to 50 ℃, more preferably from 30 to 40 ℃, most preferably 35 ℃.

In a preferred embodiment, the reaction pH of step (2) is from 6.0 to 9.0, more preferably from 7.0 to 8.5, and most preferably the pH is 7.5.

Preferably, in the step (2), the concentration of the adenosine added is 10 to 30 mmol/L; the molar concentration of the added acetyl phosphate and/or salt thereof is 1 to 6 times of the molar concentration of the added adenosine.

Preferably, in the step (2), the amount of the recombinant expression host is 1-20 g/L.

Preferably, in the step (2), the concentration of magnesium ions is 1 to 100mmol/L, more preferably 5 to 10 mmol/L; the concentration of the cation playing the role of buffering is 1-100mmol/L, and more preferably 100 mmol/L; the concentration of the buffering anion is 1 to 100mmol/L, more preferably 50 mmol/L.

Preferably, in the above technical solution, the salt of acetyl phosphate may be any suitable acetyl phosphate, preferably one or more of acetyl phosphate dilithium salt, acetyl phosphate diammonium salt; the magnesium ions may be of any suitable source, preferably from one or more of magnesium sulphate, magnesium chloride, magnesium sulphite; the potassium ions may be of any suitable source, preferably from one or more of potassium chloride, potassium sulphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate; the sodium ions may be of any suitable source, preferably from one or more of sodium chloride, sodium sulphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium tetraborate.

In another aspect, the invention provides a kit for synthesizing ATP, the kit comprising a recombinant expression host as described above. Preferably, the kit further comprises one or more of: a culture medium for culturing and expressing the recombinant expression host; acetyl phosphate and/or a salt thereof; adenosine; magnesium ions; one or more of potassium ions, sodium ions, or other suitable buffering cations; one or more of phosphate ions, tetraborate ions, or other suitable buffering anions; a pH adjusting agent; culture devices (such as tubes, bottles, etc.), instructions for use. Wherein one or more of potassium, sodium, or other suitable buffering cations, and one or more of phosphate, tetraborate, or other suitable buffering anions are capable of maintaining the fermentation system of the recombinant expression host at a suitable pH.

Through the technical scheme, the conversion rate of adenosine can reach more than 99%, and the ATP yield is improved. It will be appreciated by those skilled in the art that a conversion rate of greater than 99% means substantially complete conversion of the substrate. In addition, the recombinant expression vector containing and simultaneously expressing the three enzymes and the whole cell of the recombinant expression host are used for one-time fermentation, so that ATP is produced in one step, and the reaction efficiency is improved. In addition, the method of the invention has the following advantages: ATP or ADP or AMP is not required to be additionally supplemented for starting the reaction; acetyl phosphate or salt thereof is used as a substrate and a phosphate donor to produce ATP, so that the reaction cost is further reduced; acetate is generated after the reaction, so that the pollution is small; the reaction liquid prepared by the method has simple components and low impurity content, and is easy for subsequent purification industry. Therefore, the method can effectively reduce the operation difficulty and the production cost of the reaction, and is favorable for realizing industrial production.

Drawings

FIG. 1 is a map of a recombinant plasmid pET24a-AK-ADK-ACK constructed in accordance with a particular embodiment of the present invention.

FIG. 2 is an SDS-PAGE pattern of fermentatively expressed adenosine kinase, adenylate kinase and acetate kinase in recombinant E.coli constructed in accordance with certain embodiments of the present invention.

Detailed Description

Specific examples of the invention are described in detail below to facilitate a further understanding of the invention. It should be understood that these examples are only for illustrating the present invention and are not to be construed as limiting the present invention in any way. Any variations that may be made in the practice of the invention by those skilled in the art in light of the teachings herein will fall within the scope of the appended claims.

Unless otherwise indicated, the reagents and apparatus used in the following examples are conventional in the art and are commercially available; the methods used are conventional and the person skilled in the art can, without any doubt, carry out the examples and obtain the corresponding results from the description.

Example 1 construction of recombinant E.coli

Experimental materials and reagents:

escherichia coli BL21(DE3) (e.coil BL21(DE 3)): purchased from Invitrogen corporation;

coli DH5 α (e.coil DH5 α): purchased from Invitrogen corporation;

a saccharomyces cerevisiae strain: china center for type culture Collection, number CCTCC AY 93175;

the pET24a plasmid: for expression plasmids, having the T7 promoter, kanamycin resistance gene, available from NOVAGEN Inc., cat # 69749;

PCR primers for adenosine kinase, adenylate kinase and acetate kinase: synthesized by Suzhou Jinweizhi Biotech, Inc.;

pyrobest enzyme and 10 XPyrobest buffer: purchased from TaKaRa, cat # R005;

restriction enzyme Nde I: purchased from TaKaRa, cat # 1161A;

restriction enzyme BamH I: purchased from TaKaRa, cat # 1010A;

restriction enzyme Bgl II: purchased from TaKaRa, cat No. 1021A;

fast ligase Solution I: purchased from TaKaRa, cat # 6022;

alkaline Phosphatase (CAP): purchased from TaKaRa, cat 2250A.

In the examples of the present invention, the experimental method not specified in specific conditions was performed according to the molecular cloning instructions (third edition) (J. SammBruk, D.W. Lassel, Huangpetang, scientific Press, 2002, 8 months).

1.1 construction and transformation of recombinant plasmids, recombinant expression hosts

The genomes of E.coli BL21(DE3) and s.cerevisiae were extracted separately. The adenylate kinase gene (shown as sequence SEQ ID NO: 2) and the acetate kinase gene (shown as sequence SEQ ID NO: 3) were amplified from the E.coli BL21(DE3) genome and the adenylate kinase gene (shown as sequence SEQ ID NO: 1) was amplified from the s.cerevisiae genome as follows.

It should be noted that, in this example, the features of restriction enzymes BamH I and bglii as isocaudarner enzymes are utilized, BamH I cleavage sites are introduced when reverse primers are designed as follows, and the sticky ends cleaved with bglii are obtained by double cleavage after being linked to an expression vector, which is helpful to improve the success rate of operation. The details are as follows.

PCR primer pairs for the adenosine kinase gene were designed and synthesized:

a forward primer: 5' -aaaaaaaaaacatatgaccgcaccattggtag-3’(SEQ ID NO:4)；

Reverse primer: 5' -aaaaaaaaaaggatccctatttagagtaagat-3’(SEQ ID NO:5)。

Among the primers shown above, the underlined sequence in the forward primer "catatg"sequence underlined in the reverse primer" for Nde I cleavage site "ggatcc"is BamH I cleavage site.

PCR amplification reaction System for amplifying adenosine kinase Gene (50. mu.L): ddH₂O37.5. mu.L, 10 XPyrobest buffer 5. mu.L, dNTPs (2.5 mmol/L each) 4. mu.L, forward primer (50. mu. mol/L) (SEQ ID NO:4) 1. mu.L, reverse primer (50. mu. mol/L) (SEQ ID NO:5) 1. mu.L, template (10 ng/. mu.L) 1. mu.L, Pyrobest enzyme 0.5. mu.L.

The PCR reaction conditions for amplifying the adenosine kinase gene are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 50 ℃ for 30s, and 72 ℃ for 1 min; 10min at 72 ℃ and 1min at 16 ℃.

Designing and synthesizing PCR primer pair of adenylate kinase gene:

a forward primer: 5' -aaaaaaaaaacatatgcgtatcattctgcttg-3’(SEQ ID NO:6)；

Reverse primer: 5' -aaaaaaaaaaggatccttagccgaggattttt-3’(SEQ ID NO:7)。

PCR reaction System for amplifying adenylate kinase Gene (5)0μL)：ddH₂O37.5. mu.L, 10 XPyrobest buffer 5. mu.L, dNTPs (2.5 mmol/L each) 4. mu.L, forward primer (50. mu. mol/L) (SEQ ID NO:6) 1. mu.L, reverse primer (50. mu. mol/L) (SEQ ID NO:7) 1. mu.L, template (1 ng/. mu.L) 1. mu.L, Pyrobest enzyme 0.5. mu.L.

The PCR reaction conditions for amplifying the adenylate kinase gene are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 50 ℃ for 30s, 72 ℃ for 40 s; 10min at 72 ℃ and 1min at 16 ℃.

Design and synthesis of PCR primer pairs for the acetate kinase gene:

a forward primer: 5' -aaaaaaaaaacatatgtcgagtaagttagtac-3’(SEQ ID NO:8)；

Reverse primer: 5' -aaaaaaaaaaggatcctcaggcagtcaggcgg-3’(SEQ ID NO:9)。

PCR reaction System for amplification of acetate kinase Gene (50. mu.L): ddH₂O37.5. mu.L, 10 XPyrobest buffer 5. mu.L, dNTPs (2.5 mmol/L each) 4. mu.L, forward primer (50. mu. mol/L) (SEQ ID NO:8) 1. mu.L, reverse primer (50. mu. mol/L) (SEQ ID NO:9) 1. mu.L, template (1 ng/. mu.L) 1. mu.L, Pyrobest enzyme 0.5. mu.L.

The PCR reaction conditions for amplifying the acetate kinase gene are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 50 ℃ for 30s, and 72 ℃ for 75 s; 10min at 72 ℃ and 1min at 16 ℃.

Carrying out agarose gel electrophoresis on the coding sequence fragments of the three enzymes obtained by the method, and recovering; the recovered three enzyme gene fragments and pET24a plasmid vector were treated with restriction enzyme Nde I at 37 ℃ for 3-4 hours, and then treated with restriction enzyme BamH I at 30 ℃ for 3-4 hours. In the enzyme digestion process, the double enzyme digestion system (30 μ L) of the vector is: 10 XK buffer 3. mu.L, restriction enzyme Nde I1. mu.L, plasmid 8. mu.L, restriction enzyme BamH I1. mu.L, ddH₂O17 mu L; the double enzyme digestion system of the three enzyme gene fragments is respectively (30 mu L): 3. mu.L of 10 XK buffer, 1. mu.L of Nde I, 10. mu.L of gene fragment, 1. mu.L of BamH I, ddH₂O 15μL。

The enzyme-cleaved product was subjected to agarose gel electrophoresis, and gene fragments of the three enzymes and pET24a plasmid were recovered, respectively, by using a gel recovery kit from AXYGEN, according to the manual of the supplier.

Firstly, the pET24a plasmid treated in the last step is respectively connected with an adenosine kinase gene fragment, an adenylate kinase gene fragment and an acetate kinase gene fragment which are subjected to enzyme digestion and recovery by utilizing a fast ligase Solution I. The ligation system (12. mu.L) was: 5 mu L of gene fragment, 1 mu L of vector and 6 mu L of Solution I. The cells were ligated by incubation at 16 ℃ for 3 to 4 hours and then transferred to E.coli DH 5. alpha. competent cells to obtain recombinant plasmids pET24a-AK (a recombinant plasmid containing an adenosine kinase gene), pET24a-ADK (a recombinant plasmid containing an adenylate kinase gene) and pET24a-ACK (a recombinant plasmid containing an acetate kinase gene), respectively. The recombinant plasmid pET24a-AK is cut by restriction enzyme BamH I, and the cutting system (30 uL) is as follows: 3. mu.L of 10 XK buffer, 8. mu.L of plasmid, 1. mu.L of endonuclease BamH I, ddH₂O18. mu.L. After 3h of digestion at 30 ℃ and treatment with alkaline phosphatase (CAP) for 15min, the digestion system (35. mu.L): 10 × CAP buffer 3.5 μ L, CAP 0.5 μ L, digestion system 30 μ L, ddH₂O1. mu.L. Agarose gel electrophoresis was performed, and the digested vector containing the adenosine kinase gene (digested pET24a-AK) was recovered. pET24a-ADK was treated with BglII and BamH I in a double digestion system (30. mu.L): 3 μ L of 10 XK buffer, 8 μ L of plasmid, 1 μ L of BamH I, 1 μ L of BglII, ddH₂O17. mu.L. And (4) carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion fragment of the adenylate kinase gene. The digested fragments of pET24a-AK and adenylate kinase gene obtained as above were ligated by using a quick ligase Solution I in a ligation system (12. mu.L): cut pET24a-AK 1 uL, adenylate kinase gene cut fragment 5 uL, Solution I6 uL. Keeping the temperature at 16 ℃ for 3-4 h. The ligation product was transformed into E.coli DH 5. alpha. competent cells to obtain recombinant plasmid pET24 a-AK-ADK. The obtained recombinant plasmid pET24a-AK-ADK is cut by BamH I (the single enzyme cutting system of BamH I is the same as above), enzyme cutting is carried out for 3h at 30 ℃, alkaline phosphatase is used for processing for 15min, the cut product is processed by agarose gel electrophoresis, and the enzyme cutting is recoveredThe vector comprising AK-ADK of (digested pET24 a-AK-ADK). pET24a-ACK is subjected to double enzyme digestion treatment by BglII and BamH I (the double enzyme digestion system of BglII and BamH I is the same as above), the digestion product is subjected to agarose gel electrophoresis, and the digestion fragment of the acetate kinase gene is recovered. The enzyme-cut fragments of pET24a-AK-ADK and ACK are connected by using a quick ligase Solution I, and the connection system (12 mu L) is as follows: 1 mu L of digested pET24a-AK-ADK, 5 mu L of digested fragment of ACK, and 6 mu L of Solution I. Keeping the temperature at 16 ℃ for 3-4 h. The ligation product was transformed into E.coli DH 5. alpha. competent cells to obtain recombinant plasmid pET24 a-AK-ADK-ACK. The map of the constructed recombinant plasmid pET24a-AK-ADK-ACK is shown in FIG. 1.

The obtained recombinant plasmid pET24a-AK-ADK-ACK is transferred into a competent cell of escherichia coli BL21(DE3) by a KCM method, positive clones are screened, the successfully transformed recombinant plasmid is identified, and the expression sequences of the three enzymes are detected to be all correct by sequencing.

1.2 screening and expanded culture of high-expression Strain

The selected E.coli BL21(DE3) was inoculated into LB medium as a single clone, cultured until the logarithmic phase (OD value of 0.6-0.8) was reached, then lactose was added to a final concentration of 1%, induced overnight (about 20 hours), centrifuged at 8,000rpm for 10min using a centrifuge, and the cells were collected. The cells were suspended in a cell-breaking buffer (10mmol/L Tris, 10mmol/L EDTA, 0.1mol/L sodium chloride, 5% glycerol, pH 7.0) to a cell concentration of 50 g/L. The thalli is crushed in an ice bath by using an ultrasonicator, and the thalli is subjected to ultrasonication for 12min according to the procedures of working for 2s and pausing for 4 s. And performing SDS-PAGE electrophoresis on the whole components, the supernatant and the precipitate after the thalli are broken respectively to detect protein expression. FIG. 2 is a graph showing the expression of three enzyme proteins (total protein expression, soluble expression or not), showing an exemplary SDS-PAGE pattern of recombinant E.coli constructed as above, with lane 1 showing protein markers 14.4-116kDa (from Thermo); lane 2 is the total fraction after disruption of the cells; lane 3 is the supernatant after disruption of the cells; lane 4 shows the pellet after disruption.

Respectively fermenting and culturing different monoclonals, crushing the monoclonals according to the method, carrying out SDS-PAGE electrophoresis detection, and screening high-expression strains according to the electrophoresis detection result. The high expression strain was subjected to 5L fermentation culture as follows. Adding 1 per mill of recombinant Escherichia coli glycerol cryopreservation tube bacterial liquid into 100mL of seed culture medium containing 50 ug/mL of kanamycin, and culturing overnight at 37 ℃ and 220r/min in a shaking constant temperature incubator. Inoculating the seed culture solution containing the recombinant Escherichia coli obtained in the above manner into 5L fermentation medium according to the seed transfer amount of 2%, fermenting and culturing in a fermentation tank of 8L specification for about 3h, cooling the culture medium to 25 deg.C, adding pre-prepared and sterilized 30% lactose to a final concentration of 1% as inducer, and continuing to ferment at 25 deg.C overnight. Centrifuging at 8,000rpm/min for 10min by using a centrifuge, collecting thalli, and storing at-20 ℃ for subsequent reaction.

Wherein the seed culture medium comprises the following components: 5g/L yeast extract, 10g/L peptone, 10g/L sodium chloride and pH 7.0. The fermentation medium comprises the following components: 15g/L of peptone, 25g/L of yeast extract, 10g/L of dipotassium phosphate, 15g/L of glycerol and 6mol/L of sodium hydroxide are used for adjusting the pH to 7.5.

EXAMPLE 2 Whole cell catalytic Synthesis of ATP from recombinant E.coli

10mmol/L adenosine, 50mmol/L borax, 50mmol/L ACP and 5mmol/L magnesium sulfate were added to the reaction system (1L), 10g/L of the bacterial cells collected in example 1 as described above were added as an enzyme source, the pH of the reaction solution was adjusted to 7.5, the temperature during the reaction was controlled at 35 ℃ and the reaction was carried out at a stirring speed of 150r/min with mechanical stirring for 3 hours.

Collecting the supernatant of the reaction solution, filtering and sterilizing, and analyzing the concentration of the remaining adenosine by High Performance Liquid Chromatography (HPLC) using a high performance liquid chromatograph (Agilent 1260); for the detection of the concentration of ATP formed, the supernatant was diluted 10-fold, filter-sterilized and analyzed by HPLC.

HPLC conditions: a Welch Ultimate XB-C18 column (250 mm. times.4.6 mm I.D., 5 μm particle size, available from Asahi technology (Shanghai) Co., Ltd., cat # 00201-; sample introduction amount: 10 mu L of the solution; detection wavelength: 260 nm; column temperature: 30 ℃; flow rate: 1.0 ml/min; mobile phase: 0.2mol/L ammonium formate + 0.1% sodium octane sulfonate (formic acid adjusted pH to 2.8): methanol 95: 5; isocratic elution.

Standard curves were prepared using ATP standard (purchased from china institute for food and drug assay, cat # 140674) and adenosine standard (purchased from china institute for food and drug assay, cat # 110879), respectively, and ATP and adenosine concentrations were calculated. In this example, the concentration of ATP produced was calculated to be 5 g/L.

From the adenosine concentration calculated as above, the adenosine conversion rate was calculated using the following formula: adenosine conversion rate (1-mass concentration of adenosine peak area after completion of reaction converted from standard curve/initial adenosine concentration) 100%. The adenosine conversion was calculated to be 99% in this example.

EXAMPLE 3 Whole cell catalytic Synthesis of ATP from recombinant E.coli

Adding 30mmol/L adenosine, 50mmol/L borax, 180mmol/L ACP and 10mmol/L magnesium sulfate into a reaction system (1L), adding 5g/L thallus as an enzyme source, adjusting the pH value of a reaction solution to 7.5, controlling the temperature to be 30 ℃ in the reaction process, and reacting for 5 hours at the stirring speed of 150r/min under mechanical stirring. ATP and adenosine concentrations were measured as described in example 2. The ATP concentration produced was 15.1g/L, and the adenosine conversion rate was 99% or more, as calculated as described in example 2.

Example 4 Whole cell catalytic Synthesis of ATP in recombinant E.coli

30mmol/L adenosine, 50mmol/L borax, 135mmol/L ACP and 5mmol/L magnesium sulfate are added into a reaction system (1L), 2g/L thallus is added as an enzyme source, the pH value of a reaction liquid is adjusted to 7.5, the temperature is controlled at 35 ℃ in the reaction process, and the reaction is carried out for 3 hours at the stirring speed of 150r/min under the mechanical stirring. ATP and adenosine concentrations were measured as described in example 2. The ATP concentration produced was 15.1g/L, and the adenosine conversion rate was 99% or more, as calculated as described in example 2.

Sequence listing

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<213> Escherichia coli (Escherichia coli)

<400> 3

atgtcgagta agttagtact ggttctgaac tgcggtagtt cttcactgaa atttgccatc 60

atcgatgcag taaatggtga agagtacctt tctggtttag ccgaatgttt ccacctgcct 120

gaagcacgta tcaaatggaa aatggacggc aataaacagg aagcggcttt aggtgcaggc 180

gccgctcaca gcgaagcgct caactttatc gttaatacta ttctggcaca aaaaccagaa 240

ctgtctgcgc agctgactgc tatcggtcac cgtatcgtac acggcggcga aaagtatacc 300

agctccgtag tgatcgatga gtctgttatt cagggtatca aagatgcagc ttcttttgca 360

ccgctgcaca acccggctca cctgatcggt atcgaagaag ctctgaaatc tttcccacag 420

ctgaaagaca aaaacgttgc tgtatttgac accgcgttcc accagactat gccggaagag 480

tcttacctct acgccctgcc ttacaacctg tacaaagagc acggcatccg tcgttacggc 540

gcgcacggca ccagccactt ctatgtaacc caggaagcgg caaaaatgct gaacaaaccg 600

gtagaagaac tgaacatcat cacctgccac ctgggcaacg gtggttccgt ttctgctatc 660

cgcaacggta aatgcgttga cacctctatg ggcctgaccc cgctggaagg tctggtcatg 720

ggtacccgtt ctggtgatat cgatccggcg atcatcttcc acctgcacga caccctgggc 780

atgagcgttg acgcaatcaa caaactgctg accaaagagt ctggcctgct gggtctgacc 840

gaagtgacca gcgactgccg ctatgttgaa gacaactacg cgacgaaaga agacgcgaag 900

cgcgcaatgg acgtttactg ccaccgcctg gcgaaataca tcggtgccta cactgcgctg 960

atggatggtc gtctggacgc tgttgtattc actggtggta tcggtgaaaa tgccgcgatg 1020

gttcgtgaac tgtctctggg caaactgggc gtgctgggct ttgaagttga tcatgaacgc 1080

aacctggctg cacgtttcgg caaatctggt ttcatcaaca aagaaggtac ccgtcctgcg 1140

gtggttatcc caaccaacga agaactggtt atcgcgcaag acgcgagccg cctgactgcc 1200

tga 1203

<210> 4

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

Claims

1. A recombinant expression host comprising a recombinant expression vector; the recombinant expression vector comprises: polynucleotides encoding adenosine kinase, adenylate kinase, and acetate kinase, respectively, which are located in the same operon; wherein, the sequence of the polynucleotide for coding the adenylate kinase is shown as SEQ ID NO. 1, the sequence of the polynucleotide for coding the adenylate kinase is shown as SEQ ID NO. 2, and the sequence of the polynucleotide for coding the acetate kinase is shown as SEQ ID NO. 3; the expression vector is pET24 a; the host is Escherichia coli.

2. The recombinant expression host according to claim 1, wherein said E.coli is E.coli BL21(DE 3).

3. The recombinant expression host according to claim 1 or 2, wherein the polynucleotides encoding adenosine kinase, adenylate kinase and acetate kinase, respectively, are sequentially arranged in the same operon.

4. Use of the recombinant expression host according to any one of claims 1 to 3, or an expression product thereof, or a bacterial suspension thereof, or a disrupted product thereof, for the synthesis of adenosine triphosphate.

5. A method of synthesizing adenosine triphosphate comprising the steps of:

(1) constructing a recombinant expression host according to any one of claims 1 to 3 and inducing the expression of said adenosine kinase, said adenylate kinase and said acetate kinase;

(2) adding adenosine into the reaction system of the step (1), and simultaneously adding acetyl phosphate and/or salt thereof according to a proportion to perform catalytic synthesis, wherein the reaction system also comprises magnesium ions, and anions and cations for buffering.

6. The method of claim 5, wherein in step (1), the inducer used for induction is lactose.

7. The process according to claim 6, wherein the reaction temperature in the step (2) is 20 to 50 ℃ and the reaction pH is 6.0 to 9.0; the concentration of the added adenosine is 10-30mmol/L, and the molar concentration of the added acetyl phosphate and/or the salt thereof is 1-6 times of the molar concentration of the adenosine added in the reaction; the using amount of the thalli of the recombinant expression host is 1-20 g/L; the concentration of magnesium ions is 1-100mmol/L, the concentration of the buffering cations is 1-100mmol/L, and/or the concentration of the buffering anions is 1-100 mmol/L.

8. The method according to claim 7, wherein the reaction temperature of the step (2) is 30-40 ℃, the reaction pH is 7.0-8.5, and the magnesium ion concentration is 5-10 mmol/L; the concentration of the cation for buffering is 100 mmol/L; and/or the concentration of the buffering anion is 50 mmol/L.

9. The process of claim 8, wherein the reaction temperature of step (2) is 35 ℃; the reaction pH was 7.5.

10. The method according to any one of claims 5 to 9, wherein step (1) is preceded by: and (2) respectively amplifying the polynucleotides encoding the adenosine kinase, the adenylate kinase and the acetate kinase, further constructing a recombinant expression vector comprising the polynucleotides encoding the three enzymes, and transforming the recombinant expression vector into an expression host.

11. A kit for synthesizing ATP comprising the recombinant expression host of any one of claims 1-3.

12. The kit of claim 11, further comprising one or more of: acetyl phosphate and/or its salt, adenosine, magnesium ion, cation and anion for buffering, and pH regulator.