An adenylate cyclase mutant, recombinant bacteria and their application in cAMP synthesis
By randomly and cumulatively mutating Escherichia coli adenylate cyclase and expressing it in Escherichia coli BL21(DE3) derivatives, the problem of low efficiency in adenylate cyclase synthesis of cAMP was solved, achieving efficient and stable cAMP production, which is suitable for animal feed additives and livestock and poultry farming.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- MEIBANG MEIHE BIOTECHNOLOGY CO LTD
- Filing Date
- 2022-10-31
- Publication Date
- 2026-07-31
AI Technical Summary
In existing technologies, adenylate cyclase has low efficiency in synthesizing cAMP, and bio-fermentation and chemical synthesis methods suffer from environmental pollution and low yield. Bio-enzyme catalysis lacks an efficient expression mechanism, making it difficult to meet industrial needs.
By randomly screening and cumulatively mutating Escherichia coli adenylate cyclase, mutants with higher activity were obtained and expressed in Escherichia coli BL21(DE3) derivatives. This avoided adenylate deaminase degradation, optimized catalytic conditions, and achieved efficient cAMP synthesis.
The adenylate cyclase mutant maintains high catalytic activity under low temperature conditions, with a yield of over 95% and a maximum cAMP concentration of 20.6 g/L. This significantly improves the production efficiency and stability of the bio-enzymatic catalysis method and has promising prospects for industrial application.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of enzyme engineering technology, specifically to an adenylate cyclase mutant, a recombinant bacterium, and its application in the synthesis of cAMP. Background Technology
[0002] Cyclic adenosine monophosphate (cAMP) is an important physiologically active substance in organisms, participating in the regulation of various cellular metabolisms. It is mainly used as an animal feed additive and in the treatment of cardiovascular and cerebrovascular diseases, leukemia, liver and kidney dysfunction, diabetes, and skin diseases. Furthermore, in livestock farming, cAMP can also be used as a feed additive for livestock and poultry, greatly promoting their growth and increasing the yield of high-quality poultry products.
[0003] cAMP synthesis mainly involves chemical and biological methods. Biological methods include microbial fermentation and biocatalytic methods. Chemical synthesis of cAMP often uses various organic solvents, which are not only expensive but also mostly harmful to human health and pollute the environment, thus its prospects are not optimistic. While bio-fermentation has advantages such as environmental friendliness and low production costs, its uncertain reaction process, easy product degradation, and accumulation of byproducts result in low cAMP yields, which cannot yet meet the requirements of large-scale industrial production. Invention patent CN102899372B discloses a two-stage dissolved oxygen-controlled fermentation method for producing cyclic adenosine monophosphate (cAMP), which significantly increases cAMP yield but only achieves a yield of 7.52 g / L. Invention patent CN104342468 discloses a method for producing cAMP using Bacillus subtilis, which clones the adenylate cyclase gene into Bacillus subtilis to obtain a genetically engineered strain, but even after cultivation, the highest fermentation level achieved is only 12.1 g / L cAMP.
[0004] The enzymatic method for producing cAMP uses adenylate cyclase to catalyze ATP production. Compared to biofermentation, the enzymatic method also offers advantages such as milder conditions and no pollution. Currently, there are few reports on enzymatic cAMP production because the efficient expression mechanism of adenylate cyclase is not fully understood, its synthetic activity is low, and it is still far from true industrialization. Therefore, it is necessary to develop a more active and stable catalytic enzyme and a simpler and more efficient new process for cAMP production. Summary of the Invention
[0005] The purpose of this invention is to overcome the low efficiency of adenylate cyclase in synthesizing cAMP, and to provide new mutants of adenylate cyclase and their applications. This invention uses wild-type adenylate cyclase as the research object, selecting *Escherichia coli* (NCBI sequence number: 41182) as the original sequence. First, a random mutation followed by activity assay was used to screen for positive mutations with good activity. Then, the mutants with high activity initially screened underwent cumulative mutation, that is, positive mutations were superimposed using molecular biology techniques to improve the synthetic activity of the original adenylate cyclase. Among many mutations, eight representative positive mutants were screened. In the subsequent cumulative experiments of these eight representative mutants, it was found that mutant 6, containing two mutation sites at position 65 (T65K) and position 252 (Y252F), could increase the cAMP synthesis activity by approximately 63 times.
[0006] The first objective of this invention is to provide an adenylate cyclase mutant, which is any of the following mutants:
[0007] Mutant 1, wherein mutant 1 is based on the amino acid sequence shown in SEQ ID NO.1, wherein the 65th position Thr is mutated to Lys;
[0008] Mutant 4, wherein mutant 4 is based on the amino acid sequence shown in SEQ ID NO.1, wherein the 65th position Thr is mutated to Lys and the 207th position Glu is mutated to Ala;
[0009] Mutant 5, wherein mutant 5 is based on the amino acid sequence shown in SEQ ID NO.1, wherein the 65th position Thr is mutated to Lys and the 304th position Met is mutated to Ile;
[0010] Mutant 6, wherein mutant 6 is based on the amino acid sequence shown in SEQ ID NO.1, wherein the 65th position of Thr is mutated to Lys and the 252nd position of Tyr is mutated to Phe;
[0011] Mutant 7, wherein mutant 7 is based on the amino acid sequence shown in SEQ ID NO.1, wherein the 65th position Thr is mutated to Lys and the 393rd position Ser is mutated to Arg;
[0012] As a preferred embodiment, mutant 6 contains two mutation sites at position 65 (T65K) and position 252 (Y252F), which can increase cAMP synthesis activity by approximately 63 times, and is renamed AC-A.
[0013] A second objective of the present invention is to provide a gene encoding the aforementioned adenylate cyclase mutant.
[0014] The third objective of this invention is to provide a recombinant bacterium expressing the above-mentioned adenylate cyclase mutant, characterized in that the recombinant strain is Escherichia coli BL21(DE3), and its expression vector is pET-29a(+).
[0015] As a preferred embodiment, the host bacterium of the recombinant bacteria is a derivative bacterium derived from Escherichia coli BL21(DE3) by knocking out adenosine deaminase. This derivative bacterium can effectively avoid the degradation of substrates by adenosine deaminase in the original host bacterium during the reaction process, thereby increasing the expression level of adenosine cyclase and the high-capacity single-enzyme synthesis of cAMP.
[0016] A method for expressing an adenylate cyclase mutant includes the following steps: transforming a recombinant plasmid into an *Escherichia coli* BL21(DE3)-derived strain to obtain a recombinant strain; activating the recombinant strain and inoculating it into LB medium or fermentation medium, waiting for the bacterial cell OD... 600 When the value reaches 0.6-30, IPTG is added to induce the expression of adenylate cyclase mutant; the final concentration of IPTG in the culture medium is 0.01-1mM, the induction conditions are 16-37℃, and the induction time is 4-48h; adenylate cyclase is obtained by centrifugation.
[0017] A fourth object of the present invention is to provide the application of the adenylate cyclase mutant in the synthesis of cAMP.
[0018] A method for synthesizing cAMP catalyzed by adenylate cyclase includes the following steps: In the enzymatic reaction, the substrate ATP concentration is 5-50 g / L, the magnesium ion concentration is 5-50 mM, and the crude adenylate cyclase solution has an enzyme activity of 20-100 U / L. After stirring evenly, the reaction is carried out at pH 7.0-9.0 and a temperature of 4-30℃ for 4-6 hours to complete the enzymatic reaction of cyclic adenosine monophosphate, producing a maximum of 20.6 g / L of cAMP. The substrate, enzyme, and salt added in this invention can be added all at once or in batches according to industrial operations.
[0019] The advantages of this invention are that it uses wild-type adenylate cyclase as the research object. First, a random mutation followed by activity determination is used to screen for positive mutations with good activity. Then, the mutants with high initial activity are subjected to cumulative mutation, resulting in a series of mutants after activity screening. Compared with the wild type, the catalytic activity and stability at low temperatures of the five adenylate cyclase mutants described in this invention are significantly improved. Furthermore, the host bacterium expressing the adenylate cyclase mutants in this invention is a derivative of Escherichia coli BL21(DE3) with adenosine deaminase knocked out. This derivative bacterium can effectively avoid substrate degradation by adenosine deaminase in the original host bacterium during the reaction. Using this recombinant bacterium for whole-cell catalytic synthesis of cAMP, a maximum of 20.6 g / L of cAMP can be generated, with a yield exceeding 95%, demonstrating good industrial prospects. Attached Figure Description
[0020] Figure 1 This is a chromatogram of adenosine triphosphate (A) and cyclic adenosine monophosphate (B) standards.
[0021] Figure 2 This describes the expression of adenylate cyclase after fermentation of the recombinant strain.
[0022] Figure 3 It is a catalytic reaction in different Mg 2+ The production of cyclic adenosine monophosphate at certain concentrations.
[0023] Figure 4 This describes the formation of cyclic adenosine monophosphate (cAMP) under different temperature conditions during the catalytic reaction.
[0024] Figure 5 This describes the formation of cyclic adenosine monophosphate (cAMP) under different pH conditions during the catalytic reaction.
[0025] Figure 6 This describes the formation of cyclic adenosine monophosphate (cAMP) under different substrate concentrations during the catalytic reaction.
[0026] Figure 7 This describes the production of cyclic adenosine monophosphate (cAMP) under different enzyme solution input conditions during the catalytic reaction.
[0027] Figure 8 This is the chromatogram at the start of the catalytic reaction.
[0028] Figure 9 This is the chromatogram at the end of the catalytic reaction. Detailed Implementation
[0029] The following examples are provided to further illustrate the present invention, but are not intended to limit the invention in any way. Processes and methods not described in detail in the following examples are conventional methods known in the art, and the reagents used in the examples are commercially available or prepared by methods well known to those skilled in the art. The following examples all achieve the objectives of the present invention.
[0030] Enzyme activity is defined as the amount of enzyme required to convert 1 micromole of ATP within 1 minute under specific conditions; one unit of activity (U) is defined as such. The instrument used was an Agilent 1260 high-performance liquid chromatograph.
[0031] Enzyme activity assay conditions: The reaction was carried out in a 1 ml reaction system containing 5 g / L ATP, 50 mM magnesium sulfate heptahydrate, 20 mM Tris-HCl, and 300 μl of enzyme solution. The reaction was conducted at 10 °C for 30 min. The reaction was terminated by adding 50 μL of reaction solution to 950 μL of mobile phase, filtering through a 0.45 μm membrane, and then performing HPLC analysis.
[0032] Chromatographic method: Column: C18 4.6*250mm*5μm; Mobile phase: A: 0.05M Na₂HPO₄ + 0.05M KH₂PO₄ + 5mM tetrabutylammonium bromide (pH adjusted to 4.6 with phosphoric acid); B: Methanol; Mobile phase A:B = 80:20; Wavelength: 254nm; Flow rate: 1.2mL / min. HPLC chromatograms of ATP and cAMP standards are shown below. Figure 1 As shown.
[0033] PCR reaction conditions: (1) 95℃ pre-denaturation for 5 min; (2) 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 2 min, 30 cycles; (3) 72℃ extension for 10 min.
[0034] Example 1: Construction of a recombinant plasmid of adenylate cyclase mutant
[0035] (1) Preparation of wild-type adenylate cyclase: The entire gene of Escherichia coli species was synthesized, NCBI sequence number: 41182. Codons were optimized, primers were designed at both ends (primers are shown in Table 1), and restriction sites were introduced for PCR (reaction system is shown in Table 2) to obtain wild-type adenylate cyclase AC (amino acid sequence is shown in SEQ ID NO:1, nucleotide sequence is shown in SEQ ID NO:2), fragment size 2550bp, protein molecular weight approximately 44kD.
[0036] Table 1. Primer sequences
[0037]
[0038] Table 2. PCR reaction system
[0039]
[0040] (2) Obtaining recombinant plasmids: After recovering the PCR amplification products, they were ligated into the corresponding sites in the E. coli expression vector pET-29a. The enzyme digestion conditions were 37℃ for 60 min, and the enzyme digestion system is shown in Table 3. The ligation conditions were 7℃ for 2 h, and the ligation system is shown in Table 4. The ligation products with the correct orientation were transformed into E. coli BL21(DE3) to obtain the strain. The strain was sent to a sequencing company for sequencing. The successfully sequenced plasmid was pET29a-AC.
[0041] Table 3. Enzyme digestion system
[0042]
[0043] Table 4. Connection System
[0044]
[0045]
[0046] (3) Optimization and modification of protein expression strains: Based on E.coli BL21(DE3), adenosine deaminase was knocked out by CRISPR / Cas9-related homologous recombination gene editing technology to obtain a derivative strain that is conducive to the single accumulation of the target product.
[0047] The enzyme activity of the newly engineered bacteria was measured after the recombinant plasmid pET29a-AC was transformed into the newly engineered bacteria (as shown in Table 5). The new engineered bacteria showed higher unit enzyme activity and a lower proportion of ATP degradation into ADP and AMP compared to E. coli BL21(DE3).
[0048] Table 5. Results of enzyme activity assays of E. coli BL21(DE3) and its derivatives.
[0049]
[0050] (4) Obtaining adenylate cyclase mutants: Random mutations were introduced using a random mutation PCR kit, and the differences in cAMP synthesis capacity between mutants and wild-type were screened using HPLC. Positive mutant single-clone strains were selected, plasmids were extracted and sequenced to obtain mutation points; a total of three single-point mutations were obtained. Mutants with high initial activity were subjected to cumulative mutation, i.e., positive mutation points were superimposed using molecular biology techniques to enhance the original cAMP synthesis activity. Using the same activity screening method under the same conditions, eight representative positive mutants were screened from among many mutations, named mutants 1-8.
[0051] Example 2: Performance of adenylate cyclase mutant
[0052] (1) Enzyme activity assay of wild type and mutant
[0053] Wild-type adenylate cyclase was transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain, which was then inoculated into LB medium and cultured until the bacterial cell OD... 600 When the pH reaches 0.6, add an appropriate concentration (0.1 mM) of IPTG to induce the expression of adenylate cyclase. The induction conditions are 16-37℃. The induction time is within 20 hours. Collect the bacterial cells horizontally in the shake flask at a concentration of 3-5 g / L. The bacterial cells are then broken down to prepare a crude enzyme solution.
[0054] Adenylate cyclase mutants 1-8 were transformed into Escherichia coli BL21(DE3) to obtain recombinant strains, which were then inoculated into LB medium and cultured until the bacterial cell OD... 600 When the pH reaches 0.6, add an appropriate concentration (0.1 mM) of IPTG to induce the expression of adenylate cyclase. The induction conditions are 16-37℃. The induction time is within 20 hours. Collect the bacterial cells horizontally in the shake flask at a concentration of 4-7 g / L. The bacterial cells are then broken down to prepare a crude enzyme solution.
[0055] Under the same conditions, the enzyme activity of wild-type adenylate cyclase and its mutants was measured. As shown in Table 6, compared with the wild type, the activities and cAMP content of adenylate cyclase mutants 1, 4, 5, 6, and 7 were significantly improved. Among them, mutant 6 (renamed AC-A), which contains two mutation sites at position 65 (T65K) and position 252 (Y252F), showed an approximately 63-fold increase in cAMP synthesis activity and a cAMP content of 78%.
[0056] Table 6. Enzyme activity and cAMP percentage of adenylate cyclase mutants
[0057] wild strain ----- 0.119 10％ Mutant 1 (T65K) 5.512 77％ Mutant 2 (S106C) 1.569 25％ Mutant 3 (R188L) 2.380 23％ Mutant 4 (T65K)(E207A) 5.339 72％ Mutant 5 (T65K)(M304I) 6.545 74％ Mutant 6 (T65K)(Y252F) 7.456 78％ Mutant 7 (T65K)(S393R) 5.220 73％ Mutant 8 (Y252F)(M304I)(S106C) 3.789 36％
[0058] (2) Effects of different host bacteria on adenylate cyclase mutants
[0059] The adenylate cyclase mutant AC-A was transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain, which was then inoculated into LB medium and cultured until the bacterial cell OD... 600 When the pH reaches 0.6, add an appropriate concentration (0.1 mM) of IPTG to induce the expression of adenylate cyclase. The induction conditions are 16-37℃. The induction time is within 20 hours. Collect the bacterial cells horizontally in the shake flask at a concentration of 4-7 g / L. The bacterial cells are then broken down to prepare a crude enzyme solution.
[0060] The adenylate cyclase mutant AC-A was transformed into Escherichia coli BL21(DE3)-derived bacteria to obtain a recombinant strain, which was then inoculated into LB medium and allowed to grow until the bacterial cell OD... 600When the pH reaches 0.6, add an appropriate concentration (0.1 mM) of IPTG to induce the expression of adenylate cyclase. The induction conditions are 16-37℃. The induction time is within 20 hours. Collect the bacterial cells horizontally in the shake flask at a concentration of 4-7 g / L. The bacterial cells are then broken down to prepare a crude enzyme solution.
[0061] Enzyme activity assays were performed on adenylate cyclase mutants from different hosts under the same conditions. The results are shown in Table 7. It can be seen that the adenylate cyclase mutant AC-A, after transformation into *E. coli* BL21(DE3) derivatives, achieved an enzyme activity of 10.018 U / ml, an 84-fold increase compared to the original wild-type enzyme activity, with a cAMP content of 96%.
[0062] Table 7. Effects of different host bacteria on enzyme activity and cAMP synthesis of adenylate cyclase mutants
[0063]
[0064] (3) Stability determination of adenylate cyclase under cryopreservation
[0065] The crude enzyme solutions of wild-type adenylate cyclase and the adenylate cyclase mutant AC-A were stored at 4°C. Compared with the wild-type, the adenylate cyclase mutant AC-A still had higher catalytic activity and stronger stability under low temperature conditions for 90 days.
[0066] Table 8. Comparison of the stability of adenylate cyclase under cryopreservation
[0067]
[0068] Example 3: Fermentation culture of adenylate cyclase mutant
[0069] The recombinant plasmid pET29a-AC-A was transformed into *E. coli* BL21(DE3)-derived bacteria to obtain a recombinant strain. After activation in LB medium, the strain was inoculated into *E. coli* fermentation medium (8 g / L tryptone, 3.2 g / L yeast extract, 1.6 g / L NaCl, 0.95 g / L anhydrous Na2HPO4, 1.6 g / L anhydrous KH2PO4, 62.5 g / L glycerol, 100 mg / L Amp, with carbon source and ammonia added as needed, pH 7.2). The bacterial cell OD was then increased. 600 When the value reached 25, an appropriate concentration (1M) of IPTG was added to induce adenylate cyclase expression. The induction conditions were 16-37℃, and the induction culture time was within 48 hours. Several batches of experiments were conducted. Under fed-batch fermentation conditions, the cell concentration was 100-200 g / L, with an average cell concentration of 150 g / L. Adenylate cyclase expression was as follows... Figure 2 As shown, the bacterial cells were crushed to prepare a crude enzyme solution. HPLC analysis revealed an enzyme activity of 9-11 U / ml.
[0070] Example 4: Optimization of reaction conditions for cAMP synthesis catalyzed by adenylate cyclase mutant
[0071] The following reaction system was configured, taking a 1L reaction system as an example: adenosine triphosphate 50g / L, crude enzyme solution 80U, pH 8.0, reaction temperature 4℃, magnesium sulfate 10-50mM. After 4-6 hours, the results are as follows. Figure 3 As shown, the cAMP formation rate is better at the end of the reaction when magnesium sulfate is between 40-50 mM, with 40 mM being the optimal value.
[0072] The following reaction system was configured, taking a 1L reaction system as an example: adenosine triphosphate 50g / L, crude enzyme solution 80U, pH 8.0, magnesium sulfate 40mM, reaction temperature between 4-30℃, and results after 4-6 hours as shown. Figure 4 As shown, cAMP production is better at the end of the reaction between 4-10℃, with 4℃ being the optimal temperature.
[0073] The following reaction system was configured, taking a 1L reaction system as an example: 50g / L adenosine triphosphate, 80U crude enzyme solution, 40mM magnesium sulfate, reaction temperature 4℃, pH controlled at 7.0-9.0 during the reaction process, and results obtained after 4-6 hours are as follows. Figure 5 As shown, cAMP production was better between 7.5 and 8.5 at the end of the reaction, with 8.0 being the optimal value.
[0074] The following reaction system was configured, taking a 1L reaction system as an example: adenosine triphosphate 5-50g / L, crude enzyme solution 80U, magnesium sulfate 40mM, reaction temperature 4℃, pH controlled at 8.0 during the reaction process, and results obtained after 4-6 hours as follows. Figure 6 As shown, the cAMP generation rate can reach over 95% when the substrate concentration is between 5-50 g / L.
[0075] The following reaction system was configured, taking a 1L reaction system as an example: 50g / L adenosine triphosphate, 20-100U crude enzyme solution, 40mM magnesium sulfate, reaction temperature 4℃, pH controlled at 8.0 during the reaction process, and results obtained after 4-6 hours as follows. Figure 7 As shown, the cAMP generation rate is better when the enzyme solution input is between 60-80U, with 80U being the optimal amount.
[0076] Crude enzyme solutions of mutants 1, 4, 5, 6, and 7 were prepared under optimal reaction conditions. The reaction systems were configured, and the conversion of ATP to cAMP was completed in all cases after 4-6 hours. The initial HPLC chromatograms are shown below. Figure 8 The HPLC chromatogram after the reaction is complete is shown in [reference needed]. Figure 9 The ATP conversion rate and the quality results of the generated cAMP product are shown in Table 9.
[0077] Table 9. Effects of adenylate cyclase mutants on ATP conversion rate and quality of cAMP product.
[0078] ATP conversion rate 95.20％ 95.40％ 96.70％ 98.20％ 97.43％ cAMP 19.07 19.27 19.36 20.6 19.71
Claims
1. An adenylyl cyclase mutant, characterized in that, It is any of the following mutants: Mutant 1, wherein mutant 1 is obtained by mutating Thr at position 65 to Lys based on the amino acid sequence shown in SEQ ID NO.1; Mutant 4 is obtained by mutating Thr at position 65 to Lys and Glu at position 207 to Ala based on the amino acid sequence shown in SEQ ID NO.
1. Mutant 5 is obtained by mutating Thr at position 65 to Lys and Met at position 304 to Ile based on the amino acid sequence shown in SEQ ID NO.
1. Mutant 6 is obtained by mutating Thr at position 65 to Lys and Tyr at position 252 to Phe based on the amino acid sequence shown in SEQ ID NO.
1. Mutant 7 is obtained by mutating Thr at position 65 to Lys and Ser at position 393 to Arg based on the amino acid sequence shown in SEQ ID NO.
1.
2. The gene encoding the adenylate cyclase mutant of claim 1.
3. A recombinant bacterium expressing the adenylate cyclase mutant of claim 1, characterized in that, The host bacteria of the recombinant bacteria are derived from Escherichia coli BL21 (DE3) by knocking out adenosine deaminase.
4. The recombinant bacteria of the adenylate cyclase mutant according to claim 3, characterized in that, The expression vector for the recombinant bacteria is pET-29a(+).
5. The application of the adenylate cyclase mutant of claim 1 in the synthesis of cAMP.
6. The application according to claim 5, characterized in that, In the synthesis system, the concentration of substrate ATP is 5-50 g / L, the concentration of magnesium ions is 10-50 mM, and the crude enzyme solution of adenylate cyclase is 20-100 U / L in terms of enzyme activity; the reaction is carried out at pH 7.0-9.0, temperature 4-30℃, and time 4-6 h.