Enzyme mutant and application thereof in biosynthesis of cyclic dinucleotide cGAMP

Abstract

The invention relates to an enzyme mutant and application thereof in biosynthesis of cyclic dinucleotide cGAMP. The cGAS enzyme mutant is obtained by mutating valine (V) at the 100th site into alanine (A) on the basis of an amino acid sequence as shown in SEQ ID NO: 1. A genetically engineered bacterium for expressing the mutant is constructed, a high-copy plasmid pRSFDuet-1 is introduced as an allosteric activator, cyclic dinucleotide cGAMP is efficiently catalyzed and synthesized by utilizing endogenous ATP and GTP of escherichia coli, and the yield can reach 256 mg / L. The method has the advantages of simplicity and convenience in operation, low cost, environment friendliness and the like, and has a wide application prospect in industrial biosynthesis of cyclic dinucleotide cGAMP.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an enzyme mutant and its application in the biosynthesis of cyclic dinucleotide cGAMP. Background Technology

[0002] In mammalian cells, the presence of double-stranded DNA in the cytoplasm is a strong danger signal, typically indicating pathogen infection (such as viral or bacterial infection), cell damage, or cancer. This ectopic double-stranded DNA can allosterically activate cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), which catalyzes the synthesis of a cyclic dinucleotide 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cyclic dinucleotide cGAMP). As a messenger molecule, cGAMP binds to the interferon gene stimulator STING on the endoplasmic reticulum membrane, initiating STING oligomerization and transport to the Golgi network, thereby launching a downstream signaling cascade that ultimately leads to the secretion of type I and type III interferons. cGAMP can be readily exported extracellularly, serving as a soluble immune delivery mediator, transmitting the effects of ectopic double-stranded DNA recognition to all cells in the microenvironment, triggering a broad innate immune response. Based on this, cGAMP and its STING agonists are currently under extensive research, representing a highly promising drug molecule as mucosal adjuvants for tumor immunotherapy and anti-infective vaccines.

[0003] The chemical synthesis of cyclic dinucleotide cGAMP has been achieved through various synthetic routes. Among them, the "eight-step one-pot method" based on phosphorous amide is the most commonly used synthetic route, but it suffers from drawbacks such as low yield (approximately 5%) and the need for harmful organic solvents. As an alternative to chemical synthesis, the enzymatic biosynthesis of cyclic dinucleotide cGAMP can be achieved using cGAS enzyme heterologously expressed and purified from *E. coli*. This method uses double-stranded DNA as an allosteric activator. In HEPES buffer, cGAS enzyme catalyzes the conversion of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) into cyclic dinucleotide cGAMP, achieving a conversion rate as high as 85-90%. Enzymatic synthesis has the advantages of not requiring organic solvents and achieving high conversion rates, making it a relatively environmentally friendly process. However, the disadvantages of enzymatic biosynthesis lie in the high cost of its substrates ATP and GTP, as well as the double-stranded DNA used as an allosteric activator, and the expensive expression and purification process of cGAS enzyme, which limits its large-scale application. Summary of the Invention

[0004] Technical problem solved: This invention overcomes the shortcomings of existing cyclic dinucleotide cGAMP synthesis technology. It addresses the problems of low yield and high pollution in chemical synthesis methods, as well as the high cost and complex purification process of substrates and allosteric activators in enzymatic synthesis. It provides an enzyme mutant and its application in the biosynthesis of cyclic dinucleotide cGAMP.

[0005] Technical solution: A cGAS enzyme mutant, wherein the mutant is obtained by mutating valine (V) at position 100 to alanine (A) based on the amino acid sequence shown in SEQ ID NO:1, and the mutated amino acid sequence is shown in SEQ ID NO:3.

[0006] The coding gene of the above-mentioned cGAS enzyme mutant has a nucleotide sequence as shown in SEQ ID NO:2.

[0007] A recombinant plasmid containing the aforementioned encoding gene.

[0008] A genetically engineered bacterium containing the aforementioned recombinant plasmid.

[0009] The host of the above-mentioned genetically engineered bacteria is Escherichia coli.

[0010] The above-mentioned cGAS enzyme mutants are used in the preparation of pharmaceutical compositions or vaccine adjuvants for stimulating interferon production or enhancing immune responses.

[0011] A method for synthesizing cyclic dinucleotide cGAMP includes the following steps: using the above-mentioned cGAS enzyme mutant, catalyzing the synthesis of cGAMP from ATP and GTP in vivo in the form of recombinant cells; introducing additional plasmid DNA into the recombinant cells as an allosteric activator of the cGAS enzyme.

[0012] The aforementioned additional plasmids are high-copy plasmids.

[0013] Preferably, the high-copy plasmid is pRSFDuet-1.

[0014] The culture conditions for the recombinant cells were as follows: final IPTG concentration of 0.1 mM, fermentation temperature of 30℃, and fermentation time of 48 hours.

[0015] Beneficial Effects: The cGAS enzyme mutant obtained through site-directed mutagenesis in this invention exhibits superior catalytic activity compared to the wild type and other site-specific mutants, significantly increasing the synthesis yield of cyclic dinucleotide (cGAMP). A recombinant *E. coli* strain producing cGAMP was constructed, capable of biosynthesizing cGAMP using endogenous ATP, GTP, and double-stranded DNA from *E. coli*, and excreting it into the culture medium, greatly reducing the high cost of substrates and allosteric agents. By modifying the cGAS enzyme using protein engineering techniques and screening exogenous plasmids with different copy numbers, the limited yield of cGAMP synthesized endogenously in *E. coli* was overcome, achieving low-cost and efficient biosynthesis of cGAMP. Attached Figure Description

[0016] Figure 1This invention illustrates the effect of cGAS enzymes from different sources on the synthesis of cyclic dinucleotide cGAMP in Escherichia coli in Example 1 of this invention.

[0017] Figure 2 This describes the effect of the cGAS mutant in Example 2 of the present invention on the synthesis of cyclic dinucleotide cGAMP in Escherichia coli.

[0018] Figure 3 This describes the effect of additional plasmids with different copy numbers in Example 3 of the present invention on the synthesis of cyclic dinucleotide cGAMP in Escherichia coli. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0023] The culture medium used in the examples is:

[0024] TB medium: Weigh 12 g Tryptone, 24 g Yeast extract, 2.2 g KH2PO4, and 9.4 g K2HPO4, dissolve them in distilled water, and bring the volume to 1 L (adjust the pH to 7.0-7.5 with NaOH). Sterilize at 121℃ for 20 min.

[0025] Example 1: Discovery and screening of cGAS enzymes from different sources

[0026] The genes encoding EqcGAS (Genbank No. XP_046532466), SccGAS (Genbank No. XP_047414467), CvcGAS (Genbank No. XP_062954526), ​​and TocGAS (Genbank No. XP_037374640) were synthesized in whole and assembled into the NcoI and XhoI restriction sites of the pET-28a(+) vector. This step was performed by Sangon Biotech (Shanghai, China). A series of pET-28a(+)-cGAS plasmids were obtained. The genes carried by each plasmid are shown in Table 1. pET-28a(+) was purchased from Novagen.

[0027] Table 1

[0028]

[0029] Subsequently, a series of pET-28a(+)-cGAS plasmids were thermally transformed into E. coli BL21(DE3) using a chemical thermal shock method. The thermal shock transformation steps are as follows:

[0030] (1) Remove competent cells from a -80℃ freezer and place them on ice to thaw;

[0031] (2) Take an appropriate volume of plasmid and add it to competent cells, and place it on ice for 10 min;

[0032] (3) Place competent cells at 42℃ and heat shock them accurately for 45s, then place them on ice for 3min;

[0033] (4) Add 1 mL of SOC culture medium, and then place in a shaker at 37°C for 0.5 h to recover;

[0034] (5) Spread the bacterial solution on the corresponding antibiotic plate and incubate overnight at 37°C until single colonies grow.

[0035] The obtained strains were named Ec-EqcGAS, Ec-SccGAS, Ec-CvcGAS, and Ec-TocGAS, respectively. The plasmids contained in each strain are shown in Table 2.

[0036] Table 2

[0037]

[0038] The obtained strain was applied to the fermentation of recombinant Escherichia coli that synthesizes cyclic dinucleotide cGAMP, and the specific method is as follows:

[0039] Transformants were picked from LB solid medium and cultured overnight in LB liquid medium. They were then inoculated at a 1% inoculum onto TB medium and cultured at 37°C in a shaker until OD reached.600 The concentration was 0.6-0.8. IPTG was added to a final concentration of 0.1 mM for induced fermentation for 24 h / 48 h, with the fermentation temperature controlled at 30℃. After fermentation, the cells were collected, centrifuged at 12000 rpm, and the supernatant was collected for HPLC analysis. The results are as follows. Figure 1 As shown.

[0040] As shown in the figure, all four cGAS enzymes were active and could catalyze the synthesis of cyclic dinucleotide cGAMP from ATP and GTP in E. coli. The yield of cyclic dinucleotide cGAMP was significantly higher after 48 h of fermentation than after 24 h of fermentation. Among them, the CvcGAS source had the highest yield of cyclic dinucleotide at 48 h of fermentation, reaching 118 mg / L.

[0041] Example 2 Construction of CvcGAS enzyme mutants and screening of benign mutants

[0042] Based on the relatively high-yielding cyclic dinucleotide cGAMP recombinant strain Ec-CvcGAS, alanine scanning was performed on the potential mutation sites of CvcGAS enzyme to construct a series of site-directed mutant plasmids, and corresponding recombinant strains were constructed. The strains and plasmids used are shown in Table 3.

[0043] Table 3

[0044]

[0045] Construction method of plasmid pET-28a(+)-CvcGAS_G52A:

[0046] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-G52A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-G52A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_G52A.

[0047] Construction method of plasmid pET-28a(+)-CvcGAS_D67A:

[0048] Using pET-28a(+)-CvcGAS plasmid as a template, fragment 1 was obtained by PCR amplification using primers F-D67A / R-Kana, and fragment 2 was obtained by PCR amplification using primers F-Kana / R-D67A. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_D67A.

[0049] Construction method of plasmid pET-28a(+)-CvcGAS_V100A:

[0050] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-V100A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-V100A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_V100A.

[0051] Construction method of plasmid pET-28a(+)-CvcGAS_P121A:

[0052] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-P121A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-P121A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_P121A.

[0053] Construction method of plasmid pET-28a(+)-CvcGAS_E182A:

[0054] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-E182A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-E182A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_E182A.

[0055] Construction method of plasmid pET-28a(+)-CvcGAS_D306A:

[0056] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-D306A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-D306A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_D306A.

[0057] Construction method of plasmid pET-28a(+)-CvcGAS_E413A:

[0058] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-E413A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-E413A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_E413A.

[0059] Construction method of plasmid pET-28a(+)-CvcGAS_F479A:

[0060] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-F479A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-F479A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_F479A.

[0061] Construction method of plasmid pET-28a(+)-CvcGAS_G502A:

[0062] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-G502A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-G502A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_G502A.

[0063] Construction method of plasmid pET-28a(+)-CvcGAS_K516A:

[0064] Using pET-28a(+)-CvcGAS plasmid as a template, PCR amplification was performed using primers F-K516A / R-Kana to obtain fragment 1, and PCR amplification was performed using primers F-Kana / R-K516A to obtain fragment 2. Fragments 1 and 2 were then assembled using seamless cloning to obtain plasmid pET-28a(+)-CvcGAS_K516A.

[0065] The primers required to construct the above plasmids are shown in Table 4.

[0066] Table 4

[0067]

[0068] Different CvcGAS mutant plasmids were introduced into *E. coli* BL21 (DE3) using a chemical heat shock method. The heat shock transformation steps are as follows:

[0069] (1) Remove competent cells from a -80℃ freezer and place them on ice to thaw;

[0070] (2) Take an appropriate volume of plasmid and add it to competent cells, and place it on ice for 10 min;

[0071] (3) Place competent cells at 42℃ and heat shock them accurately for 45s, then place them on ice for 3min;

[0072] (4) Add 1 mL of SOC medium, and then place in a shaker at 37°C for 0.5 h to recover;

[0073] (5) The bacterial suspension was spread onto the corresponding antibiotic plates and incubated overnight at 37°C until single colonies grew. The following strains were obtained: Ec-CvcGAS_G52A, Ec-CvcGAS_D67A, Ec-CvcGAS_V100A, Ec-CvcGAS_P121A, Ec-CvcGAS_E182A, Ec-CvcGAS_D306A, Ec-CvcGAS_E413A, Ec-CvcGAS_F479A, Ec-CvcGAS_G502A, and Ec-CvcGAS_K516A. The plasmids contained in each strain are shown in Table 2.

[0074] The recombinant strain Ec-CvcGAS containing wild-type CvcGAS enzyme was used in the fermentation of recombinant Escherichia coli to synthesize cyclic dinucleotide cGAMP, and the specific method was as follows:

[0075] Transformants were picked from LB solid medium and cultured overnight in LB liquid medium. They were then inoculated at a 1% inoculum onto TB medium and cultured at 37°C with a shaker until OD reached [value missing]. 600 The concentration was 0.6-0.8. IPTG was added to a final concentration of 0.1 mM and fermented for 48 h at 30℃. After fermentation, the cells were collected, centrifuged at 12000 rpm, and the supernatant was analyzed by HPLC. The results are as follows. Figure 2 As shown.

[0076] As shown in the figure, after 48 hours of fermentation, the yield of cyclic dinucleotide (cGAMP) in most mutants decreased to varying degrees compared with the wild type. The yield of the CvcGAS_K516A mutant was 124 mg / L, which was the same as that of the wild type. However, the yields of the CvcGAS_D67A, CvcGAS_V100A, and CvcGAS_E413A mutants increased to varying degrees compared with the wild type, at 143, 187, and 165 mg / L, respectively. The results indicate that site-directed mutagenesis of CvcGAS, which screens for benign mutants, significantly improves the yield of cGAMP. The CvcGAS_V100A mutant showed the highest yield of cGAMP, approximately 187 mg / L.

[0077] Example 3: Effect of extra plasmid copy number on de novo synthesis of cyclic dinucleotide cGAMP

[0078] cGAS enzymes require double-stranded DNA as an allosteric activator to perform their catalytic function. Based on the screening and identification of the superior mutant CvcGAS_V100A, in order to further improve its production of cyclic dinucleotide cGAMP, three compatible plasmids with different copy numbers were introduced into the Ec-CvcGAS_V100A strain.

[0079] The strains and plasmids used are shown in Table 5.

[0080] Table 5

[0081]

[0082] The strains Ec-CvcGAS_V100A, Ec-CvcGAS_V100A_low, Ec-CvcGAS_V100A_medium, and Ec-CvcGAS_V100A_high were applied to the fermentation of recombinant E. coli for synthesizing cyclic dinucleotide cGAMP. The specific method is as follows:

[0083] Transformants were picked from LB solid medium and cultured overnight in LB liquid medium. They were then inoculated at a 1% inoculum onto TB medium and cultured at 37°C with a shaker until OD reached [value missing]. 600 The concentration was 0.6-0.8. IPTG was added to a final concentration of 0.1 mM for induction fermentation for 48 h. After fermentation was complete, the cells were collected, centrifuged at 12000 rpm, and the supernatant was analyzed by HPLC. The results are as follows. Figure 3 As shown.

[0084] As shown in the figure, the introduction of exogenous plasmids with different copy numbers into strain Ec-CvcGAS_V100A can increase the yield of cyclic dinucleotide cGAMP. The yield of cyclic dinucleotide cGAMP shows an increasing trend with the increase of plasmid copy number. After fermentation for 48 h, the strain Ec-CvcGAS_V100A_high containing the high copy number plasmid pRSFDuet-1 has the highest yield of cyclic dinucleotide cGAMP, which is 256 mg / L, 1.37 times that of the control strain Ec-CvcGAS_V100A.

Claims

1. A cGAS enzyme mutant, characterized in that, The mutant was obtained by mutating valine at position 100 to alanine based on the amino acid sequence shown in SEQ ID NO:

1. The mutated amino acid sequence is shown in SEQ ID NO:

3.

2. The encoding gene of the cGAS enzyme mutant as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

3. A recombinant plasmid, characterized in that, It includes the coding gene as described in claim 2.

4. A genetically engineered bacterium, characterized in that, It contains the recombinant plasmid as described in claim 3.

5. The genetically engineered bacteria as described in claim 4, characterized in that, The host of the genetically engineered bacteria is Escherichia coli. (Escherichia coli) .

6. The use of the cGAS enzyme mutant of claim 1 in the preparation of pharmaceutical compositions or vaccine adjuvants for stimulating interferon production or enhancing immune responses.

7. A method for synthesizing cyclic dinucleotide cGAMP, characterized in that, Includes the following steps: Using the cGAS enzyme mutant as described in claim 1, cGAMP is synthesized in vivo from ATP and GTP in recombinant cells; additional plasmid DNA is introduced into the recombinant cells as an allosteric activator of the cGAS enzyme.

8. The method as described in claim 7, characterized in that, The additional plasmid is a high-copy plasmid.

9. The method as described in claim 8, characterized in that, The high-copy plasmid is pRSFDuet-1.

10. The method as described in claim 7, characterized in that, The culture conditions for the recombinant cells included: a final IPTG concentration of 0.1 mM, a fermentation temperature of 30°C, and a fermentation time of 48 hours.

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