A diguanylate cyclase mutant, engineered bacterium and its application

Abstract

The present invention relates to the field of biosynthesis technology, and particularly relates to a diguanylate cyclase mutant, an engineered bacterium and their applications. In the present invention, the diguanylate cyclase gene is derived from Pseudothermotoga subterranea, and its amino acid sequence is as shown in SEQ ID NO.1, and its nucleotide sequence is as shown in SEQ ID NO.2; the single mutants of diguanylate cyclase are obtained by single mutations at positions 262, 264, 265, and 280 of the amino acid sequence shown in SEQ ID NO.1, and the double mutants are obtained by double mutations at positions 262 and 264, and 262 and 265 of the amino acid sequence shown in SEQ ID NO.2. Among them, the enzyme activity of the 262 / 264 double mutant screened is 4.12 times that of the original enzyme. The engineered bacterial strain of the diguanylate cyclase mutant constructed in the present invention effectively solves the problem of product inhibition and realizes the green production of cyclic diguanylate.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and particularly relates to a diguanylate cyclase mutant, an engineered bacterium and their applications. Background Art

[0002] Cyclic diguanylic acid (c-di-GMP) is a second messenger widely present in bacteria, and participates in regulating various physiological functions, including regulating the formation of biofilms, bacterial motility and influencing the formation of virulence factors, etc. Benziman et al. first discovered this substance, c-di-GMP, when studying the cellulose biosynthesis pathway of Acetobacter xylinum subsp. Gluconicum, and it has an allosteric activation function on cellulose synthase [Ross P, Weinhouse H, Aloni Y, et al. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid [J]. Nature, 1987, 325(6101): 279-281.]. c-di-GMP is a cyclic dinucleotide formed by the condensation of two molecules of GTP, and its synthesis and degradation metabolism in cells are regulated by diguanylate cyclase (DGC) and phosphodiesterase (PDE), respectively.

[0003] At present, c-di-GMP can be prepared by two synthetic methods: chemical synthesis and biosynthesis. In 2003, Kawai R et al. reported an improved method for the chemical synthesis of c-di-GMP [Rie, Kawai, Reiko, et al. A new synthetic approach to cyclic bis(3′→5′)diguanylic acid[J]. Nucleic Acids Symp Ser, 2003.]. In 2010, Barbara L et al. reported a one-pot method for the synthesis of c-di-GMP, which involves a total of eight chemical reactions [Gaffney B L, Veliath E, Zhao J, et al. One-flask syntheses of c-di-GMP and the [Rp,Rp] and [Rp,Sp] thiophosphate analogues.[J]. Organic Letters, 2010, 12(14):3269.]. However, these chemical synthesis methods have problems such as cumbersome steps, low yield, and environmental unfriendliness. Compared with chemical synthesis methods, the method of DGC catalyzing GTP to synthesize c-di-GMP is simple and efficient, involving only one condensation of two GTP molecules.Reported DGCs include PleD [Paul, R. Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain [J]. Genes & Development, 2004, 18(6): 715-727.], VCA0956 [Hunter J L, Severin G B, Koestler B J, et al. The Vibrio cholerae diguanylate cyclase VCA0965 has an AGDEF active site and synthesizes cyclic di-GMP [J]. Bmc Microbiology, 2014, 14(1): 1-10.] and WspR [Rao F, Yang Y, Qi Y, et al. Catalytic mechanism of cyclic di-GMP-specific phosphodiesterase: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa. [J]. Journal of bacteriology, 2008, 190(10): 3622-3631.], etc. However, all of the above DGCs show strong product inhibition, and the ki values are all in the micromolar range.

[0004] In 2007, Christen M et al. studied different DGC mutants and also found that there is an I-site (RxxD motif) at the N-terminus of some GG(D / E)EF domains, which can bind to c-di-GMP [Christen B, Christen M, Paul R, et al. Allosteric Control of Cyclic di-GMP Signaling [J]. Journal of Biological Chemistry, 2006, 281(42): 32015-32024.]. When the concentration of c-di-GMP in the body is too high, this binding mechanism will inhibit the enzyme activity of DGC and play a feedback regulation function. In 2009, Rao F et al. reported that a tDGC derived from the thermophilic microorganism Thermotoga maritima (T. maritima) was used for the enzymatic synthesis of c-di-GMP. This enzyme has high thermal stability and low product inhibition, and this low product inhibition was obtained by mutating the conserved residues at the I-site [Rao F, Pasunooti S, Ng Y, et al. Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase [J]. Analytical Biochemistry, 2009, 389(2): 138-142.]. In 2011, Spehr V et al. reported a method for the large-scale production of c-di-GMP, in which a mutant form of diguanylate cyclase from Caulobacter crescentus (C. crescentus) was used to provide DGC in the form of inclusion bodies [Spehr V, Warrass R, Hcherl K, et al. Large-Scale Production of the Immunomodulator c-di-GMP from GMP and ATP by an Enzymatic Cascade [J]. Applied biochemistry and biotechnology, 2011, 165(3-4): 761-775.]. Patent CN111647611A provides a modified gene of diguanylate cyclase with high expression in Escherichia coli. Through codon optimization, the product inhibition phenomenon was improved, but the conversion rate of the catalytic reaction of the enzyme provided by this method is still not ideal enough. Therefore, when synthesizing c-di-GMP by enzymatic method, the DGC enzyme and its enzyme activity that can reduce product inhibition and improve the expression level at the same time are still the key problems to be solved. Summary of the Invention

[0005] To solve the above problems, the object of the present invention is to provide a diguanylate cyclase mutant, an engineered bacterium and their applications.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] The first object of the present invention is to provide a diguanylate cyclase mutant, wherein the diguanylate cyclase mutant comprises a mutant obtained by single mutation at positions 262, 264, 265, and 280 of the amino acid sequence shown in SEQ ID NO.1, or a mutant obtained by double mutation at positions 262 and 264, and positions 262 and 265 of the amino acid sequence shown in SEQ ID NO.1.

[0008] In one embodiment of the present invention, the single mutation at position 262 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of arginine at position 262 to phenylalanine, tyrosine or threonine;

[0009] The single mutation at position 264 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of asparagine at position 264 to aspartic acid, lysine or glycine;

[0010] The single mutation at position 265 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of aspartic acid at position 265 to valine, tryptophan or arginine;

[0011] The single mutation at position 280 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of tyrosine at position 280 to arginine.

[0012] In one embodiment of the present invention, the double mutation at positions 262 and 264 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of arginine at position 262 to threonine, and the mutation of asparagine at position 264 to lysine;

[0013] The double mutation at positions 262 and 265 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of arginine at position 262 to threonine, and the mutation of aspartic acid at position 265 to tryptophan.

[0014] The second object of the present invention is to provide a coding gene, which encodes the coding gene of the diguanylate cyclase mutant.

[0015] The third object of the present invention is to provide an engineered bacterium, which contains the above coding gene.

[0016] The fourth object of the present invention is to provide an application of an engineered bacterium in catalyzing the synthesis of cyclic diguanosine monophosphate from guanosine triphosphate. Guanosine triphosphate is used as a substrate, an activator and a catalyst are added, and after mixing in a buffer system, cyclic diguanosine monophosphate is obtained through a cyclization reaction;

[0017] Among them, the catalyst is selected from one of the engineered bacterium cells or the protein purified from the engineered bacterium cells.

[0018] In one embodiment of the present invention, the engineered bacterium cells are obtained by centrifugation after the engineered bacterium is induced and cultured.

[0019] In one embodiment of the present invention, the final concentration ratio of guanosine triphosphate, activator, and catalyst is 2000:1000:1.

[0020] In one embodiment of the present invention, the activator is selected from one of magnesium chloride, manganese sulfate, manganese chloride, cobalt chloride, and calcium chloride;

[0021] The buffer system is selected from one of Tris-HCl buffer, HEPES buffer, phosphate buffer, and acetate-sodium acetate buffer.

[0022] In one embodiment of the present invention, during the cyclization reaction, the temperature is 30-90 °C, the time is 0.5-2 h, and the pH is 7.0-8.0.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention performs enzyme modification on the diguanosine cyclase derived from Pseudocaldithrix subterranea at the molecular level, and screens five single mutants and two double mutants with significantly improved enzyme activity. The highest activity is increased by 4.12 times, and the product inhibition phenomenon is eliminated. At a substrate concentration of 20 mM, the conversion rate of GTP is greater than 95%, and the product yield reaches 85.3%, which is suitable for the large-scale and low-cost production of cyclic diguanosine monophosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the protein electrophoresis pattern of the diguanosine cyclase isolated and purified in Example 2; among them, 1-3 are the whole cell lysate, precipitate, and supernatant, 4 is the effluent from the column, and 5-7 are the eluates;

[0026] Figure 2 It is the high performance liquid chromatography pattern of the reaction solution in Example 3;

[0027] Figure 3 It is the mass spectrometry pattern of cyclic diguanosine monophosphate in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides a diguanylate cyclase mutant, which includes mutants obtained by single mutations at positions 262, 264, 265, and 280 of the amino acid sequence shown in SEQ ID NO.1, or mutants obtained by double mutations at positions 262 and 264, and positions 262 and 265 of the amino acid sequence shown in SEQ ID NO.1.

[0029] In one embodiment of the present invention, the single mutation at position 262 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of arginine at position 262 to phenylalanine, tyrosine, or threonine;

[0030] The single mutation at position 264 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of asparagine at position 264 to aspartic acid, lysine, or glycine;

[0031] The single mutation at position 265 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of aspartic acid at position 265 to valine, tryptophan, or arginine;

[0032] The single mutation at position 280 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of tyrosine at position 280 to arginine.

[0033] In one embodiment of the present invention, the double mutation at positions 262 and 264 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of arginine at position 262 to threonine, and the mutation of asparagine at position 264 to lysine;

[0034] The double mutation at positions 262 and 265 of the amino acid sequence shown in SEQ ID NO.1 is specifically the mutation of arginine at position 262 to threonine, and the mutation of aspartic acid at position 265 to tryptophan.

[0035] The present invention provides a coding gene, which is a coding gene encoding a diguanylate cyclase mutant.

[0036] The present invention provides an engineered bacterium, which contains the above-mentioned coding gene.

[0037] The present invention provides an application of an engineered bacterium in catalyzing the synthesis of cyclic diguanylate from guanosine triphosphate. Using guanosine triphosphate as a substrate, after adding an activator and a catalyst, it is mixed evenly in a buffer system, and cyclic diguanylate is obtained through a cyclization reaction;

[0038] Among them, the catalyst is selected from one of the engineered bacterium cells or the protein purified from the engineered bacterium cells.

[0039] In one embodiment of the present invention, the engineered bacterial cells are obtained by centrifugation after induced culture of the engineered bacteria.

[0040] In one embodiment of the present invention, the final concentration ratio of guanosine triphosphate, activator, and catalyst is 2000:1000:1.

[0041] In one embodiment of the present invention, the activator is selected from one of magnesium chloride, manganese sulfate, manganese chloride, cobalt chloride, and calcium chloride;

[0042] The buffer system is selected from one of Tris-HCl buffer, HEPES buffer, phosphate buffer, and acetate-sodium acetate buffer.

[0043] In one embodiment of the present invention, during the cyclization reaction, the temperature is 30-90 °C and the time is 0.5-2 h.

[0044] The present invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0045] In the following examples, unless otherwise specified, the reagents used are commercially available reagents, and the detection means and methods used are conventional detection means and methods in the art.

[0046] In the primer sequences of the present application, "N" refers to A, C, G, or T; "M" refers to A or C; "K" refers to G or T.

[0047] Example 1

[0048] This example provides the construction of an engineered bacterium of Pseudoalteromonas thermohalophila PhDgc and mutants.

[0049] Based on the gene database of Pseudoalteromonas thermohalophila PhDgc (P. hypogea, NCBI ACCESSION: WP_031505526), the amino acid sequence shown in SEQ ID NO.1 was artificially synthesized (Shanghai Qingke Co., Ltd.) after codon optimization for Escherichia coli, and the gene PhDgc encoding diguanylate cyclase was obtained (as shown in SEQ ID NO.2).

[0050] (1) The plasmid vector pET28a was digested with BamHI and XhoI double enzymes. After the digestion product was recovered using a gel recovery kit (Omega, USA), it was ligated with the gene PhDgc encoding diguanylate cyclase through T4 ligase (ThermoFisher, USA) to obtain a ligation product.

[0051] (2) Transform 10 μL of the ligation product prepared in step (1) into competent cells of the expression host BL21(DE3), incubate on ice for about 25 min, heat shock in a 42 °C water bath for 90 s, immediately incubate on ice for 2 min, then add 800 μL of antibiotic-free LB liquid medium, and incubate and resuscitate on a shaker at 37 °C for 50 min. Centrifuge at 4000 rpm for 2 min, pour off 700 μL of the supernatant, resuspend the bacterial cells, spread the remaining bacterial solution on a kanamycin-resistant plate, and culture overnight at 37 °C.

[0052] After positive clones were verified by colony PCR using the primers phdgc-F (shown in SEQ ID NO.3) and phdgc-R (shown in SEQ ID NO.4) as shown in Table 1, sequencing verification was carried out. After successful sequencing, an engineered bacterium containing the pET28a-PhDgc recombinant plasmid (expressing diguanylate cyclase) was obtained.

[0053] Using the recombinant vector pET28a-PhDgc as a template, site-directed saturation mutagenesis was carried out on sites such as R174, E220, R262, S263, N264, D265, and Y280 of PhDgc by the whole plasmid PCR method. The primers for each saturation mutagenesis used NNK degenerate primers (specifically shown in Table 1). After purification of the PCR products, seamless cloning was used to circularize the linear plasmid by self-ligation. The ligation product was transformed into the expression host BL21(DE3). After positive clones were verified by colony PCR and enzyme digestion, sequencing verification was carried out. After successful sequencing, the pET28a-PhDgc mutant recombinant plasmid and the single mutant engineered bacterium were obtained. The double mutant engineered bacterium was obtained by combining mutations at two sites on the basis of the single mutant engineered bacterium.

[0054] Among them, the amino acid sequence shown in SEQ ID NO.1 is as follows:

[0055] MSKFETCVEILQYLGEIASKLLSGQTENVYQDVLEKAMKIVPGAQAGSILV

[0056] RENDRFVYVAAVGYELEELQKVSFTVEEEEEWVGRDRSYAIVLREDIERFDEAL

[0057] LKSDKRVGILANFGGIKKIKATLIIPVRIKNELALVLNLDNFERSDAFNEDSIVLA

[0058] KVLANVLGIIFNRLELENQLRVKNQLLEYMSYHDTLTNLPNRRLLEEFAEKMLK

[0059] LAKRENKPLSILFMDLDKFKPINDTYGHQVGDEVMKLVAARLERFTRSNDMVS

[0060] RFGGDEFVISAYDCSKQDAKAFERLIKAMEEPMKIDQLTLQLSASVGIATFPEDG

[0061] DELTKLIRVADERLYMAKKSGSKIVTQG

[0062] The nucleotide sequence shown in SEQ ID NO.2 is as follows:

[0063]

[0064] Table 1 Summary Table of Primers

[0065]

[0066] Example 2

[0067] This example provides the screening of mutants.

[0068] High-activity mutant strains were screened from the obtained saturated mutant libraries of R174, E220, R262, S263, N264, D265, and Y280.

[0069] The specific screening method is as follows: The engineered bacteria constructed in Example 1 were inoculated into a 5 mL LB test tube containing kanamycin resistance and cultured overnight in a shaker at 37 °C to obtain a primary seed solution. Then, it was inoculated into a 200 mL LB flask containing kanamycin resistance at an inoculation amount of 1%, and cultured in a shaker at 37 °C until the OD 600 reached between 0.6 and 0.8. IPTG was added for low-temperature induction for 16 - 20 h, and then the cells were collected. The cells were sonicated and centrifuged to obtain the supernatant. The supernatant was purified through a nickel column to obtain purified diguanylate cyclase and mutants. The enzyme activity was measured, and the protein purification electrophoresis pattern is as shown in Figure 1 shown.

[0070] Enzyme activity assay system: A 1 mL reaction system contains substances with the following final concentrations: 1 mM GTP, 10 mM MgCl₂·6H₂O, 50 mM Tris-HCl buffer system (pH = 7.5), 5 μL of pure enzyme solution. The reaction was carried out at 70 °C for 30 min and immediately terminated by boiling. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze 1 μM GTP or generate 1 μM c-di-GMP per minute.

[0071] Five high-activity diguanylate cyclase single mutant strains and two double mutant strains were screened, namely PhDgc-E220R, PhDgc-R262F, PhDgc-N264D, PhDgc-D265F, PhDgc-Y280R, PhDgc-R262T / N264K, and PhDgc-R262T / D265W; the relative enzyme activities of their mutants were 2.76 times, 3.67 times, 2.31 times, 2.04 times, 3.55 times, 4.12 times, and 3.42 times that of the wild type, respectively. Among them, the relative enzyme activity of the PhDgc-R262T / N264K mutant was 4.12 times that of the wild type and could effectively eliminate product inhibition.

[0072] Example 3

[0073] In this example, the diguanylate cyclase mutant (PhDgc-R262T / N264K) was used to catalyze the production of c-di-GMP.

[0074] Take a 250 mL beaker and prepare a 200 mL reaction system: the final concentration of GTP is 20 mM, the final concentration of MgCl2 . 6H2O is 10 mM, the final concentration of Tris-HCl (pH = 7.5) is 50 mM, and the final concentration of the diguanylate cyclase mutant is 10 μM; the cyclization reaction is carried out in a 70 °C water bath for 1 h, and the reaction is terminated by boiling to obtain c-di-GMP.

[0075] The diluted reaction solution was quantitatively analyzed by high performance liquid chromatography. HPLC conditions: the chromatographic column was a Zorbax xb-aq column (4.6 mm × 250 mm, 5 μm), 10 mM ammonium acetate-acetonitrile (98:2; V / V) was used as the mobile phase, the flow rate was 1 mL / min, the detection wavelength was 260 nm, and the column temperature was 35 °C; the liquid chromatogram was as Figure 2 shown, where the GMP peak was at 2.763 min and the c-di-GMP peak was at 6.591 min, and the product ratio was 89%. The product c-di-GMP was detected and analyzed by mass spectrometry, and the mass spectrum was as Figure 3 shown.

[0076] Example 4

[0077] In this example, the diguanylate cyclase mutant (PhDgc-R262T / N264K) engineering bacteria were used to catalyze the production of c-di-GMP.

[0078] Take a 250 mL beaker and prepare a 200 mL reaction system: the final concentration of GTP is 20 mM, the final concentration of MgCl2·6H2O is 10 mM, the final concentration of Tris-HCl (pH = 7.5) is 50 mM, and the final concentration of the mutant wet cells is 15 g / L; the cyclization reaction is carried out in a 70 °C water bath for 1 h, and the reaction is terminated by boiling to obtain c-di-GMP.

[0079] Centrifuge at 8000 rpm for 20 minutes, precipitate the supernatant with ethanol or acetone, filter by suction, and vacuum dry the solid to obtain 6.21 g of white solid. Quantitative analysis by HPLC showed a conversion rate of 95.1%, a purity of c-di-GMP of 92%, and a yield of 85.3%.

[0080] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. Obviously, those who are familiar with the technology in this field can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the interpretation of the present invention should be within the protection scope of the present invention.

Claims

1. A diguanylate cyclase mutant, characterized in that, The diguanylate cyclase mutant is one of the following: (1) a single mutation occurs at position 262 of the amino acid sequence shown in SEQ ID NO.1, and the arginine at position 262 is mutated to phenylalanine; (2) double mutations occur at positions 262 and 264 of the amino acid sequence shown in SEQ ID NO.1, the arginine at position 262 is mutated to threonine, and the asparagine at position 264 is mutated to lysine; (3) double mutations occur at positions 262 and 265 of the amino acid sequence shown in SEQ ID NO.1, the arginine at position 262 is mutated to threonine, and the aspartic acid at position 265 is mutated to tryptophan.

2. A coding gene, characterized in that, The encoding gene encodes the diguanylate cyclase mutant described in claim 1.

3. An engineered bacterium, characterized in that, The engineered bacterium contains the encoding gene described in claim 2.

4. Use of an engineered bacterium in catalyzing the synthesis of cyclic diguanosine monophosphate from guanosine triphosphate, characterized in that, Using guanosine triphosphate as a substrate, after adding an activator and a catalyst, it is mixed evenly in a buffer system, and cyclic diguanosine monophosphate is obtained through a cyclization reaction; wherein, the catalyst is selected from one of the engineered bacterium cells described in claim 3 or a protein purified from the engineered bacterium cells; The protein purified from the engineered bacterium cells is the diguanylate cyclase mutant described in claim 1.

5. Use of an engineered bacterium according to claim 4 in the catalysis of guanosine triphosphate to synthesize cyclic diguanosine monophosphate, characterized in that, The engineered bacterium cells are obtained by centrifugation after the engineered bacterium is induced to culture.

6. Use of an engineered bacterium according to claim 4 in the catalytic synthesis of cyclic diguanosine monophosphate from guanosine triphosphate, characterized in that, The final concentration ratio of guanosine triphosphate, activator, and catalyst is 2000:1000:

1.

7. Use of an engineered bacterium according to claim 4 in the catalytic synthesis of cyclic diguanosine monophosphate from guanosine triphosphate, characterized in that, The activator is selected from one of magnesium chloride, manganese sulfate, manganese chloride, cobalt chloride, and calcium chloride; The buffer system is selected from one of Tris-HCl buffer, HEPES buffer, phosphate buffer, and acetate-sodium acetate buffer.

8. Use of an engineered bacterium according to claim 4 in the catalytic synthesis of cyclic diguanosine monophosphate from guanosine triphosphate, characterized in that, During the cyclization reaction, the temperature is 30~90 °C and the time is 0.5~2 h.

Images