阅读说明：本技术 一种双鸟苷酸环化酶改造基因、编码蛋白、工程菌及其构建方法和应用 (Modified gene of diguanylate cyclase, encoded protein, engineering bacterium, construction method and application thereof ) 是由 李晓燕 王宏 傅得响 张燕 于 2020-07-02 设计创作，主要内容包括：本发明公开了一种双鸟苷酸环化酶改造基因、编码蛋白、工程菌及其构建方法和应用；该双鸟苷酸环化酶改造基因,其核苷酸序列如SEQ ID NO.1所示；该双鸟苷酸环化酶改造基因编码蛋白氨基酸序列如SEQ ID NO.2所示；其工程菌由克隆有SEQ ID NO.1所示基因的重组质粒转入大肠杆菌中获得,还公开了上述工程菌的构建方法及应用该工程菌生产双鸟苷酸环化酶,所得的双鸟苷酸环化酶进一步应用于转化三磷酸鸟苷生成环鸟苷二磷酸的方法。该双鸟苷酸环化酶改造基因在大肠杆菌中的表达量高,根据双鸟苷酸环化酶改造基因编码的双鸟苷酸环化酶耐热性极强,最适反应温度为90℃,在70℃下保存6h保留70％的活性,该酶的比活力高、不容易出现产物抑制作用。(The invention discloses a modified gene of diguanylate cyclase, a coded protein, an engineering bacterium, a construction method and application thereof; the nucleotide sequence of the modified gene of the diguanylate cyclase is shown as SEQ ID NO. 1; the amino acid sequence of the modified gene coding protein of the diguanylate cyclase is shown as SEQ ID NO. 2; the engineering bacterium is obtained by transferring the recombinant plasmid cloned with the gene shown in SEQ ID NO.1 into escherichia coli, and also discloses a construction method of the engineering bacterium and a method for producing diguanylate cyclase by applying the engineering bacterium, wherein the obtained diguanylate cyclase is further applied to converting guanosine triphosphate to generate cyclic guanosine diphosphate. The modified gene of the diguanylate cyclase has high expression level in escherichia coli, the diguanylate cyclase coded by the modified gene of the diguanylate cyclase has extremely strong heat resistance, the optimal reaction temperature is 90 ℃, 70% of activity is reserved after the gene is stored for 6 hours at 70 ℃, the specific activity of the enzyme is high, and the product inhibition effect is not easy to occur.)

1. A modified gene of diguanylate cyclase is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. The protein encoded by the modified diguanylate cyclase gene of claim 1 and having the amino acid sequence shown as SEQ ID No. 2.

3. The engineering bacterium is obtained by transforming recombinant plasmids into expression host escherichia coli, wherein the recombinant plasmids are obtained by cloning genes shown in SEQ ID NO.1 onto a plasmid vector.

4. The engineered bacterium of claim 3, wherein the Escherichia coli is one of BL21(DE3), Rosetta (DE3), BSR (DE3) and Shuffle T7; the plasmid vector is one of pET28a, pET32a and pET39 a.

5. A method for constructing and expressing a modified gene engineering bacterium of diguanylate cyclase is characterized by comprising the following steps:

s1: artificially synthesizing the nucleotide sequence shown in SEQ ID NO.1 to obtain a diguanylate cyclase modifying gene;

s2: adopting NcoI and XhoI double enzyme digestion diguanylate cyclase to modify gene and plasmid vector, and carrying out gel recovery on enzyme digestion products;

s3: connecting the enzyme digestion products by using T4 ligase, and transforming the connection products into an expression host escherichia coli;

s4: selecting a single colony, inoculating the single colony into a test tube filled with an LB liquid culture medium, and culturing for 8-12h at 37 ℃ and 220 rpm; extracting recombinant plasmids;

s5: enzyme digestion verification is carried out, the recombinant plasmid containing the diguanylate cyclase modification gene is subjected to sequencing verification, and a strain with the correct diguanylate cyclase modification gene is selected, namely the gene engineering bacterium for expressing the diguanylate cyclase modification.

6. A method for producing diguanylate cyclase by using the engineering bacterium as claimed in claim 3, which comprises the following steps:

s1: inoculating the constructed engineering bacteria into an LB flat plate containing kanamycin for culture at 37 ℃;

s2: picking single bacterial colony to an LB culture medium containing kanamycin for culture to form seed liquid;

s3: transferring the seed liquid into LB culture medium containing kanamycin, and culturing until OD is reached_600nm0.6, 1.7 and 3.2;

s4: adding IPTG, inducing for 2, 4, 6 and 8 hours, and collecting thalli;

s5: crushing and centrifuging the thallus, taking supernatant, and purifying to obtain pure diguanylic acid cyclase liquid.

7. The method for producing diguanylate cyclase by using the engineering bacteria as claimed in claim 6, which comprises the following specific steps:

s1: inoculating the constructed engineering bacteria into an LB flat plate containing 50mg/L kanamycin, and culturing at 37 ℃ for 12-16 h;

s2: picking single colony to LB culture medium containing 50mg/L kanamycin, culturing at 37 deg.C and 220rpm for 8-12h to form seed liquid;

s3: the seed liquid was inoculated at an inoculum size of 2% to LB medium containing 50mg/L kanamycin, cultured at 37 ℃ and 220rpm to OD_600nm0.6, 1.7 and 3.2;

s4: adding IPTG with final concentration of 0.05, 0.1, 0.5 and 1.0mmol/L, respectively, inducing fermentation at 22, 26, 30 and 34 deg.C for 2, 4, 6 and 8h, collecting thallus, and washing with 50mM Tris, pH 8.0;

s5: and (3) crushing the thalli, centrifuging, collecting supernatant, and purifying by a nickel column to obtain purified diguanylic acid cyclase liquid.

8. A method for producing cyclic guanosine diphosphate by using the diguanylate cyclase of claim 7, comprising the steps of:

s1: preparing reaction liquid containing guanosine triphosphate, diguanylate cyclase, magnesium chloride and Tris-HCl buffer solution in a reaction vessel;

s2: reacting the reaction solution at 35-70 ℃, and adjusting the pH of the reaction solution to 7.0 by using sodium hydroxide;

s3: after the reaction, the reaction mixture was boiled for 5min to terminate the reaction.

9. The method for producing cyclic guanosine diphosphate according to claim 8, wherein the concentration of guanosine triphosphate in said step S1 is 2 to 100 mmol/L; the concentration of the diguanylate cyclase is 1 mIU/mL-50 mIU/mL; the final concentration of the magnesium chloride is 5-20 mmol/L; the pH of the Tris-HCl buffer solution is 6.0-8.0.

10. The method for producing cyclic guanosine diphosphate according to claim 8, wherein said step S2 is performed by placing the reaction vessel in a water bath, controlling the reaction temperature, reacting the reaction solution at 35-70 ℃, and adjusting the pH of the reaction solution to 7.0 with sodium hydroxide every 2h for 2-48 h.

Technical Field

The invention relates to the technical field of biological engineering and chemical engineering, in particular to a modified gene of diguanylate cyclase, a modified gene coding protein, an expression modified gene engineering bacterium of diguanylate cyclase and a construction method thereof, and application of the engineering bacterium in producing diguanylate cyclase and synthesizing c-di-GMP by biocatalysis of diguanylate cyclase.

Background

Cyclic guanosine diphosphate (c-di-GMP) is a small molecule second messenger widely existing in various bacteria, and the second messenger plays an important role in regulating biological processes such as biofilm formation, virulence factor production and environmental adaptation. Cyclic guanosine diphosphate is utilized by most bacteria and is involved in a variety of physiological processes; cyclic guanosine diphosphate regulated cellular functions include induction of cell-cell signal transduction, biofilm formation, motility, and virulence, among others; cyclic guanosine diphosphate exerts its regulatory role by binding to different receptors including Pi1Z, degenerate GGDEF, EAL, HD-GYP domains, transcription factors and riboswitches. Among many bacteria, intracellular high concentrations of cyclic guanosine diphosphate promote biofilm formation, while low concentrations of cyclic guanosine diphosphate promote pathogen movement and the synthesis of pathogenic agents; with the deep research of the regulation and control mechanism of cyclic guanosine diphosphate, the cyclic guanosine diphosphate has very wide market and application prospects in the field of medicine.

At present, the cyclic guanosine diphosphate is produced mainly by a chemical method and a biological enzyme method. Kawai R et al disclose a method for chemically synthesizing c-di-GMP, which takes 3'-O- [ (diisopropylamino) (2-cyanoethoxy) phosphino ] -5' -O- (4,4 '-dimethoxytrityl) -2' -O-tert-butyldimethylchlorosilane-N2-isobutyrylguanosine (S1) as a raw material, and carries out hydrolysis, beta elimination and detritylation reactions on the raw material to obtain an intermediate S2; performing intermolecular esterification reaction on the 3 '-phosphate group and the 5' -hydroxyl group of S2, and removing trityl to obtain an intermediate S4; s4 forms a cyclic intermediate S5 through intramolecular esterification; s6 is obtained by beta elimination reaction of S5; s6 is subjected to final deprotection reaction, crystallization and drying to obtain a cyclic guanosine diphosphate finished product S7, the total yield is about 30 percent, the method has multiple synthesis steps and low yield, and relates to a large amount of highly toxic organic solvents; other synthetic methods that have been disclosed are time consuming, expensive, inefficient multistep synthetic routes, and suffer from the use of large amounts of hazardous chemicals, are environmentally unfriendly, and are not suitable for large-scale industrial production.

The synthesis of c-di-GMP is controlled by diguanylate cyclase, and the biological enzyme method mainly uses diguanylate cyclase (DGC, EC 2.7.7.65) to catalyze Guanosine Triphosphate (GTP) to generate c-di-GMP; the activity of DGC is located in a GGDEF domain, the domain contains a conserved Gly-Gly-Asp-Glu-Phe amino acid sequence, the three-stage structure of 27 DGCs or similar proteins in 11 bacterial species is identified at present, and the enzyme method for producing c-di-GMP has the advantages of simple process, short period, low energy consumption, strong specificity, high conversion rate and the like, but the existing diguanylate cyclase has the problems of low expression level, low specific activity, high cost, product inhibition and the like, for example, the diguanylate cyclase produced by Thermotoga maritima is easy to precipitate and is easy to inhibit products; therefore, in the process of producing cyclic guanosine diphosphate by an enzyme method, how to obtain the diguanylate cyclase which has low cost, high expression level and difficult product inhibition is a key problem at present.

Disclosure of Invention

In view of the above problems, a first object of the present invention is to provide a modified gene of diguanylate cyclase, which is obtained from a hypothalamus by using a virtual screening means such as genomic database mining and homologous sequence comparison, combining a molecular biology means to obtain a gene sequence of the diguanylate cyclase from the hypothalamus, and simultaneously modifying the gene sequence of the diguanylate cyclase from the hypothalamus by using a molecular biology and process optimization means to obtain the modified gene of diguanylate cyclase, wherein the nucleotide sequence of the modified gene of diguanylate cyclase is shown in SEQ ID No.1, and the expression level of the modified gene of diguanylate cyclase in escherichia coli is high.

The second purpose of the invention is to provide the protein coded by the modified gene of the diguanylate cyclase, wherein the protein is the diguanylate cyclase, the diguanylate cyclase has extremely strong heat resistance, the optimal reaction temperature is 90 ℃, and 70% of activity is reserved after the diguanylate cyclase is stored for 6 hours at 70 ℃.

The third purpose of the invention is to provide a modified gene engineering bacterium for expressing diguanylate cyclase, which can efficiently express diguanylate cyclase and solve the problems of low expression level, low specific activity and easy product inhibition of diguanylate cyclase in the prior art.

The fourth purpose of the invention is to provide a construction method of the engineering bacteria.

The fifth purpose of the invention is to provide a method for producing diguanylate cyclase by applying the engineering bacteria, and the specific activity of the diguanylate cyclase obtained by the method can reach 0.047IU/mg, which is far higher than that of the diguanylate cyclase reported in the prior art.

The sixth purpose of the invention is to provide a method for producing cyclic guanosine diphosphate by applying the diguanylate cyclase, the method uses the diguanylate cyclase as a catalyst to convert guanosine triphosphate to produce the cyclic guanosine diphosphate, the accumulated amount of c-di-GMP is 6.10mmol/L when the substrate concentration is 15mmol/L at 50 ℃, the conversion rate reaches 81.33%, and the substrate inhibition effect in the prior art is greatly overcome.

The first technical scheme adopted by the invention is as follows: the nucleotide sequence of the modified gene of diguanylate cyclase is shown as SEQ ID NO. 1.

The second technical scheme adopted by the invention is as follows: a modified gene coding protein of diguanylate cyclase has an amino acid sequence shown in SEQ ID NO. 2.

The third technical scheme adopted by the invention is as follows: a gene engineering bacterium for modifying diguanylate cyclase is obtained by transforming a recombinant plasmid into an expression host escherichia coli, wherein the recombinant plasmid is obtained by cloning a gene shown by SEQ ID NO.1 onto a plasmid vector.

Further, the Escherichia coli is one of BL21(DE3), Rosetta (DE3), BSR (DE3) and Shuffle T7; the plasmid vector is one of pET28a, pET32a and pET39 a.

The fourth technical scheme adopted by the invention is as follows: a method for constructing a modified genetic engineering bacterium for expressing diguanylate cyclase comprises the following steps:

s1: artificially synthesizing the nucleotide sequence shown in SEQ ID NO.1 to obtain a diguanylate cyclase modifying gene;

s2: adopting NcoI and XhoI double enzyme digestion diguanylate cyclase to modify gene and plasmid vector, and carrying out gel recovery on enzyme digestion products;

s3: connecting the enzyme digestion products by using T4 ligase, and transforming the connection products into an expression host escherichia coli;

s4: selecting a single colony, inoculating the single colony into a test tube filled with an LB liquid culture medium, and culturing for 8-12h at 37 ℃ and 220 rpm; extracting recombinant plasmids;

s5: enzyme digestion verification is carried out, the recombinant plasmid containing the diguanylate cyclase modification gene is subjected to sequencing verification, and a strain with the completely correct diguanylate cyclase modification gene is selected, namely the gene engineering bacterium for expressing the diguanylate cyclase modification.

The fifth technical scheme adopted by the invention is as follows: a method for producing diguanylate cyclase by applying the engineering bacteria comprises the following steps:

s1: inoculating the constructed engineering bacteria into an LB flat plate containing kanamycin for culture at 37 ℃;

s2: picking single bacterial colony to an LB culture medium containing kanamycin for culture to form seed liquid;

s3: transferring the seed liquid into LB culture medium containing kanamycin, and culturing until OD is reached_600nm0.6, 1.7 and 3.2;

s4: adding IPTG, inducing for 2, 4, 6 and 8 hours, and collecting thalli;

s5: crushing and centrifuging the thallus, taking supernatant, and purifying to obtain pure diguanylic acid cyclase liquid.

Further, the method for producing the diguanylate cyclase by the engineering bacteria comprises the following specific steps:

s1: inoculating the constructed engineering bacteria into an LB flat plate containing 50mg/L kanamycin, and culturing at 37 ℃ for 12-16 h;

s2: picking single colony to LB culture medium containing 50mg/L kanamycin, culturing at 37 deg.C and 220rpm for 8-12h to form seed liquid;

s3: the seed liquid was inoculated at an inoculum size of 2% to LB medium containing 50mg/L kanamycin, cultured at 37 ℃ and 220rpm to OD_600nm0.6, 1.7 and 3.2;

s4: adding IPTG with final concentration of 0.05, 0.1, 0.5 and 1.0mmol/L, respectively, inducing fermentation at 22, 26, 30 and 34 deg.C for 2, 4, 6 and 8h, collecting thallus, and washing with 50mM Tris, pH 8.0;

s5: and (3) crushing the thalli, centrifuging, collecting supernatant, and purifying by a nickel column to obtain purified diguanylic acid cyclase liquid.

The sixth technical scheme adopted by the invention is as follows: a method for producing cyclic guanosine diphosphate by using the diguanylate cyclase comprises the following steps:

s1: preparing reaction liquid containing guanosine triphosphate, diguanylate cyclase, magnesium chloride and Tris-HCl buffer solution in a reaction vessel;

s2: reacting the reaction solution at 35-70 ℃, and adjusting the pH of the reaction solution to 7.0 by using sodium hydroxide;

s3: after the reaction, the reaction mixture was boiled for 5min to terminate the reaction.

Further, the concentration of guanosine triphosphate in the step S1 is 2-100 mmol/L; the concentration of the diguanylate cyclase is 1 mIU/mL-50 mIU/mL; the final concentration of the magnesium chloride is 5-20 mmol/L; the pH of the Tris-HCl buffer solution is 6.0-8.0.

Further, the step S2 is to put the reaction vessel in a water bath to regulate the reaction temperature, so that the reaction solution reacts at 35-70 ℃, adjust the pH of the reaction solution to 7.0 with sodium hydroxide every 2 hours, and control the reaction time to 2-48 hours.

The beneficial effects of the above technical scheme are that:

(1) the invention obtains the sequence of the diguanylate cyclase gene from the pseudomonas mandibulae (Pseudothermoga hypogea) by a genome database mining, homologous sequence comparison and other virtual screening means in combination with a molecular biology means, and simultaneously modifies the sequence of the diguanylate cyclase gene from the Pseudothermoga hypogea by a molecular biology and process optimization means to obtain the modified gene of the diguanylate cyclase, wherein the nucleotide sequence of the modified gene is shown as SEQ ID NO. 1; the modified gene of the diguanylate cyclase can be smoothly and efficiently expressed in later-stage escherichia coli engineering bacteria, and the activity of the diguanylate cyclase produced by the engineering bacteria is improved.

(2) The diguanylate cyclase modified gene has extremely strong heat resistance, the optimal reaction temperature is 90 ℃, 70 percent of activity is reserved after the diguanylate cyclase is stored for 6 hours at 70 ℃, and the stability is good; the specific activity of the alpha-beta-gamma-.

(3) The crude enzyme liquid obtained by fermentation is purified to obtain pure diguanylate cyclase, the pure diguanylate cyclase is used for converting and producing cyclic guanosine diphosphate (c-di-GMP), the components of a reaction system are clear, and the separation and purification of subsequent products are facilitated.

(4) The diguanylate cyclase encoded by the diguanylate cyclase modification gene is used as a catalyst to convert guanosine triphosphate to produce cyclic guanosine diphosphate, the accumulated amount of c-di-GMP is 6.10mmol/L when the substrate concentration is 15mmol/L at 50 ℃, the conversion rate reaches 81.33%, and the problem of substrate inhibition of the diguanylate cyclase in the prior art is greatly solved.

Drawings

FIG. 1 is a protein electrophoresis chart showing separation and purification of diguanylate cyclase of the present invention (where SM is a sample solution, FT is an upper column permeation solution, and E is an eluent);

FIG. 2 is a graph showing the effect of temperature on the catalytic activity of the bisguanylate cyclase;

FIG. 3 shows the stability of diguanylate cyclase at different temperatures;

FIG. 4 is a structural formula of cyclic guanosine diphosphate;

FIG. 5 is a high performance liquid chromatogram of the transformation liquid of the present invention.

Detailed Description

The present invention is further illustrated by the following specific examples, it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.