CN118147107A - A phosphotransferase mutant, a genetically engineered bacterium expressing the same, and applications thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a phosphotransferase mutant, genetically engineered bacteria expressing the phosphotransferase mutant and application of the phosphotransferase mutant. The phosphotransferase mutant is obtained by mutating the wild type phosphotransferase of the enterobacter aerogenes, and the genetically engineered bacteria expressing the phosphotransferase mutant can be used for catalyzing and producing 5' -taste nucleotide by whole cells. The low endotoxin escherichia coli provided by the invention is recombinant bacteria obtained by knocking out endotoxin synthesis related genes of escherichia coli strain BL21 (DE 3), the expression of a phosphotransferase mutant is not influenced, the conversion rate is higher when the low endotoxin escherichia coli is applied to whole cell catalysis production of 5' -flavor nucleotide, and meanwhile, the product has no endotoxin residue risk.

Description

A phosphotransferase mutant, a genetically engineered bacterium expressing the same, and applications thereof

Technical Field

The invention belongs to the field of biotechnology, and specifically relates to a phosphotransferase mutant, a genetic engineering bacterium expressing the mutant and an application thereof.

Background technique

5'-inosinic acid (IMP) is a substrate for conversion to other purine nucleotides and is also an important flavor nucleotide, widely used in food, medicine, agriculture, defense and other industries. Its flavoring effect when added to glutamate at a mass fraction of 5% to 12% is about 8 times higher than that of sodium glutamate alone, and it is known as "powerful MSG". At present, the content of 5'-inosinic acid is used as an important indicator for measuring the umami taste of meat internationally.

In the prior art, methods for synthesizing 5'-nucleotides include chemical synthesis, microbial fermentation, and whole cell biocatalysis. The reagents required for chemical synthesis are expensive and have certain toxicity. The direct microbial fermentation method for producing 5'-inosinic acid mainly utilizes the biosynthetic pathway of microbial strains. Limited by the characteristics of microorganisms, the process is complex, the cycle is long, the cost is high, and the indicators are difficult to effectively improve. The whole cell biocatalysis method has the advantages of efficient and mild reaction, easy control, no need for protective groups, simple steps, strong reaction specificity, few side reactions, and easy post-processing, so industrial production has been achieved.

The whole-cell biocatalysis method generally uses Escherichia coli engineered bacteria expressing phosphotransferase. After the reaction is completed, 5'-inosinic acid or 5'-guanylic acid is obtained through separation and purification. Existing research results show that under the conditions of Escherichia coli whole-cell catalytic reaction, when the concentrations of inosine and pyrophosphate are 100 mM and 120 mM respectively, the inosine conversion rate is 64%-75% after 6 hours of reaction, and this conversion rate still has room for improvement. Increasing the concentration ratio of pyrophosphate to inosine can increase the conversion rate, but it will increase the cost of pyrophosphate use and affect the overall benefits.

On the other hand, when whole E. coli cells are used for catalysis and subsequent separation and extraction of products, their cell fragments contain endotoxins (lipopolysaccharide, LPS) that must be avoided in the food industry. These endotoxins can activate the immune response of human cells and cause septic shock. Therefore, if products produced with E. coli as carriers need to be used in the food or pharmaceutical fields, they must undergo extremely strict purification steps to reduce the LPS content. In order to eliminate this hidden danger, the downstream purification and testing costs of inosinic acid have to be increased. However, in industrial-scale purification production, the effect of endotoxin removal processes is difficult to be satisfactory.

Summary of the invention

In view of the above problems, the present invention provides a phosphotransferase mutant, a genetically engineered bacterium expressing the same, and an application thereof. The genetically engineered bacterium can improve the product conversion rate in the whole cell catalytic process under the condition of low pyrophosphate dosage, and the endotoxin content in the conversion liquid is far lower than the limit value, which can avoid the endotoxin removal step in the purification process, and has important significance for improving the economic benefits, green manufacturing and food safety of the whole cell catalytic preparation of 5'-inosinic acid and 5'-guanylic acid.

In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

The first aspect of the present invention provides a phosphotransferase mutant, which is obtained by mutating the wild-type phosphotransferase of Enterobacter aerogenes whose nucleotide sequence is shown in SEQ ID NO.1 and whose amino acid sequence is shown in SEQ ID NO.2, wherein the mutation includes mutating the leucine at position 81 to glutamine, the alanine at position 83 to glutamine, the glutamate at position 84 to alanine, the asparagine at position 87 to aspartic acid, the serine at position 89 to alanine, the alanine at position 90 to phenylalanine, the glycine at position 92 to aspartic acid, the threonine at position 153 to lysine, the glutamate at position 154 to aspartic acid and the isoleucine at position 171 to threonine.

In order to improve the conversion rate and reduce the amount of pyrophosphate used, the present invention mutates the wild-type phosphotransferase (AP/PTase, gene name pho, genebank accession number: AB044338.1) of Enterobacter aerogenes to obtain a phosphotransferase mutant. After the above-mentioned phosphotransferase mutant provided by the present invention is transformed into a host bacterium through an expression plasmid, a genetically engineered bacterium expressing the phosphotransferase mutant can be obtained. The genetically engineered bacterium can be used for whole-cell catalytic production of 5'-flavor nucleotides (5'-inosinic acid and 5'-guanylic acid). Compared with the phosphotransferase in the prior art and the wild-type phosphotransferase of Enterobacter aerogenes, the phosphotransferase mutant has significantly improved inosine and guanosine conversion rates.

Preferably, the nucleotide sequence of the phosphotransferase mutant is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4. Compared with the catalytic ability of the wild-type phosphotransferase, the conversion rate of the phosphotransferase mutant to inosine can be increased to more than 86%, and the conversion rate of the guanosine can be increased to more than 80%.

Preferably, the mutation further comprises mutating lysine at position 133 to arginine. This mutation can further increase the conversion rate of the phosphotransferase mutant to inosine to more than 93%.

Further preferably, the nucleotide sequence of the phosphotransferase mutant is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO.6.

Preferably, the mutation further comprises mutating the valine at position 212 to threonine. This mutation can further increase the conversion rate of the phosphotransferase mutant to guanosine to more than 86%.

More preferably, the nucleotide sequence of the phosphotransferase mutant is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO.8.

The second aspect of the present invention provides a recombinant plasmid containing the nucleotide sequence of the above-mentioned phosphotransferase mutant.

The third aspect of the present invention provides a genetically engineered bacterium constructed using the above-mentioned recombinant plasmid.

The fourth aspect of the present invention provides a low-endotoxin Escherichia coli constructed using the above-mentioned recombinant plasmid, wherein at least one gene among lpxL, lpxM, pagP, lpxP and eptA is knocked out. The above-mentioned genes are endotoxin synthesis genes, and knocking out the above-mentioned genes can reduce the endotoxin content in the reaction system and reduce the process cost of removing endotoxins during purification.

Further preferably, the low-endotoxin E. coli has the eptA gene knocked out (GenBank ID: 948629, full length 1644 bp). The eptA gene knocked out E. coli not only has greatly reduced endotoxins compared to the original strain, but also has a certain change in cell membrane permeability compared to the original strain, thereby improving the efficiency of whole cell catalysis, so that the nucleoside conversion rate after expressing phoC -K133R and phoC- V212T is improved compared to the original strain.

The fifth aspect of the present invention provides a method for constructing the above-mentioned low endotoxin Escherichia coli, which specifically comprises the following steps:

S1. Design upstream and downstream homology arm primers for the sequence of the knocked-out gene, and obtain the upstream and downstream DNA fragments of the eptA gene by PCR amplification. In addition, a Kan resistance gene with FRT invertase sites on both sides is amplified from the pKD4 plasmid by PCR. The upstream and downstream DNA fragments of the target gene and the resistance gene containing FRT invertase sites are connected by overlap extension PCR (Overlap PCR) technology to obtain the donor DNA fragment of homology arm + FRT resistance gene;

S2, transform the knockout strain E. coli BL21 (DE3) with the pKD46 plasmid expressing the homologous recombinase, select the pKD46 positive colonies on the Amp resistance medium, culture for 12-16 hours and induce the expression of the recombinase with arabinose after transfer; prepare the induced bacteria into a competent cell suspension, add 3-4 µg of the donor DNA fragment obtained in S1 to every 50 mL of the competent cell suspension, mix, and add to the electroporation cup for electrotransformation; obtain positive transformants by screening on the resistance plate;

S3. Inoculate the positive transformants obtained in S2 into LB culture tubes and culture at 42°C for 12-16 hours to eliminate the temperature-sensitive plasmid pKD46; prepare the knockout strain that eliminated the plasmid pKD46 into competent state, and then transform it into the pCP20 plasmid to eliminate the Kan resistance gene (Amp and Cm resistance, temperature-sensitive), and culture at 30°C;

S4. Cultivate the knockout strain carrying the pCP20 plasmid obtained in S3 at 42°C to eliminate pCP20 and obtain a knockout strain without resistance.

The construction method may also include identifying whether the gene is successfully knocked out by Red recombination by PCR on the positive transformants obtained in S2, and streaking the knockout strain obtained in S4 on a non-resistance, Amp or Cm plate to identify whether the resistance is successfully eliminated.

The sixth aspect of the present invention provides the use of the above-mentioned genetically engineered bacteria or low-endotoxin Escherichia coli in the whole-cell catalytic production of 5'-flavor nucleotides, wherein the 5'-flavor nucleotides include 5'-inosinic acid and 5'-guanylic acid.

The seventh aspect of the present invention provides a method for whole cell catalytic production of 5'-flavor nucleotides, wherein the 5'-flavor nucleotides include 5'-inosinic acid and 5'-guanylic acid, and the method specifically comprises the following steps:

Step 1, culturing and activating the above-mentioned genetically engineered bacteria or low-endotoxin Escherichia coli, inducing expression with isopropyl-β-D-thiogalactoside (IPTG), collecting the cells by centrifugation, and washing with sodium acetate buffer to obtain wet cells;

Step 2: After precooling the wet cells at 4°C, react with inosine or guanosine and disodium dihydrogen pyrophosphate in a sodium acetate buffer at 25-40°C for 5-7 h, wherein the concentration of inosine or guanosine is 80-120 mM, the concentration of disodium dihydrogen pyrophosphate is 120-220 mM, and the concentration of the wet cells is 20-100 g/L.

There is no particular limitation on the culture medium for culturing genetically engineered bacteria or low-endotoxin Escherichia coli in step 1 of the method, and the culture medium conventionally used in the art can be used.

Preferably, the conditions for inducing expression are: culturing on a shaking platform at 37°C and 200 rpm; adding isopropyl-β-D-thiogalactoside for induction when _OD600 reaches 0.6±0.1, and cooling to 30°C for 12±2h of induction culture.

Preferably, the concentration of the sodium acetate buffer is 80-120 mM, and the pH is 4.7-4.9.

Preferably, the wet cells are precooled at 4° C. for 6 to 8 h.

The beneficial effects of the present invention are as follows: the present invention obtains a new phosphotransferase mutant by mutation modification of multiple amino acid sites of wild-type phosphotransferase of Enterobacter aerogenes. After the phosphotransferase mutant gene is connected to an expression plasmid and transformed into a host bacterium, a genetically engineered bacterium expressing the phosphotransferase mutant can be obtained, and the conversion rate of catalytic production of 5'-inosinic acid or 5'-guanylic acid is significantly improved compared with the prior art. The present invention also knocks out the endotoxin gene of the host bacterium, connects the above-mentioned phosphotransferase mutant gene to an expression plasmid and transforms it into a host cell in which the endotoxin gene has been knocked out, and a whole cell biocatalyst with high enzyme activity and low endotoxin can be obtained. The whole cell biocatalyst uses inosine/guanosine and a relatively low concentration of disodium dihydrogen pyrophosphate as substrates, and can efficiently and safely prepare 5'-inosinic acid or 5'-guanylic acid, which is of great significance for food safety and green biomanufacturing of inosinic acid and guanylic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a map of plasmid pET28a-phoC in Example 1;

FIG2 is a HPLC detection spectrum of the reaction solution of the whole cell catalytic production of 5'-inosinic acid reaction for 7 hours in Example 3;

FIG3 is a HPLC detection spectrum of the reaction solution of the whole cell catalytic production of 5'-guanylate in Example 3 for 4 hours.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work fall within the scope of protection of the present invention.

In the prior art, the whole-cell biocatalytic method for synthesizing 5'-nucleotides generally uses an engineered Escherichia coli expressing a phosphotransferase, and after the reaction is completed, 5'-inosinic acid or 5'-guanylic acid is obtained by separation and purification. At present, the conversion rate of this method still has room for improvement. The conversion rate can be increased by increasing the concentration ratio of pyrophosphate to inosine, but the cost of pyrophosphate use will increase, affecting the overall benefits. Therefore, it is necessary to further improve the whole-cell strain so that it can achieve a high conversion rate of the substrate under a lower concentration ratio of pyrophosphate to nucleoside.

On the other hand, when E. coli whole cells are used for catalysis and subsequent separation and extraction of products, the endotoxins contained in its cell fragments increase the cost of downstream purification and detection of inosinic acid. From the perspective of the source of E. coli strains, removing endotoxin synthesis genes through genetic modification is a technology with more application prospects. By knocking out related genes, the acyl chain of LPS can be reduced, so that LPS is converted into lipid IVA, which does not trigger an endotoxin reaction. However, after the endotoxin gene is knocked out, E. coli will undergo changes in the cell wall structure, which may cause changes in growth and substrate and product metabolism, thereby affecting actual production. In the process of E. coli whole cell catalytic production of inosinic acid, the knockout application of endotoxin genes also has similar problems. Therefore, there is no research on the application of endotoxin gene knockout E. coli in the whole cell catalytic preparation of 5'-inosinic acid in the prior art.

To address this problem, the present invention obtains a phosphotransferase mutant by modifying the wild-type phosphotransferase of Enterobacter aerogenes. The genetically engineered bacteria expressing the phosphotransferase mutant can be used for whole-cell catalytic production of 5'-inosinic acid or 5'-guanylic acid, and the conversion rate is significantly improved compared with the phosphotransferase in the prior art and the wild-type phosphotransferase of Enterobacter aerogenes.

The embodiments of the present invention also provide a recombinant plasmid containing the nucleotide sequence of the mutant and a genetically engineered bacterium constructed using the recombinant plasmid.

The embodiments of the present invention also provide low-endotoxin Escherichia coli with endotoxin synthesis genes knocked out and a construction method thereof.

The present invention also provides the use of the genetically engineered bacteria and low-endotoxin Escherichia coli in whole-cell catalytic production of 5'-inosinic acid or 5'-guanylic acid.

The scheme of the present invention is described below through specific embodiments.

Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents, etc. used in the following examples can be obtained from commercial sources, among which the original host bacteria E. coli BL21 (DE3) and the expression plasmid vector pET28a are all products of commercial companies. pKD46, pKD4 and pCP20 plasmids are all commercial plasmids. The horseshoe crab kit was purchased from GenScript Biotech Co., Ltd.

Example 1

This embodiment provides a phosphotransferase mutant AP/PTase- phoC , which is obtained by subjecting the wild-type phosphotransferase of Enterobacter aerogenes ( Enterobacter aerogenes ) to the following mutations: mutating the leucine at position 81 to glutamine, the alanine at position 83 to glutamine, the glutamic acid at position 84 to alanine, the asparagine at position 87 to aspartic acid, the serine at position 89 to alanine, the alanine at position 90 to phenylalanine, the glycine at position 92 to aspartic acid, the threonine at position 153 to lysine, the glutamic acid at position 154 to aspartic acid, and the isoleucine at position 171 to threonine. The nucleotide sequence of the phosphotransferase mutant AP/PTase- phoC is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

The phosphotransferase mutant AP/PTase- phoC provided in this example was directly synthesized by Beijing Qingke Biotechnology Co., Ltd. after codon optimization.

Example 2

This embodiment provides a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO. 3 and a construction method thereof, as well as a genetically engineered bacterium constructed using the recombinant plasmid.

1. Construction of recombinant plasmid pET28a- phoC

The nucleotide sequence shown in SEQ ID NO. 3 was cloned into the expression plasmid pET28a containing a 6×His tag to obtain the recombinant plasmid pET28a- phoC (see Figure 1 for the plasmid map).

2. Construction of Escherichia coli BL21(DE3)/pET28a- phoC

Add the synthesized pET28a- phoC plasmid powder to sterile water to a concentration of 30 ng/μL. Thaw the competent cells (100 μL) of E. coli BL21 (DE3) stored at -80 ℃ on ice, slowly add the 30 ng/μL pET28a- phoC plasmid solution to the completely thawed competent cells, and place on ice for 30 min. Heat shock in a constant temperature water bath at 42 ℃ for 90 s, immediately place on ice for 3-5 min, then add 1 mL of LB medium (without antibodies), and culture at 37 ℃, 200 rpm shaking for 1 h. At the end of the culture, centrifuge at 4°C and 4000 rpm for 5 min, remove the supernatant and keep about 100 μL of the resuspended bacteria, spread on an LB solid plate containing Kan resistance, and culture in a 37°C constant temperature incubator for 12-14 h. After identification, the genetically engineered bacteria Escherichia coli BL21(DE3)/pET28a- phoC (hereinafter referred to as E. coli BL21(DE3)/pET28a- phoC ) were obtained.

Example 3

This example provides the use of the genetically engineered bacteria E. coli BL21 (DE3) / pET28a- phoC in Example 2 in whole cell catalytic production of 5'-inosinic acid or 5'-guanylic acid:

1. Induced expression of E. coli BL21(DE3)/pET28a- phoC and wet cell preparation

Pick a single colony of the genetically engineered bacteria E. coli BL21(DE3)/pET28a- phoC on the above LB solid plate (Kan) and inoculate it into LB liquid medium (100 µg/mL Kan), and culture it in a shaker at 37 ℃ and 200 rpm for 8-12 h to obtain seed liquid. Transfer the inoculum to 150 mL LB liquid medium with the same kanamycin concentration at a volume ratio of 2%, and culture the bacterial cell concentration to about OD ₆₀₀ at 37 ℃ and 200 rpm. Add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.2 mM, and induce it in a shaker at 30 ℃ and 200 rpm for 12 h. After the induction is completed, pour the bacterial liquid into a centrifuge cup, centrifuge it in a high-speed refrigerated centrifuge at 4 ℃ and 8000 rpm for 10 min, and discard the supernatant. Resuspend the bacterial precipitate with 100 mM sodium acetate buffer (pH 4.6), centrifuge repeatedly at 4°C, 8000 rpm for 10 min, discard the supernatant, and obtain the precipitate, which is the E. coli BL21(DE3)/pET28a- phoC wet bacterial cell for whole cell catalytic production of 5'-inosinic acid and 5'-guanylic acid. Store it in a -20°C refrigerator for later use.

2. Whole cell catalysis produces 5'-inosinic acid or 5'-guanylic acid

(1) Preparation of 5'-inosinic acid

Take out the frozen E. coli BL21(DE3)/pET28a- phoC wet cells from step 1 and precool them at 4℃ for 6~8 hours. Prepare the whole cell catalytic reaction system: 0.02 g/mL E. coli BL21(DE3)/pET28a- phoC wet cells, 100mM inosine, 180mM disodium dihydrogen pyrophosphate, 10mM magnesium sulfate, and sodium acetate buffer (pH 4.8) to make up to 10~200mL, and react in a shaker at 30℃ and 150 rpm for 7 hours. After the reaction, take 1mL of the reaction solution into a 1.5mL EP tube, add 100μL of 2M HCl to terminate the reaction, and let it stand for 10 minutes. Dilute the reaction solution 20 times, take 200μL of the dilution solution and add it to the liquid phase bottle, detect the changes in the content of inosine and inosinic acid by HPLC and calculate the conversion rate.

(2) Preparation of 5'-guanylate

The frozen wet cells in Example 1 were taken out and precooled at 4°C for 6-8 hours. The whole cell catalytic reaction system was prepared: 0.02g/mL E. coli BL21(DE3)/pET28a- phoC wet cells, 100 mM guanosine, 220 mM disodium dihydrogen pyrophosphate, 10 mM magnesium sulfate, and sodium acetate buffer (pH 4.8) was made up to 10-200 mL, and the reaction was carried out in a shaker at 30°C and 150 rpm for 7 hours. The termination reaction operation was the same as the preparation of 5'-inosinic acid.

3. HPLC Analysis of Inosinic and Guanylic Acids

Chromatographic conditions:

Mobile phase: Phase A: Weigh 6.46 g of potassium dihydrogen phosphate, add 950 mL of ultrapure water to dissolve, and filter with an aqueous filter membrane; Phase B: Measure 50 mL of chromatographic grade acetonitrile and filter with an organic filter membrane. Mix phase A and phase B evenly and ultrasonicate in an ultrasonic cleaner for 30 min.

Chromatographic column model: Welchrom ®C18 (250 × 4.6 mm/5 µm) column;

Mobile phase flow rate: 1 mL/min; detection wavelength: 245 nm; detection temperature: 30 ℃.

The HPLC detection spectrum of the reaction solution catalyzed by whole cells to produce 5'-inosinic acid is shown in Figure 2, and the HPLC detection spectrum of the reaction solution catalyzed by whole cells to produce 5'-guanylic acid is shown in Figure 3.

4. Phosphotransferase activity assay

A 10 mL reaction system containing 0.02 g/mL bacterial cells, 100 mM inosine, and 180 mM disodium dihydrogen pyrophosphate was prepared in a 50 mL conical flask with sodium acetate buffer (pH 4.8). The reaction was terminated after shaking in a water bath at 30°C for 1 h, and the amount of 5'-inosinic acid generated was determined.

A 10 mL reaction system containing 0.02 g/mL bacterial cells, 100 mM guanosine, and 220 mM disodium dihydrogen pyrophosphate was prepared in a 50 mL conical flask with sodium acetate buffer (pH 4.8). The reaction was terminated after shaking in a water bath at 30°C for 1 h, and the amount of 5'-guanylic acid generated was determined.

Enzyme activity definition: The amount of enzyme required to generate 1 μmol of 5'-inosinic acid (or 5'-guanylic acid) per minute at 30°C is 1 enzyme activity unit (U). Specific enzyme activity definition: The enzyme activity units contained in 1 g of dry bacteria, expressed as U/g DCW.

Substrate conversion rate = product mole number/substrate mole number × 100%.

Results: The wild-type phosphotransferase of Enterobacter aerogenes and three batches of genetically engineered bacteria E. coli BL21(DE3)/pET28a- phoC were used to prepare 5'-inosinic acid according to the above method, and the changes in the content of inosine and inosinic acid were detected, and the average conversion rate was calculated. The results showed that the conversion rate of wild-type phosphotransferase of Enterobacter aerogenes to produce inosinic acid was 41.95%, and the conversion rate of guanylic acid was 21.45%. The average conversion rate of three batches of genetically engineered bacteria E. coli BL21(DE3)/pET28a- phoC to produce 5'-inosinic acid was 89.54%, and the average conversion rate of 5'-guanylic acid was 86.81%, which were significantly higher than those of wild-type phosphotransferase of Enterobacter aerogenes.

Example 4

This example provides a phosphotransferase mutant AP/PTase- phoC- K133R, which is obtained by mutating the lysine at position 133 to arginine based on the phosphotransferase mutant AP/PTase- phoC in Example 1. Its nucleotide sequence is shown in SEQ ID NO.5 and its amino acid sequence is shown in SEQ ID NO.6.

Example 5

This embodiment provides a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO.5 and a construction method thereof, as well as a genetically engineered bacterium constructed using the recombinant plasmid.

The genetically engineered bacteria E. coli BL21 (DE3) / pET28a- phoC in Example 2 were streaked on LB solid plates (Kan) and cultured in a 37 ° C incubator for 12 to 14 h. Then, a single colony was picked and inoculated into LB liquid culture medium (Kan), cultured in a 37 ° C shaker for 8 to 12 h, 2 mL of the cultured bacterial solution was drawn into a 2.0 mL EP tube, and the plasmid was extracted using a plasmid extraction kit. The extracted pET28a- phoC plasmid was used as a template and mutated using the inverse PCR technique. The primers are shown in Table 1.

Table 1 Primers for whole plasmid PCR (targeting site K133)

Note: The underlined part of the primer represents the mutation site

After adding dd H ₂ O, plasmid template, upstream and downstream primers in sequence, high-fidelity enzyme was finally added and mixed on ice. The amplification procedure of PCP reaction mutation is shown in Table 2 .

Table 2 PCR amplification procedure of fragments

3 µL of PCR product was taken for agarose gel electrophoresis verification, and a bright band at 8000 bp was visible under ultraviolet light, which was consistent with the theoretical value of the plasmid. Then 1 µL of Dpn I restriction endonuclease was added to the reaction solution, and the reaction was carried out at 37℃ for 1 h for digestion. After removing the methylated template, a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO.5 was obtained.

The recombinant plasmid was directly transformed into the host bacterium E. coli BL21 (DE3). The transformation plate was identified by colony PCR and sequenced to confirm that it was a correct mutant. The engineered bacterium was the genetically engineered bacterium E. coli BL21 (DE3) / pET28a- phoC- K133R expressing the phosphotransferase mutant AP/PTase- phoC- K133R.

Example 6

This embodiment provides the use of the genetically engineered bacteria E. coli BL21 (DE3) / pET28a- phoC- K133R in Example 5 in whole cell catalytic production of 5'-inosinic acid:

According to the method of Example 3, E. coli BL21 (DE3) / pET28a- phoC- K133R was used for enzyme expression and whole cell catalysis to produce 5'-inosinic acid. The conversion rate was 94.84%.

Example 7

This example provides a phosphotransferase mutant AP/PTase- phoC- V212T, which is obtained by mutating the valine at position 212 to threonine based on the phosphotransferase mutant AP/PTase- phoC in Example 1. Its nucleotide sequence is shown in SEQ ID NO.7 and its amino acid sequence is shown in SEQ ID NO.8.

Example 8

This embodiment provides a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO.7 and a construction method thereof, as well as a genetically engineered bacterium constructed using the recombinant plasmid.

The genetically engineered bacteria E. coli BL21 (DE3) / pET28a- phoC in Example 2 were streaked on LB solid plates (Kan) and cultured in a 37 ° C incubator for 12 to 14 h. Then, a single colony was picked and inoculated into LB liquid culture medium (Kan), cultured in a 37 ° C shaker for 8 to 12 h, 2 mL of the cultured bacterial solution was drawn into a 2.0 mL EP tube, and the plasmid was extracted using a plasmid extraction kit. The extracted pET28a- phoC plasmid was used as a template and mutated using the inverse PCR technique. The primers are shown in Table 3.

Table 3 Whole plasmid PCR primers (targeting site V212)

Note: The underlined part of the primer represents the mutation site

After adding dd H ₂ O, plasmid template, upstream and downstream primers in sequence, high-fidelity enzyme was finally added and mixed on ice. The amplification procedure of PCP reaction mutation is shown in Table 4.

Table 4 PCR amplification procedure of fragments

3 µL of PCR product was taken for agarose gel electrophoresis verification, and a bright band at 8000 bp was visible under ultraviolet light, which was consistent with the theoretical value of the plasmid. Then 1 µL of Dpn I restriction endonuclease was added to the reaction solution, and the reaction was carried out at 37℃ for 1 h for digestion. After removing the methylated template, a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO.7 was obtained.

The recombinant plasmid was directly transformed into the host bacterium E. coli BL21 (DE3). The transformation plate was identified by colony PCR and sequenced to confirm that it was a correct mutant. The engineered bacterium was the genetically engineered bacterium E. coli BL21 (DE3)/pET28a- phoC- V212T expressing the phosphotransferase mutant AP/PTase- phoC- V212T.

Example 9

This example provides the use of the genetically engineered bacteria E. coli BL21 (DE3) / pET28a- phoC- V212T in Example 8 in whole cell catalytic production of 5'-guanylate:

According to the method of Example 3, E. coli BL21(DE3)/pET28a- phoC- V212T was used for enzyme expression and whole cell catalysis to produce 5'-guanylate. The conversion rate was 87.88%.

Example 10

This example provides a series of genetically engineered bacteria expressing phosphotransferase mutants and their use in whole cell catalytic production of 5'-inosinic acid or 5'-guanylic acid. The phosphotransferase mutants expressed by each genetically engineered bacteria are mutated at sites P165, G167, H168, G198, H207, and D211 based on the phosphotransferase mutant AP/PTase- phoC in Example 1.

The genetically engineered bacteria E. coli BL21 (DE3) / pET28a- phoC in Example 2 were streaked on LB solid plates (Kan) and cultured in a 37 ° C incubator for 12 to 14 h. Then, a single colony was picked and inoculated into LB liquid culture medium (Kan), cultured in a 37 ° C shaker for 8 to 12 h, 2 mL of the cultured bacterial solution was drawn into a 2.0 mL EP tube, and the plasmid was extracted using a plasmid extraction kit. The extracted pET28a- phoC plasmid was used as a template and mutated using the inverse PCR technique. The primers are shown in Table 5.

Table 5 Primers for whole plasmid PCR

Note: The underlined part of the primer represents the mutation site

After adding dd H ₂ O, plasmid template, upstream and downstream primers in sequence, high-fidelity enzyme was finally added and mixed on ice. The amplification procedure of PCP reaction mutation is shown in Table 6.

Table 6 PCR amplification procedure of fragments

Note: The above annealing temperature is adjusted according to primer design.

3 µL of PCR product was taken for agarose gel electrophoresis verification. A bright band at 8000 bp was visible under ultraviolet light, which was consistent with the theoretical value of the plasmid. Then 1 µL of Dpn I restriction endonuclease was added to the reaction solution, and the reaction was carried out at 37°C for 1 h for digestion. After removing the methylated template, the host bacteria E. coli BL21 (DE3) was directly transformed. The transformation plate was identified by colony PCR and sequenced and compared to confirm the correct mutant. The obtained engineered bacteria are genetically engineered bacteria that express each phosphotransferase mutant.

According to the method of Example 3, the above genetically engineered bacteria were subjected to enzyme expression and whole-cell catalysis to produce 5'-inosinic acid or 5'-guanylic acid. After testing, the conversion rates of the genetically engineered bacteria were shown in Table 7.

Table 7 Transformation rate of each genetically engineered bacteria

From the above results, it can be seen that the conversion rate of 5'-inosinic acid or 5'-guanylic acid produced by the genetically engineered bacteria expressing the mutants containing the mutation sites in the mutants of Example 1 is also better than that of the wild-type phosphotransferase of Enterobacter aerogenes, but the conversion rate of 5'-inosinic acid is lower than that of the genetically engineered bacteria E. coli BL21(DE3)/pET28a- phoC- K133R in Example 5, and the conversion rate of 5'-guanylic acid is lower than that of the genetically engineered bacteria E. coli BL21(DE3)/pET28a- phoC- V212T in Example 8.

Embodiment 11

This example provides the construction of a low-endotoxin Escherichia coli strain, the expression of a phosphotransferase mutant, and its catalytic application.

Lambda-Red homologous recombination technology was used to knock out the endotoxin synthesis-related gene eptA (GenBank ID: 948629, full length 1644 bp) of E. coli BL21 (DE3), and its nucleotide sequence is shown in SEQ ID NO. 9. The pKD46, pKD4 and pCP20 plasmids used for knockout are all commercial plasmids.

1. Obtain Donor DNA fragments

E. coli BL21 (DE3) and DH5α-pKD4 strains were streaked on LB plates with no resistance and Kan resistance, respectively, and inverted at 37°C for 12-14 hours. To obtain the upstream and downstream DNA fragments of the endotoxin gene, primers were designed based on the E. coli BL21 (DE3) genome sequence, and homology arms were added to one end of the upstream and downstream fragments by PCR. The pKD4 plasmid was extracted as a template, and the Kan resistance gene with FRT reverse enzyme sites on both sides was obtained by PCR amplification. The designed primers are shown in Table 8.

Table 8 Target gene primer sequences

Note: The homology arm of the downstream primer of the upstream DNA fragment of the target gene is ATCTTGTTCAATCAT; the homology arm of the downstream primer of the downstream DNA fragment is GTCAGCCGTTAAGTG; both are added at the 5' end.

Using the upstream and downstream DNA fragments of the target gene and the Kan gene containing the FRT invertase site as templates, the three DNA fragments were connected by overlapping extension PCR to obtain donor DNA. The PCR amplification system is shown in Table 9.

Table 9 Overlap extension PCR amplification system

The obtained Donor DNA was mixed with 385 µL of anhydrous ethanol and 54 µL of 3M sodium chloride per 100 µL, and placed at -20 °C for 12 h. Then, the mixture was centrifuged at 4 °C and 8000 rpm for 30 min, the supernatant was discarded, 300 µL of 70% ethanol was added, and the mixture was centrifuged at 4 °C and 8000 rpm for 5 min, the supernatant was discarded, and the mixture was placed in a metal bath at 50-60 °C. After the ethanol evaporated completely, 30 µL of ddH ₂ O was added to dissolve the mixture to obtain a high concentration of Donor DNA.

2. λ-Red Homologous Recombination

Take out the strain DH5α/pKD46, streak it on an LB plate with Amp resistance, and place it in a 37 ℃ constant temperature incubator for 12~14h. Then pick a single colony and inoculate it in LB liquid medium (Amp), culture it in a shaker at 37 ℃ and 200 rpm for 12 h, take 2mL of the cultured bacterial liquid into a 2.0 mL EP tube, and extract the plasmid using a plasmid extraction kit. Transform the extracted pKD46 plasmid into E. coli BL21(DE3) competent state at a transformation temperature of 30 ℃ to obtain E. coli BL21(DE3)/pKD46. Pick a single colony of E. coli BL21(DE3)/pKD46 in LB liquid medium (Amp), and culture it in a shaker at 30 ℃ and 200 rpm for 12~16h. Take the overnight culture seed liquid, inoculate it into 50 mL LB liquid medium (Amp) at a ratio of 1:50, and continue to shake culture until OD ₆₀₀ is 0.2, add 1M L-arabinose to make the final concentration 30 mM, continue to shake culture until OD ₆₀₀ reaches about 0.6, take out the bacterial liquid and put it on ice for 30 minutes. Wash the competent state with ddH ₂ O for about 5 times, and resuspend the competent state with 30% glycerol for the last time, and store it at -80℃.

Dissolve the above-mentioned E. coli BL21(DE3)/pKD46 competent cells on ice, take 50 µL, add 3 µg of the above-mentioned DonorDNA, mix well, quickly add to the pre-cooled 0.1 mm electroporation cup, electrotransform at 1.8 KV, and then immediately add LB recovery solution to shake for 2 h. After the recovery, take the bacterial solution and spread it on the LB solid plate (Amp). Colony PCR identifies whether each transformant has successfully knocked out the corresponding endotoxin gene through Red recombination.

3. Elimination of resistance genes

The transformants with successful knockout were inoculated into LB liquid medium without resistance and cultured at 42 °C and 200 rpm for 12-16 h in a shaking incubator to eliminate the temperature-sensitive plasmid pKD46. The bacterial solution was then streaked on Amp and knockout corresponding resistance plates (Cm or Kana), and the pKD46 elimination strain did not grow on Amp plates, but could grow on Cm or Kana plates.

The DH5α-pCP20 strain was cultured and the pCP20 plasmid was extracted. The temperature-sensitive pCP20 plasmid can express flipase recombination enzyme (FLP) under 30 ℃ culture conditions, eliminating a FRT site and the kanamycin resistance gene. The successfully identified pKD46 elimination strain was prepared as competent, transformed into the pCP20 plasmid (Amp and Cm resistance, temperature-sensitive), and cultured at 30 ℃. The plate method was used to identify the strains with successful elimination of the FRT site and kanamycin resistance gene. The knockout strain carrying the pCP20 plasmid was further cultured at 42 ℃ to eliminate pCP20, becoming a knockout strain without resistance. The strain was streaked on a non-resistance, Amp or Cm plate to identify whether the resistance was successfully eliminated.

The endotoxin gene knockout strain E. coli BL21(DE3) △ eptA was obtained through the above three steps.

4. Expression of phosphotransferase mutants in endotoxin gene knockout strains and their catalytic applications

The “Bacterial Endotoxin Detection Method” in Part 4 of the 2020 edition of the Chinese Pharmacopoeia was used to detect the endotoxin content of the above-mentioned endotoxin gene knockout strains and the original strain E. coli BL21 (DE3).

The above-mentioned endotoxin gene knockout E. coli BL21 (DE3) strain was made into a competent state, and then the plasmids pET28a- phoC- K133R and pET28a- phoC- V212T were respectively transferred to obtain low-endotoxin E. coli strains E. coli BL21 (DE3) △ eptA /pET28a- phoC- K133R and E. coli BL21 (DE3) △ eptA /pET28a- phoC- V212T expressing phosphotransferase mutants. According to the method of Example 3, the E. coli BL21 (DE3) strain and the above-mentioned strains were subjected to enzyme expression and whole-cell catalytic production of 5'-inosinic acid or 5'-guanylic acid, and the amount of 5'-inosinic acid or 5'-guanylic acid generated was determined and the conversion rate was calculated, and the endotoxin content was calculated at the same time.

After testing, when the E. coli BL21 (DE3) strain produced 5'-inosinic acid or 5'-guanylic acid, the endotoxin content in the transformation liquid was 101.87EU/L. The relative endotoxin content and conversion rate of E. coli BL21 (DE3) △ eptA /pET28a- phoC- K133R and E. coli BL21 (DE3) △ eptA /pET28a- phoC- V212T when producing 5'-inosinic acid and 5'-guanylic acid are shown in Table 10.

Table 10 Endotoxin content and catalytic application results

Note: The above is the average value of three parallel experiments.

Example 12

This example provides a series of low endotoxin Escherichia coli strains and their construction methods

The low endotoxin E. coli strain is basically the same as in Example 11, except that the primer sequences for obtaining the Donor DNA fragment are shown in Table 9.

Table 9 Target gene primer sequences

After three steps of obtaining Donor DNA fragment, λ-Red homologous recombination and resistance gene elimination, the endotoxin gene knockout strains E. coli BL21(DE3) △ pagP , E. coli BL21(DE3) △ LpxL , E. coli BL21(DE3) △ LpxP and E. coli BL21(DE3) △ LpxM were obtained respectively.

The “Bacterial Endotoxin Detection Method” in Part 4 of the 2020 edition of the Chinese Pharmacopoeia was used to detect the endotoxin content of the above-mentioned endotoxin gene knockout strains. The method was the same as in Example 11. The endotoxin content, inosine conversion rate, and guanosine conversion rate of each strain are shown in Table 11, respectively.

Table 11

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (13)

1. A phosphotransferase mutant is characterized in that the mutant is obtained by mutating a wild type phosphotransferase of enterobacter aerogenes with a nucleotide sequence shown as SEQ ID NO.1 and an amino acid sequence shown as SEQ ID NO. 2, wherein the mutation comprises the steps of mutating leucine at position 81 into glutamine, alanine at position 83 into glutamine, glutamic acid at position 84 into alanine, asparagine at position 87 into aspartic acid, serine at position 89 into alanine, alanine at position 90 into phenylalanine, glycine at position 92 into aspartic acid, threonine at position 153 into lysine, glutamic acid at position 154 into aspartic acid and isoleucine at position 171 into threonine.

2. The phosphotransferase mutant according to claim 1, wherein the nucleotide sequence of the phosphotransferase mutant is shown in SEQ ID NO.3 and the amino acid sequence is shown in SEQ ID NO. 4.

3. The phosphotransferase mutant according to claim 1 or 2, wherein the mutation further comprises mutating lysine at position 133 to arginine.

4. The phosphotransferase mutant according to claim 3, wherein the nucleotide sequence of the phosphotransferase mutant is shown in SEQ ID NO.5 and the amino acid sequence is shown in SEQ ID NO. 6.

5. The phosphotransferase mutant according to claim 1 or 2, wherein the mutation further comprises a mutation of valine at position 212 to threonine.

6. The phosphotransferase mutant according to claim 5, wherein the nucleotide sequence of the phosphotransferase mutant is shown in SEQ ID NO.7 and the amino acid sequence is shown in SEQ ID NO. 8.

7. A recombinant plasmid comprising the nucleotide sequence of the phosphotransferase mutant according to any one of claims 1 to 6.

8. A genetically engineered bacterium constructed using the recombinant plasmid of claim 7.

9. The low endotoxin escherichia coli engineering bacteria constructed by the recombinant plasmid of claim 7, wherein at least one gene of lpxL, lpxM, pagP, lpxP and eptA is knocked out by the low endotoxin escherichia coli engineering bacteria.

10. The low endotoxin escherichia coli engineering bacterium of claim 9, wherein the low endotoxin escherichia coli gene is knocked out eptA.

11. The method for constructing the low endotoxin escherichia coli engineering bacteria as defined in claim 10, which is characterized by comprising the following steps:

S1, designing upstream and downstream homology arm primers aiming at the sequence of the knocked-out gene, performing PCR amplification to obtain upstream and downstream DNA fragments of eptA genes, amplifying Kan resistance genes containing FRT invertase sites at two sides from a pKD4 plasmid through PCR, and connecting the upstream and downstream DNA fragments of a target gene with the resistance genes containing FRT invertase sites through overlap extension PCR technology to obtain donor DNA fragments of homology arm+FRT resistance genes;

S2, transforming a strain E.coli BL21 (DE 3) to be knocked out by using a pKD46 plasmid for expressing homologous recombinase, screening a pKD46 positive colony on an Amp resistance culture medium, culturing for 12-16 h, and transferring and then inducing expression of the recombinase by using arabinose; preparing induced thalli into competent cell suspension, adding 3-4 mu g S1 of the obtained donor DNA fragment into each 50mL of competent cell suspension, mixing, and adding into a electric shock cup for electric transformation; positive transformants were obtained by resistance plate screening;

S3, inoculating the positive transformant obtained in the S2 into an LB culture tube, and culturing for 12-16 hours at 42 ℃ to eliminate the temperature sensitive plasmid pKD46; preparing competent strain of knockout strain of eliminating plasmid pKD46, then transforming into pCP20 plasmid to eliminate Kan resistance gene, culturing at 30 deg.C;

S4, culturing the knocked-out strain carrying the pCP20 plasmid obtained in the S3 at 42 ℃ to eliminate the pCP20, and obtaining the knocked-out strain without resistance.

12. Use of the genetically engineered bacterium of claim 8 or the low endotoxin e.coli engineered bacterium of claim 9 or 10 in whole cell catalyzed production of 5 '-taste nucleotides, wherein the 5' -taste nucleotides comprise 5 '-inosinic acid and 5' -guanylic acid.

13. A method for whole-cell catalytic production of 5 '-taste nucleotides, characterized in that the 5' -taste nucleotides comprise 5 '-inosinic acid and 5' -guanylic acid, the method specifically comprising the steps of:

Step 1, after culturing and activating the genetically engineered bacterium of claim 8 or the low endotoxin escherichia coli engineering bacterium of claim 9 or 10, performing induced expression by isopropyl-beta-D-thiogalactoside, centrifugally collecting thalli, and washing with sodium acetate buffer solution to obtain wet thalli;

and 2, after precooling the wet thalli at 4 ℃, reacting the wet thalli with inosine or guanosine and disodium dihydrogen pyrophosphate in a sodium acetate buffer solution at 25-40 ℃ for 5-7 hours, wherein the concentration of the inosine or guanosine is 80-120 mM, the concentration of the disodium dihydrogen pyrophosphate is 120-220 mM, and the concentration of the wet thalli is 20-100 g/L.