A phosphotransferase mutant, a genetically engineered bacterium expressing the same and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a phosphotransferase mutant, a genetically engineered bacterium expressing the same, and an application thereof. The phosphotransferase mutant is obtained by mutating the wild-type phosphotransferase of Enterobacter aerogenes, and the genetically engineered bacterium expressing the phosphotransferase mutant can be used for whole cell catalytic production of 5'-flavor nucleotides. The low-endotoxin Escherichia coli provided by the present invention is a recombinant bacterium obtained by knocking out endotoxin synthesis-related genes of the Escherichia coli strain BL21 (DE3), which does not affect the expression of the phosphotransferase mutant and has a higher conversion rate when applied to whole cell catalytic production of 5'-flavor nucleotides, and the product has no risk of endotoxin residues.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a phosphotransferase mutant, a genetic engineering bacterium expressing the mutant and an application thereof. Background Art

[0002] 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 glutamic acid at a mass fraction of 5% to 12% is about 8 times higher than that of monosodium glutamate, 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.

[0003] In the prior art, the 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. Due to the limitations of microbial characteristics, 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.

[0004] 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 by 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.

[0005] 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 content of LPS. 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

[0006] 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.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme:

[0008] The first aspect of the present invention provides a phosphotransferase mutant, which is a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2 of Enterobacter aerogenes ( Enterobacter aerogenes ) wild-type phosphotransferase is mutated, wherein the mutations include 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.

[0009] In order to improve the conversion rate and reduce the amount of pyrophosphate used, the present invention treats Enterobacter aerogenes ( Enterobacter aerogenes ) Wild-type phosphotransferase (AP / PTase, gene name pho,[[]END]] The invention discloses a phosphotransferase mutant, wherein the phosphotransferase mutant is ...

[0010] 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 to guanosine can be increased to more than 80%.

[0011] Preferably, the mutation further comprises mutating the lysine at position 133 to arginine. This mutation can further increase the conversion rate of the phosphotransferase mutant to inosine to more than 93%.

[0012] 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.

[0013] 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%.

[0014] Further 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.

[0015] The second aspect of the present invention provides a recombinant plasmid containing the nucleotide sequence of the above-mentioned phosphotransferase mutant.

[0016] The third aspect of the present invention provides a genetically engineered bacterium constructed using the above-mentioned recombinant plasmid.

[0017] The fourth aspect of the present invention provides a low-endotoxin Escherichia coli constructed using the above-mentioned recombinant plasmid, wherein the low-endotoxin Escherichia coli has lpxL, lpxM, pagP, lpxP and eptA The above gene is an endotoxin synthesis gene. Knocking out the above gene can reduce the endotoxin content in the reaction system and reduce the process cost of removing endotoxin during purification.

[0018] Further preferably, the low endotoxin Escherichia coli is knocked out eptA Gene (GenBank ID: 948629, full length 1644bp). Knockout eptA The E. coli strain with the gene not only has a greatly reduced endotoxin compared to the original strain, but also has a certain change in its cell membrane permeability compared to the original strain, which improves the efficiency of the whole cell catalysis and makes it express phoC- K133R and phoC- The nucleoside conversion rate after V212T was higher than that of the original strain.

[0019] 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:

[0020] S1. Design upstream and downstream homology arm primers for the sequence of the knocked-out gene, and obtain eptAThe upstream and downstream DNA fragments of the gene, and the Kan resistance gene with FRT invertase sites on both sides are 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;

[0021] S2. Transform the knockout strain with the pKD46 plasmid expressing the homologous recombinase E. coli BL21(DE3), screen pKD46 positive colonies on Amp resistance medium, culture for 12-16 hours and induce the expression of recombinant enzyme with arabinose after transfer; prepare the induced bacteria into competent cell suspension, add 3-4 µg of the donor DNA fragment obtained in S1 per 50 mL of competent cell suspension, mix, add to the electroporation cup for electrotransformation; screen the positive transformants through resistance plate;

[0022] 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;

[0023] 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.

[0024] 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.

[0025] The sixth aspect of the present invention provides the use of the above-mentioned genetically engineered bacteria or low-endotoxin Escherichia coli in whole-cell catalytic production of 5'-flavor nucleotides, wherein the 5'-flavor nucleotides include 5'-inosinic acid and 5'-guanylic acid.

[0026] 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:

[0027] 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;

[0028] 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.

[0029] 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.

[0030] Preferably, the conditions for inducing expression are: culturing in a shaking incubator at 37°C and 200 rpm; 600 When the concentration reached 0.6±0.1, isopropyl-β-D-thiogalactoside was added for induction, and the temperature was lowered to 30°C and induced for 12±2h.

[0031] Preferably, the concentration of the sodium acetate buffer is 80-120 mM, and the pH is 4.7-4.9.

[0032] Preferably, the wet cells are precooled at 4° C. for 6 to 8 h.

[0033] 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 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

[0034] Figure 1 is the map of plasmid pET28a-phoC in Example 1;

[0035] Figure 2 The HPLC detection spectrum of the reaction solution when the whole cell catalyzes the production of 5'-inosinic acid for 7 hours in Example 3;

[0036] Figure 3 This is the HPLC detection spectrum of the reaction solution when the whole cell catalyzes the production of 5'-guanylate for 4 hours in Example 3. DETAILED DESCRIPTION

[0037] 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 making creative work fall within the scope of protection of the present invention.

[0038] 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 using pyrophosphate 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.

[0039] On the other hand, when the whole Escherichia coli cell is used for catalysis and the subsequent separation and extraction of the product, the endotoxin contained in its cell fragments increases the cost of downstream purification and detection of inosinic acid. From the perspective of the source of the Escherichia coli strain, removing the endotoxin synthesis gene through genetic modification is a technology with more application prospects. By knocking out the relevant 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, the cell wall structure of Escherichia coli will change, which may cause changes in growth and substrate and product metabolism, thereby affecting actual production. In the process of catalytic production of inosinic acid by whole Escherichia coli cells, the knockout application of endotoxin genes also has similar problems. Therefore, there is no research on the application of endotoxin gene knockout Escherichia coli in the whole cell catalytic preparation of 5'-inosinic acid in the prior art.

[0040] 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.

[0041] 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.

[0042] The embodiments of the present invention also provide low-endotoxin Escherichia coli with endotoxin synthesis genes knocked out and a construction method thereof.

[0043] 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.

[0044] The scheme of the present invention is described below through specific embodiments.

[0045] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials and reagents used in the following examples can be obtained from commercial channels, among which the original host bacteria E. coli BL21(DE3) and expression plasmid vector pET28a are products of commercial companies. pKD46, pKD4 and pCP20 plasmids are commercial plasmids. Limulus test kit was purchased from GenScript Biotech Co., Ltd.

[0046] Example 1

[0047] This example provides a phosphotransferase mutant AP / PTase- phoC The mutant is a mutant of Enterobacter aerogenes ( Enterobacter aerogenes ) wild-type phosphotransferase is obtained by 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 phosphotransferase mutant AP / PTase- phoC The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0048] The phosphotransferase mutant AP / PTase- phoC It was directly synthesized by Beijing Qingke Biotechnology Co., Ltd. after codon optimization.

[0049] Example 2

[0050] 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.

[0051] 1. Recombinant plasmid pET28a- phoC Construction

[0052] The nucleotide sequence shown in SEQ ID NO.3 was cloned into the expression plasmid pET28a containing 6×His tag to obtain the recombinant plasmid pET28a- phoC (Plasmid map see Figure 1 ).

[0053] 2. Escherichia coli BL21(DE3) / pET28a- phoC Construction

[0054] The synthesized pET28a- phoC Add the plasmid powder to sterile water to make the concentration 30 ng / μL. E. coli BL21(DE3) competent cells (100 μL) were thawed on ice, and pET28a- phoC The plasmid solution was slowly added to the completely melted competent medium and placed on ice for 30 min. Heat shock was performed in a constant temperature water bath at 42°C for 90 s, and then immediately placed on ice for 3-5 min. Then, 1 mL of LB medium (without antibiotics) was added and cultured in a shaking incubator at 37°C and 200 rpm for 1 h. At the end of the culture, centrifuged at 4°C and 4000 rpm for 5 min, the supernatant was removed and about 100 μL of the resuspended bacteria was spread on a solid LB plate containing Kan resistance, and inverted in a constant temperature incubator at 37°C for 12-14 h. After identification, the genetically engineered bacteria were obtained. Escherichia coli BL21(DE3) / pET28a- phoC (hereinafter referred to as E. coli BL21(DE3) / pET28a- phoC ).

[0055] Example 3

[0056] This example provides the genetically engineered bacteria in Example 2 E. coli BL21(DE3) / pET28a- phoC Application in whole cell catalytic production of 5'-inosinic acid or 5'-guanylic acid:

[0057] 1. E. coli BL21(DE3) / pET28a- phoC Inducible expression and wet bacterial cell preparation

[0058] Pick the genetically engineered bacteria on the above LB solid plate (Kan) E. coli BL21(DE3) / pET28a- phoCA single colony was inoculated into LB liquid medium (100 µg / mL Kan) and cultured in a shaker at 37°C and 200 rpm for 8-12 h to obtain seed solution. A 2% volume ratio inoculation was transferred to 150 mL LB liquid medium with the same kanamycin concentration and cultured at 37°C and 200 rpm to a bacterial concentration of OD 600 To about 0.6, add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.2 mM, and induce in a shaker at 30 ℃ and 200 rpm for 12 h. After the induction, pour the bacterial solution 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), repeat the centrifugation at 4 ℃ and 8000 rpm for 10 min, discard the supernatant, and the precipitate obtained is used for whole cell catalysis to produce 5'-inosinic acid and 5'-guanylic acid. E. coli BL21(DE3) / pET28a- phoC Wet bacteria were placed in a -20 ℃ refrigerator for later use.

[0059] 2. Whole cell catalysis produces 5'-inosinic acid or 5'-guanylic acid

[0060] (1) Preparation of 5'-inosinic acid

[0061] Frozen in step 1 E. coli BL21(DE3) / pET28a- phoC After the wet cells are taken out, they are precooled at 4°C 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, sodium acetate buffer (pH 4.8) are added to 10-200mL, and the mixture is shaken at 30℃ and 150rpm for 7h. After the reaction, 1mL of the reaction solution is placed in a 1.5mL EP tube, 100µL of 2M HCl is added to terminate the reaction, and the mixture is allowed to stand for 10min. The reaction solution is diluted 20 times, 200µL of the dilution solution is added to a liquid phase bottle, and the changes in the content of inosine and inosinic acid are detected by HPLC and the conversion rate is calculated.

[0062] (2) Preparation of 5'-guanylate

[0063] The frozen wet cells in Example 1 were taken out and precooled at 4°C for 6-8 hours. Preparation of whole cell catalytic reaction system: 0.02 g / mL E. coli BL21(DE3) / pET28a- phoCWet the cells, add 100 mM guanosine, 220 mM disodium dihydrogen pyrophosphate, 10 mM magnesium sulfate, and sodium acetate buffer (pH 4.8) to make up to 10-200 mL, and react in a shaker at 30 °C and 150 rpm for 7 h. The termination reaction operation is the same as the preparation of 5'-inosinic acid.

[0064] 3. HPLC Analysis of Inosinic and Guanylic Acids

[0065] Chromatographic conditions:

[0066] 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.

[0067] Chromatographic column model: Welchrom ®C18 (250 × 4.6 mm / 5 µm) column;

[0068] Mobile phase flow rate: 1 mL / min; detection wavelength: 245 nm; detection temperature: 30 ℃.

[0069] The HPLC detection spectrum of the reaction solution of whole cell catalytic production of 5'-inosinic acid is as follows Figure 2 As shown, the HPLC detection spectrum of the reaction solution of whole cell catalytic production of 5'-guanylate is as follows Figure 3 shown.

[0070] 4. Phosphotransferase activity assay

[0071] 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.

[0072] 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.

[0073] 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.

[0074] Substrate conversion rate = product mole number / substrate mole number × 100%.

[0075] Results: Wild-type phosphotransferase from Enterobacter aerogenes and three batches of genetically engineered bacteria E. coli BL21(DE3) / pET28a- phoC According to the above method, 5'-inosinic acid was prepared 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%. E. coli BL21(DE3) / pET28a- phoC The average conversion rates of 5'-inosinic acid and 5'-guanylic acid were 89.54% and 86.81%, respectively, which were significantly higher than those of wild-type phosphotransferase of Enterobacter aerogenes.

[0076] Example 4

[0077] This example provides a phosphotransferase mutant AP / PTase- phoC- K133R, this mutant is the phosphotransferase mutant AP / PTase- phoC The lysine at position 133 is mutated to arginine on the basis of , and its nucleotide sequence is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6.

[0078] Example 5

[0079] 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.

[0080] The genetically engineered bacteria in Example 2 E. coli BL21(DE3) / pET28a— phoC Streak on LB solid plate (Kan) and culture in 37 ℃ incubator for 12~14 h. Then pick a single colony and inoculate it in LB liquid medium (Kan), culture it in 37 ℃ shaker for 8~12 h, draw 2 mL of cultured bacterial liquid into 2.0 mL EP tube, and extract plasmid using plasmid extraction kit. phoC The plasmid was used as a template to mutate the plasmid using inverse PCR technology. The primers are shown in Table 1.

[0081] Table 1 Primers for whole plasmid PCR (targeting site K133)

[0082]

[0083] Note: The underlined part of the primer represents the mutation site

[0084] After adding dd H2O, 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.

[0085] Table 2 PCR amplification procedure of fragments

[0086]

[0087] 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.

[0088] The recombinant plasmid was directly transformed into the host bacterium Escherichia coli E. coli BL21(DE3). The transformed plate was identified by colony PCR and sequenced to confirm the correct mutant. The engineered bacteria is the one expressing the phosphotransferase mutant AP / PTase- phoC- K133R genetically engineered bacteria E. coli BL21(DE3) / pET28a- phoC- K133R.

[0089] Example 6

[0090] This embodiment provides the genetically engineered bacteria in Example 5. E. coli BL21(DE3) / pET28a- phoC- Application of K133R in whole cell catalytic production of 5'-inosinic acid:

[0091] 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%.

[0092] Example 7

[0093] This example provides a phosphotransferase mutant AP / PTase- phoC- V212T, which is a mutant of the phosphotransferase mutant AP / PTase- phoC The 212th valine was mutated to threonine on the basis of , and its nucleotide sequence is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.8.

[0094] Example 8

[0095] 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.

[0096] The genetically engineered bacteria in Example 2 E. coli BL21(DE3) / pET28a- phoC Streak on LB solid plate (Kan) and culture in 37 ℃ incubator for 12~14 h. Then pick a single colony and inoculate it in LB liquid medium (Kan), culture it in 37 ℃ shaker for 8~12 h, draw 2 mL of cultured bacterial liquid into 2.0 mL EP tube, and extract plasmid using plasmid extraction kit. phoC The plasmid was used as a template to perform mutation using inverse PCR. The primers are shown in Table 3.

[0097] Table 3 Whole plasmid PCR primers (targeting site V212)

[0098]

[0099] Note: The underlined part of the primer represents the mutation site

[0100] After adding dd H2O, 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.

[0101] Table 4 PCR amplification procedure of fragments

[0102]

[0103] 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.

[0104] The recombinant plasmid was directly transformed into the host bacterium Escherichia coli E. coli BL21(DE3). The transformed plate was identified by colony PCR and sequenced to confirm the correct mutant. The engineered bacteria is the one expressing the phosphotransferase mutant AP / PTase- phoC- Genetically engineered bacteria V212T E. coli BL21(DE3) / pET28a- phoC- V212T.

[0105] Example 9

[0106] This example provides the genetically engineered bacteria in Example 8 E. coli BL21(DE3) / pET28a- phoC- Application of V212T in whole cell catalytic production of 5'-guanylate:

[0107] 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%.

[0108] Example 10

[0109] 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 respectively the phosphotransferase mutant AP / PTase- phoC On the basis of this, sites P165, G167, H168, G198, H207, and D211 were mutated.

[0110] The genetically engineered bacteria in Example 2 E. coli BL21(DE3) / pET28a- phoC Streak on LB solid plate (Kan) and culture in 37 ℃ incubator for 12~14 h. Then pick a single colony and inoculate it in LB liquid medium (Kan), culture it in 37 ℃ shaker for 8~12 h, draw 2 mL of cultured bacterial liquid into 2.0 mL EP tube, and extract plasmid using plasmid extraction kit. phoC The plasmid was used as a template to mutate the plasmid using the inverse PCR technique. The primers are shown in Table 5.

[0111] Table 5 Primers for whole plasmid PCR

[0112]

[0113] Note: The underlined part of the primer represents the mutation site

[0114] After adding dd H2O, 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.

[0115] Table 6 PCR amplification procedure of fragments

[0116]

[0117] Note: The above annealing temperature is adjusted according to primer design.

[0118] 3 µL of PCR product was taken for agarose gel electrophoresis verification. A bright band at 8000 bp was visible under UV 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, the host bacteria Escherichia coli was directly transformed. E. coli BL21 (DE3). The transformation plate was identified by colony PCR and sequenced to confirm the correct mutant. The obtained engineered bacteria are genetically engineered bacteria that express the phosphotransferase mutants.

[0119] 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.

[0120] Table 7 Transformation rate of each genetically engineered bacteria

[0121]

[0122] 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 in Example 5. E. coli BL21(DE3) / pET28a- phoC- K133R, the conversion rate of producing 5'-guanylate is not as good as that of the genetically engineered bacteria in Example 8 E. coli BL21(DE3) / pET28a- phoC- V212T.

[0123] Embodiment 11

[0124] This embodiment provides the construction of a low-endotoxin Escherichia coli strain, the expression of a phosphotransferase mutant, and its catalytic application.

[0125] Knockout using Lambda-Red homologous recombination technology E. coli Endotoxin synthesis-related genes in BL21(DE3) eptA (GenBank ID: 948629, full length 1644 bp), and its nucleotide sequence is shown in SEQ ID NO. 9. The pKD46, pKD4 and pCP20 plasmids used for knockout are all commercial plasmids.

[0126] 1. Obtain Donor DNA fragments

[0127] WillE. coli BL21(DE3) and DH5α-pKD4 strains were streaked on LB plates without resistance and with Kan resistance, respectively, and inverted and cultured at 37°C for 12-14 h. E. coli Primers were designed based on the 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 invertase sites on both sides was obtained by PCR amplification. The designed primers are shown in Table 8.

[0128] Table 8 Target gene primer sequences

[0129]

[0130] 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.

[0131] 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.

[0132] Table 9 Overlap extension PCR amplification system

[0133]

[0134] 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 ddH2O was added to dissolve the mixture to obtain a high concentration of Donor DNA.

[0135] 2. λ-Red Homologous Recombination

[0136] Take out the strain DH5α / pKD46, streak it on the LB plate with Amp resistance, and place it in a 37 ℃ constant temperature incubator for 12-14 hours. 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 hours, draw 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 intoE. coli BL21(DE3) competent state, transformation temperature is 30 ℃, obtain E. coli BL21(DE3) / pKD46. Pick E. coli A single colony of BL21(DE3) / pKD46 was cultured in LB liquid medium (Amp) at 30°C and 200 rpm in a shaker for 12-16 hours. The overnight culture seed solution was inoculated into 50 mL of LB liquid medium (Amp) at a ratio of 1:50 and continued to be cultured in a shaker until OD 600 0.2, add 1M L-arabinose to make the final concentration 30 mM, and continue to shake and culture OD 600 When the concentration reaches about 0.6, take out the bacterial solution and place it on ice for 30 min. Wash the competent cells with ddH2O for about 5 times, and resuspend them with 30% glycerol for the last time and store them at -80℃.

[0137] The above E. coli After BL21(DE3) / pKD46 competent cells were dissolved on ice, 50 µL was taken, 3 µg of the above-mentioned DonorDNA was added, mixed evenly, and quickly added to a pre-cooled 0.1 mm electroporation cup, electroporated at 1.8 KV, and then immediately added to the LB recovery solution for 2 h on a shaker. After the recovery, the bacterial solution was spread on an LB solid plate (Amp). Colony PCR was used to identify whether each transformant had successfully knocked out the corresponding endotoxin gene through Red recombination.

[0138] 3. Elimination of resistance genes

[0139] 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.

[0140] 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.

[0141] Obtain endotoxin gene knockout strain through the above 3 steps E. coli BL21(DE3) △ eptA.[[]END]]

[0142] 4. Expression of phosphotransferase mutants in endotoxin gene knockout strains and their catalytic applications

[0143] The above endotoxin gene knockout strains and original strains were tested using the "Bacterial Endotoxin Detection Method" in Part 4 of the Chinese Pharmacopoeia 2020 Edition. E. coli Endotoxin content was detected in BL21(DE3).

[0144] The above endotoxin gene knockout E. coli BL21(DE3) strain was made competent and then transformed into plasmid pET28a- phoC- K133R and pET28a- phoC- V212T, obtaining a low-endotoxin E. coli strain expressing a phosphotransferase mutant E. coli BL21(DE3) △ eptA / pET28a- phoC- K133R and E. coli BL21(DE3) △ eptA / pET28a- phoC- V212T. E. coli The BL21(DE3) strain and the above strains were used for enzyme expression and whole cell catalysis to produce 5'-inosinic acid or 5'-guanylic acid, the generated amount of 5'-inosinic acid or 5'-guanylic acid was determined and the conversion rate was calculated, and the endotoxin content was also calculated.

[0145] After testing, E. coli When the BL21(DE3) strain produced 5'-inosinic acid or 5'-guanylic acid, the endotoxin content in the transformation liquid was 101.87EU / L. E. coliBL21(DE3) △ eptA / pET28a- phoC- K133R and E. coli BL21(DE3)△ eptA / pET28a- phoC- The relative endotoxin content and conversion rate of V212T when producing 5'-inosinic acid and 5'-guanylic acid are shown in Table 10.

[0146] Table 10 Endotoxin content and catalytic application results

[0147]

[0148] Note: The above is the average value of three parallel experiments.

[0149] Example 12

[0150] This example provides a series of low endotoxin Escherichia coli strains and their construction methods

[0151] 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.

[0152] Table 9 Target gene primer sequences

[0153]

[0154] Endotoxin gene knockout strains were obtained through three steps: obtaining Donor DNA fragments, λ-Red homologous recombination, and eliminating resistance genes. E. coli BL21(DE3) △ pagP , E. coli BL21(DE3) △ LpxL , E. coli BL21(DE3) △ LpxP , E. coli BL21(DE3) △ LpxM .

[0155] 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.

[0156] Table 11

[0157]

[0158] 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

1. A phosphotransferase mutant, characterized in that The mutant 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 is that the leucine at position 81 is mutated to glutamine, the alanine at position 83 is mutated to glutamine, the glutamate at position 84 is mutated to alanine, the asparagine at position 87 is mutated to aspartic acid, the serine at position 89 is mutated to alanine, the alanine at position 90 is mutated to phenylalanine, the glycine at position 92 is mutated to aspartic acid, the threonine at position 153 is mutated to lysine, the glutamate at position 154 is mutated to aspartic acid, and the isoleucine at position 171 is mutated to threonine.

2. A phosphotransferase mutant, characterized in that: The mutant is obtained by mutating the lysine at position 133 of the phosphotransferase mutant according to claim 1 to arginine.

3. A phosphotransferase mutant, characterized in that: The mutant is a mutant in which the valine at position 212 of the phosphotransferase mutant according to claim 1 is mutated to threonine.

4. A recombinant plasmid comprising the nucleotide sequence of the phosphotransferase mutant according to any one of claims 1 to 3.

5. A genetically engineered bacterium constructed using the recombinant plasmid according to claim 4.

6. The low endotoxin Escherichia coli engineered bacteria constructed using the recombinant plasmid according to claim 4, characterized in that: The low endotoxin Escherichia coli engineered bacteria have been knocked out lpxL, lpxM, pagP, lpxP or eptA Any gene in the gene.

7. The low endotoxin Escherichia coli engineered bacteria according to claim 6, characterized in that: The low endotoxin Escherichia coli knockout eptA Gene.

8. The method for constructing the low endotoxin Escherichia coli engineered bacteria according to claim 7, characterized in that: The specific steps include: S1. Design upstream and downstream homology arm primers for the sequence of the knocked-out gene, and obtain eptA The upstream and downstream DNA fragments of the gene, and the Kan resistance gene with FRT invertase sites on both sides are amplified from the pKD4 plasmid by PCR, and the upstream and downstream DNA fragments of the target gene and the resistance gene containing FRT invertase sites are connected by overlapping extension PCR technology to obtain the donor DNA fragment of homology arm + FRT resistance gene; S2. Transform the knockout strain with the pKD46 plasmid expressing the homologous recombinase E. coli BL21 DE3, screen pKD46 positive colonies on Amp resistance medium, culture for 12-16 hours and induce the expression of recombinant enzyme with arabinose after transfer; prepare the induced bacteria into competent cell suspension, add 3-4 µg of the donor DNA fragment obtained in S1 per 50 mL of competent cell suspension, mix, add to the electroporation cup for electrotransformation; screen the positive transformants through 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, 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.

9. Use of the genetically engineered bacteria according to claim 5 or the low-endotoxin Escherichia coli engineered bacteria according to claim 6 or 7 in whole-cell catalytic production of 5'-flavor nucleotides, characterized in that: The genetically engineered bacteria are constructed by a recombinant plasmid containing the nucleotide sequence of the phosphotransferase mutant according to claim 1, and the 5'-flavor nucleotides are 5'-inosinic acid and 5'-guanylic acid; or The genetically engineered bacteria are constructed by a recombinant plasmid containing the nucleotide sequence of the phosphotransferase mutation according to claim 2, and the 5'-flavor nucleotide is 5'-inosinic acid; or The genetically engineered bacteria are constructed from a recombinant plasmid containing the nucleotide sequence of the phosphotransferase mutation according to claim 3, and the 5'-flavor nucleotide is 5'-guanylate.

10. A method for whole cell catalytic production of 5'-flavor nucleotides, characterized in that: The method specifically comprises the following steps: Step 1, culturing and activating the genetically engineered bacteria according to claim 5 or the low-endotoxin Escherichia coli engineered bacteria according to claim 6 or 7, inducing expression with isopropyl-β-D-thiogalactoside, 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; The genetically engineered bacteria is constructed from a recombinant plasmid containing the nucleotide sequence of the phosphotransferase mutant according to claim 1, and the 5'-flavor nucleotides are 5'-inosinic acid and 5'-guanylic acid; or the genetically engineered bacteria is constructed from a recombinant plasmid containing the nucleotide sequence of the phosphotransferase mutant according to claim 2, and the 5'-flavor nucleotide is 5'-inosinic acid; or the genetically engineered bacteria is constructed from a recombinant plasmid containing the nucleotide sequence of the phosphotransferase mutant according to claim 3, and the 5'-flavor nucleotide is 5'-guanylic acid.

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