Escherichia coli for efficiently producing 5'-inosinic acid and its application

Abstract

The present invention relates to an Escherichia coli that efficiently produces 5'-inosinic acid and its application, belonging to the field of bioengineering technology. In order to reduce the degradation of 5'-inosinic acid in Escherichia coli, four phosphatase encoding genes were knocked out using the CRISPR / Cas9 system, and it was found that knocking out the UMP phosphatase encoding gene nagD and the 5'-nucleotidase encoding gene ushA respectively can increase the production of 5'-inosinic acid. Subsequently, in order to inhibit the further conversion of 5'-inosinic acid into guanylate and adenylate, the 5'-inosinic acid dehydrogenase encoding gene guaB was knocked out alone. Finally, in order to enhance the supply and utilization of formyltetrahydrofolate required in the purine synthesis process, the promoter of the serine hydroxymethyltransferase encoding gene glyA was replaced with P tac The recombinant Escherichia coli produced 2113.12 mg / L of 5'-inosinic acid in 48 hours of fermentation, and no antibiotics were required during the fermentation process, indicating that the product has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, in particular to an Escherichia coli for efficiently producing 5'-inosinic acid and its application. Background Art

[0002] 5'-Inosinic acid is the first purine nucleotide synthesized during the de novo synthesis of various purine nucleotides in organisms. It is widely present in meat and is produced by the degradation of ATP. It participates in the synthesis of RNA and DNA. Because its sodium salt has a specific fresh fish flavor and is easily soluble in water, it is widely used in the preparation of high-efficiency compound MSG.

[0003] Currently, the main methods for preparing 5'-inosinic acid include chemical methods using inosine as a substrate and chemical catalysts to produce 5'-inosinic acid. This method produces byproducts, 2'-inosinic acid or 3'-inosinic acid. The generation of these byproducts not only reduces the purity of the target product, but also leads to waste of raw materials and reduced conversion rate. Furthermore, the catalysts and reaction conditions used in chemical methods may pollute the environment, increasing production costs and environmental pressures. Enzymatic methods use inosine as a substrate and convert it to 5'-inosinic acid using phosphorylase. Although enzymatic methods offer the advantages of mild reaction conditions and high specificity, their conversion rate is limited by the activity of the phosphorylase and the accessibility of the substrate. Insufficient enzyme activity or insufficient binding between the substrate and the enzyme can lead to a decrease in conversion rate. Furthermore, enzymatic methods require enzyme isolation and purification, which increases operational complexity and cost. Microbial fermentation methods utilize a production strain to directly produce 5'-inosinic acid, but the fermentation process is affected by various factors, such as strain performance, culture medium composition, and fermentation conditions. If these factors are not optimized, problems such as slow bacterial growth and metabolic pathways deviating from the target product can result, thereby reducing conversion rates. Furthermore, microbial fermentation methods also require consideration of how to effectively separate and extract the target product from the fermentation broth, which increases process complexity and cost.

[0004] To increase the yield of 5'-inosinic acid (5'-IP) produced by microorganisms, researchers often increase or decrease the activity or expression of enzymes in the metabolic pathways involved in 5'-IP synthesis. However, existing production strains either express key genes via plasmids, requiring the addition of antibiotics required to maintain plasmid stability during fermentation, limiting the application of 5'-IP. Alternatively, 5'-IP production strains are selected through mutagenesis breeding, but the required mutagenesis cycle and unclear mechanism limit their application. Therefore, an industrial production strain capable of stably and efficiently expressing 5'-IP is urgently needed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an Escherichia coli that efficiently produces 5'-inosinic acid. In order to reduce the degradation of 5'-inosinic acid in Escherichia coli BLI7, four phosphatase encoding genes were knocked out using the CRISPR / Cas9 system, and it was found that knocking out the UMP phosphatase encoding gene nagD and the 5'-nucleotidase encoding gene ushA respectively can increase the production of 5'-inosinic acid. Subsequently, in order to inhibit the further conversion of 5'-inosinic acid into guanylate and adenylate, the 5'-inosinic acid dehydrogenase encoding gene guaB was knocked out alone. Finally, in order to enhance the supply and utilization of formyltetrahydrofolate required in the purine synthesis process, the promoter of the serine hydroxymethyltransferase encoding gene glyA was replaced with P tac .

[0006] The first object of the present invention is to provide Escherichia coli for efficiently producing 5'-inosinic acid, wherein the 6-phosphogluconolactonase encoding gene pgl is heterologously introduced into the Escherichia coli genome, the codon encoding the 128th aspartic acid of phosphoribosyl pyrophosphokinase is replaced by an alanine codon, the codon encoding the 326th lysine of glutamate amine phosphoribosyl pyrophosphotransferase is replaced by a glutamine codon, and the codon encoding the 410th proline is replaced by a tryptophan codon, the repressor protein encoding gene purR and the 6-phosphofructokinase encoding gene pfkA are knocked out or silenced, the phosphoribosyl pyrophosphokinase encoding gene prs and the glutamate amine phosphoribosyl pyrophosphotransferase encoding gene purF are overexpressed, and the UMP phosphatase encoding gene nagD and the 5'-inosinic acid dehydrogenase encoding gene guaB are knocked out or silenced.

[0007] Furthermore, the serine hydroxymethyltransferase encoding gene glyA was overexpressed in the Escherichia coli genome.

[0008] A second object of the present invention is to provide a method for constructing the above-mentioned Escherichia coli, comprising steps S1-S6 or S1-S7:

[0009] S1, integrating the 6-phosphogluconolactonase encoding gene pgl into the flgG site of the starting Escherichia coli genome;

[0010] S2, replacing the codon encoding aspartic acid at position 128 of phosphoribosylpyrophosphokinase in the genome of the strain obtained in S1 with an alanine codon;

[0011] S3, replacing the codon encoding lysine at position 326 of the strain obtained in S2 with a glutamine codon, and replacing the codon encoding proline at position 410 with a tryptophan codon;

[0012] S4, knocking out or silencing the repressor protein encoding gene purR and 6-phosphofructokinase encoding gene pfkA in the genome of the strain obtained from S3;

[0013] S5, the phosphoribosylpyrophosphokinase encoding gene prs in the genome of the strain obtained in S4 was D128A and purF, the gene encoding glutamate phosphoribosylpyrophosphate amidotransferase K326W / D410W The promoter was replaced by P tac promoter;

[0014] S6. Knock out or silence the UMP phosphatase encoding gene nagD and the 5'-inosinic acid dehydrogenase encoding gene guaB in the genome of the strain obtained in S5;

[0015] S7, in the genome of the strain obtained in S6, the promoter of the serine hydroxymethyltransferase encoding gene glyA was replaced with P tac Promoter.

[0016] Furthermore, the nucleotide sequence of the gene pgl is shown as SEQ ID NO.1.

[0017] Furthermore, the starting strain is Escherichia coli BL21 (DE3).

[0018] Furthermore, the CRISPR / Cas9 system was used for gene editing.

[0019] The third object of the present invention is to provide a method for increasing the production of 5'-inosinic acid in Escherichia coli, wherein the UMP phosphatase encoding gene nagD or the 5'-nucleotidase encoding gene ushA is knocked out in the genome of the starting strain.

[0020] A fourth object of the present invention is to provide a microbial preparation comprising the above-mentioned Escherichia coli.

[0021] The fifth object of the present invention is to provide a method for producing 5'-inosinic acid, wherein the above-mentioned Escherichia coli or the above-mentioned microbial preparation is added to the fermentation system.

[0022] Furthermore, the fermentation substrate is glucose.

[0023] Preferably, the concentration of glucose is 24 g / L.

[0024] Preferably, the fermentation time is 48 hours.

[0025] Beneficial effects of the present invention:

[0026] The Escherichia coli constructed by the present invention for efficiently producing 5'-inosinic acid uses glucose as a substrate. After 48 hours of fermentation culture, the yield of 5'-inosinic acid reaches 2113.12 mg / L, which is significantly improved compared with the yield of 434.19 mg / L of the starting strain, accounting for 479.51% of the starting strain. It is a recombinant Escherichia coli that can stably and efficiently express 5'-inosinic acid, and no antibiotics need to be added during the fermentation production process, which has prospects for industrial production. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0028] Example 1: Knockout of the UMP phosphatase encoding gene nagD

[0029] In order to reduce the degradation of 5'-inosinic acid, Escherichia coli BL21 (DE3) was used as the starting strain, and the UMP phosphatase encoding gene nagD was knocked out to obtain the recombinant strain BLI8.

[0030] Based on the sequence information of Escherichia coli, primers nagD-ff, nagD-fr, nagD-rf, and nagD-rr were designed. In the first round of amplification, 500 bp sequences before and after the nagD coding region were amplified from Escherichia coli BL21 (DE3). The primers were set with additional sequences, and the number of amplification cycles was 34-36, so that the fragment had a common sequence of about 30 bp after amplification. The agarose gel electrophoresis verification results showed that the fragment length was 500 bp; the fragment obtained in the first round of amplification was purified, and in the second round of amplification, only two fragments were added without primers, and the number of amplification cycles was changed to 10-15 rounds; the product of the second round of amplification did not need to be purified or verified by agarose gel electrophoresis; using this product as a template, primers nagD-ff and nagD-rr were added in the third round of amplification, the number of amplification cycles was 34-36, and agarose gel electrophoresis was performed to verify that the fragment length should be about 1000 bp, that is, the △nagD band. If there are impurity bands, the gel needs to be cut and recovered. If there are no impurity bands, the fragments can be directly purified for later use.

[0031] The ptarget plasmid is used to express the 20-bp sgRNA required for gene editing. Based on the ptarget plasmid information, primers n20-nagD-f and n20-nagD-r were designed. These primers carry the 20-bp sgRNA consensus sequence and bind to ptarget. This primer pair was used to amplify the ptarget vector using the ptarget template to obtain a linearized vector. The linearized vector was purified and transformed into E. coli DH5α. Colony PCR and sequencing were performed to verify the correct plasmid, which was identified as ptarget-nagD. The plasmid was then isolated and used for later use.

[0032] The plasmid pcas9 expressing the Cas9 protein was transformed into Escherichia coli BL21 (DE3), and the successfully transformed strains were screened using kanamycin resistance plates. The pcas9-transformed strain was then prepared into an electroporation competent state, and △nagD and ptarget-nagD were co-transformed into the competent state. After screening with kanamycin and spectinomycin, single colonies were picked from the plate and colony PCR was performed using primers nagD-cxf and nagD-cxr. A band size of about 1200bp indicated a successful knockout. Single colonies with correct verification results were picked into seed culture medium, kanamycin and rhamnose were added, and cultured for 12 hours to eliminate the ptarget plasmid; cultured in seed culture medium supplemented with glucose for 12 hours, and streaked on a plate supplemented with sucrose to eliminate the pcas9 plasmid. The strains that completed plasmid elimination were used for subsequent fermentation verification.

[0033] Table 1 BLI8 primer sequences

[0034]

[0035] Example 2: Knockout of the broad-specificity phosphatase encoding gene surE

[0036] In order to reduce the degradation of 5'-inosinic acid, according to the method provided in Example 1, BL21 (DE3) was used as the starting strain, and the broad specificity phosphatase encoding gene surE was knocked out to obtain the recombinant strain BLI9.

[0037] Table 2 BLI9 primer sequences

[0038]

[0039]

[0040] Example 3: Knockout of the purine nucleotidase encoding gene yrfG

[0041] In order to reduce the degradation of 5'-inosinic acid, according to the method provided in Example 1, BL21 (DE3) was used as the starting strain, and the purine nucleotidase encoding gene yrfG was knocked out to obtain the recombinant strain BLI10.

[0042] Table 3 BLI10 primer sequences

[0043]

[0044] Example 4: Knockout of 5'-nucleotidase encoding gene ushA

[0045] In order to reduce the degradation of 5'-inosinic acid, according to the method provided in Example 1, BL21 (DE3) was used as the starting strain, and the 5'-nucleotidase encoding gene ushA was knocked out to obtain the recombinant strain BLI11.

[0046] Table 4 BLI11 primer sequences

[0047]

[0048] Example 5: Knockout of the UMP phosphatase encoding gene nagD

[0049] Using BL21 (DE3) as the starting strain, the 6-phosphogluconolactonase encoding gene pgl (nucleotide sequence shown in SEQ ID NO.1) was knocked out at the flgG site, the 128th aspartic acid of phosphoribosylpyrophosphokinase prs was replaced with alanine, the 326th lysine of glutamine phosphoribosylpyrophosphotransferase purF was replaced with glutamine, and the 410th proline was replaced with tryptophan, the repressor protein encoding gene purR was knocked out, the 6-phosphofructokinase encoding gene pfkA was knocked out, and the promoter of phosphoribosylpyrophosphokinase prs was replaced with P tac Promoter, replace the promoter of glutamate phosphoribosyl pyrophosphate transamidase purF with P tac The promoter was expressed in the recombinant strain BLI7, and the UMP phosphatase encoding gene nagD was knocked out to obtain the recombinant strain BLI12. The specific steps are as follows:

[0050] (1) At the flgG site, the 6-phosphogluconolactonase encoding gene pgl

[0051] Based on the Escherichia coli sequence information, primers flgG-ff, flgG-fr, flgG-rf, and flgG-rr were designed. In the first round of amplification, 500 bp sequences before and after the flgG site were amplified from Escherichia coli BL21 (DE3). Additional primer sequences were set to introduce the pgl gene sequence. The number of amplification cycles was 34-36, so that the fragment had a consensus sequence of about 30 bp after amplification. Agarose gel electrophoresis verification results showed that the fragment length was 500 bp; the fragment obtained in the first round of amplification was purified, and in the second round of amplification, only two fragments were added without primers, and the number of amplification cycles was changed to 10-15 cycles; the product of the second round of amplification did not need to be purified or verified by agarose gel electrophoresis; using this product as a template, primers flgG-ff and flgG-rr were added in the third round of amplification, the number of amplification cycles was 34-36, and agarose gel electrophoresis was performed to verify that the fragment length should be about 1000 bp, that is, the flgG band. If there are impurity bands, the gel needs to be cut and recovered. If there are no impurity bands, the fragments can be directly purified for later use.

[0052] The ptarget plasmid is used to express the 20-bp sgRNA required for gene editing. Based on the ptarget plasmid information, primers n20-flgG-f and n20-flgG-r were designed. These primers carry the 20-bp sgRNA consensus sequence and bind to ptarget. This primer pair was used to amplify the ptarget template to obtain a linearized vector. The linearized vector was purified and transformed into E. coli DH5α. Colony PCR and sequencing were performed to verify the plasmid. The correct plasmid was used as the ptarget-flgG extraction plasmid for future use.

[0053] The plasmid pcas9 expressing the Cas9 protein was transformed into Escherichia coli BL21 (DE3), and the successfully transformed strains were screened using a kanamycin resistance plate. The pcas9-transformed strain was then prepared into an electroporation competent state, and flgG and ptarget-flgG were co-transformed into the competent state. The strains were screened by kanamycin and spectinomycin, and single colonies were picked from the plate. Colony PCR was performed using primers flgG-cxf and flgG-cxr. A band size of about 2200bp indicated successful integration. The single colony with the correct verification result was picked into the seed culture medium, kanamycin and rhamnose were added, cultured for 12 hours, and the ptarget plasmid was eliminated; the culture was cultured in a seed culture medium supplemented with glucose for 12 hours, and streaked on a plate supplemented with sucrose to eliminate the pcas9 plasmid;

[0054] (2) Replace aspartic acid at position 128 of prs with alanine, replace lysine at position 326 of purF with glutamine, and replace proline at position 410 with tryptophan

[0055] Based on the sequence information of E. coli, primers 128-ff, 128-fr, 128-rf, and 128-rr were designed. In the first round of amplification, 500 bp of the sequence before and after the aspartic acid codon 128 in the prs coding region were amplified from E. coli BL21 (DE3). The primers were set with additional sequences to skip the aspartic acid codon 128 and introduce the alanine codon. The number of amplification cycles was 34-36, so that the fragments had a consensus sequence of about 30 bp after amplification. The results were confirmed by agarose gel electrophoresis. The results indicate a fragment length of 500 bp. Purify the fragment obtained from the first round of amplification. In the second round of amplification, only two fragments need to be added without primers, and the number of amplification cycles should be changed to 10-15. The product from the second round of amplification does not need to be purified or verified by agarose gel electrophoresis. Using this product as a template, add primers 128-ff and 128-rr in the third round of amplification, perform 34-36 amplification cycles, and verify by agarose gel electrophoresis. The fragment length should be approximately 1000 bp, indicating the D128A band. If there are any contaminants, excise the gel for recovery. If there are no contaminants, purify the fragment directly for future use.

[0056] The ptarget plasmid is used to express the 20-bp sgRNA required for gene editing. Based on the ptarget plasmid information, primers n20-128-f and n20-128-r were designed. These primers carry the 20-bp sgRNA consensus sequence and bind to ptarget. This primer pair was used to amplify the ptarget vector using the ptarget template to obtain a linearized vector. The linearized vector was purified and transformed into E. coli DH5α. Colony PCR and sequencing were performed to verify the correct plasmid, which was identified as ptarget-D128A. This plasmid was then extracted and used for future use.

[0057] The plasmid pcas9 expressing the Cas9 protein was transformed into Escherichia coli BL21 (DE3), and the successfully transformed strains were screened using kanamycin resistance plates. The pcas9-transformed strain was then prepared into an electroporation competent state, and D128A and ptarget-D128A were co-transformed into the competent state. After screening with kanamycin and spectinomycin, single colonies were picked from the plate and colony PCR was performed using primers 128-cxf and 128-cxr. A band size of about 1200bp indicated a successful knockout. Single colonies with correct verification results were picked into seed culture medium, kanamycin and rhamnose were added, and cultured for 12 hours to eliminate the ptarget plasmid; cultured in seed culture medium supplemented with glucose for 12 hours, and streaked on a plate supplemented with sucrose to eliminate the pcas9 plasmid.

[0058] The lysine at position 326 of purF was replaced by glutamine, and the proline at position 410 was replaced by tryptophan according to the above method.

[0059] (3) The repressor protein encoding gene purR was knocked out, the 6-phosphofructokinase encoding gene pfkA was knocked out, and the UMP phosphatase encoding gene nagD was knocked out according to the method provided in Example 1.

[0060] (4) Replace the promoter of glutamate phosphoribosyl pyrophosphate transamidase purF with P tac Promoter, replace the promoter of glutamate phosphoribosyl pyrophosphate transamidase purF with P tac promoter

[0061] Based on the sequence information of E. coli, primers tcprs-ff, tcprs-fr, tcprs-rf, and tcprs-rr were designed. In the first round of amplification, the first 500 bp sequence of the prs coding region and a 500 bp sequence with a certain interval (the fragment should not contain the cleavage site of the Cas9 protein) were amplified from E. coli BL21 (DE3). The primers were set with additional sequences to introduce P tacThe fragments obtained from the first round of amplification were purified. In the second round of amplification, only two fragments were added without primers, and the number of amplification cycles was changed to 10-15 cycles. The product from the second round of amplification did not require purification or agarose gel electrophoresis verification. Using this product as a template, the primers TCPRS-FF and TCPRS-RR were added in the third round of amplification. The number of amplification cycles was 34-36, and agarose gel electrophoresis was performed to verify that the fragment length was approximately 1000 bp, which was the TCPRS band. If there were any mixed bands, the gel was cut and recovered. If there were no mixed bands, the fragment was purified directly for later use.

[0062] The ptarget plasmid is used to express the 20-bp sgRNA required for gene editing. Based on the ptarget plasmid information, primers n20-pprs-f and n20-pprs-r were designed. These primers carry the 20-bp sgRNA consensus sequence and bind to ptarget. This primer pair was used to amplify the ptarget template to obtain a linearized vector. The linearized vector was purified and transformed into E. coli DH5α. Colony PCR and sequencing were performed to verify the plasmid. The correct plasmid was identified as ptarget-pprs, which was then extracted and used for future use.

[0063] The plasmid pcas9 expressing the Cas9 protein was transformed into Escherichia coli BL21 (DE3), and the successfully transformed strains were screened using kanamycin resistance plates. The pcas9-transformed strain was then prepared into an electroporation competent state, and tcprs and ptarget-pprs were co-transformed into the competent state. After screening with kanamycin and spectinomycin, single colonies were picked from the plate and colony PCR was performed using primers pprs-cxf and pprs-cxr. A band size of about 1200bp indicated a successful knockout. Single colonies with correct verification results were picked into seed culture medium, kanamycin and rhamnose were added, and cultured for 12 hours to eliminate the ptarget plasmid; cultured in seed culture medium supplemented with glucose for 12 hours, and streaked on a plate supplemented with sucrose to eliminate the pcas9 plasmid.

[0064] According to the above method, the promoter of glutamate phosphoribosyl pyrophosphate transamidase purF was replaced with P tac promoter.

[0065] Example 6: Knockout of the 5'-inosinic acid dehydrogenase encoding gene guaB

[0066] According to the method provided in Example 1, using BLI12 as the starting strain, the 5'-inosinic acid dehydrogenase encoding gene guaB was knocked out to obtain the recombinant strain BLI13.

[0067] Table 5 BLI13 primer sequences

[0068]

[0069] Example 7: Knockout of the succinate adenosine synthase encoding gene purA

[0070] According to the method provided in Example 1, BLI13 was used as the starting strain and the succinate adenosine synthase encoding gene purA was used to obtain the recombinant strain BLI14.

[0071] Table 6 BLI14 primer sequences

[0072]

[0073]

[0074] Example 8: Replacement of the Serine Hydroxymethyltransferase Promoter

[0075] According to the method provided in Example 5, BLI12 was used as the starting strain, and the promoter of the serine hydroxymethyltransferase encoding gene glyA was replaced with P tac promoter, and obtained the recombinant bacterium BLI15.

[0076] Table 7 BLI15 primer sequences

[0077]

[0078] Example 9: Replacement of the methylenetetrahydrofolate dehydrogenase promoter

[0079] According to the method provided in Example 8, BLI15 was used as the starting strain, and the promoter of the bifunctional methylenetetrahydrofolate dehydrogenase encoding gene folD was replaced with P tac promoter, and obtained the recombinant bacterium BLI16.

[0080] Table 8 BLI16 primer sequences

[0081]

[0082]

[0083] Example 10: Replacement of the AICAR formylase promoter

[0084] According to the method provided in Example 8, the recombinant strain BLI16 was used as the starting strain, and the promoter of the bifunctional AICAR formylase encoding gene purH was replaced with P tac promoter, and obtained the recombinant bacterium BLI17.

[0085] Table 9 BLI17 primer sequences

[0086]

[0087] Table 10 BLI7-17 strain genotype

[0088]

[0089]

[0090] Example 11: Fermentation production of 5'-inosinic acid using strain BLI7-15

[0091] (1) Seed solution preparation

[0092] The starting strain E. coli BL21 (DE3) and the strain BLI7-17 constructed in Example 1-10 were streaked on plates and cultured for 18 hours. Single colonies were picked from the plates and inoculated into seed culture medium and cultured for 12 hours.

[0093] (2) Fermentation culture

[0094] Fermentation medium was inoculated with a 1% inoculum and cultured at 37°C and 220 rpm for 48 hours. 5'-inosinic acid (5'-IP) in the fermentation supernatant was determined by HPLC. The results are shown in Table 11. As shown in Table 11, BLI15 produced the highest 5'-inosinic acid yield, reaching 2113.12 mg / L after 48 hours of fermentation using glucose as the substrate, nearly five times the yield of the starting strain.

[0095] Judging from the fermentation results of the other recombinant strains, knocking out the UMP phosphatase encoding gene nagD and the 5'-nucleotidase encoding gene ushA can increase the 5'-inosinic acid production of the starting strain, but knocking out the two together can reduce the 5'-inosinic acid production. Knocking out the 5'-inosinic acid dehydrogenase encoding gene guaB alone can increase the 5'-inosinic acid production, while further knocking out the succinate adenosine synthase encoding gene purA will affect the production. Replacing the promoter of the serine hydroxymethyltransferase encoding gene glyA with P tac The promoter of the bifunctional methylenetetrahydrofolate dehydrogenase encoding gene folD and the promoter of the bifunctional AICAR formylase encoding gene purH were replaced with P tac The promoter will cause a small decrease in yield.

[0096] Table 11 BLI7-17 strain fermentation results

[0097]

[0098]

[0099] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An Escherichia coli for efficiently producing 5'-inosinic acid, characterized by: Heterologously introducing a 6-phosphogluconolactonase encoding gene into the Escherichia coli genome pgl , replace the codon encoding aspartic acid at position 128 of phosphoribosylpyrophosphate kinase with an alanine codon, replace the codon encoding lysine at position 326 of glutamate phosphoribosylpyrophosphate transamidase with a glutamine codon, and replace the codon encoding proline at position 410 with a tryptophan codon, and knock out or silence the repressor protein encoding gene purR and 6-phosphofructokinase encoding genes pfq , overexpression of the gene encoding ribose pyrophosphate kinase prs and glutamate phosphoribosylpyrophosphate amidotransferase encoding genes purF , knockout or silencing of UMP phosphatase encoding genes nagD and 5'-inosinic acid dehydrogenase encoding genes guaB; Overexpression of a serine hydroxymethyltransferase encoding gene in the Escherichia coli genome glyA ; The overexpression was performed using P tac Promoter for expression; The gene pgl The nucleotide sequence is shown in SEQ ID NO.1; The starting strain was Escherichia coli BL21 (DE3).

2. A method for constructing the Escherichia coli according to claim 1, characterized in that: Steps include S1-S7: S1. Integrate the 6-phosphogluconolactonase encoding gene into the flgG site of the starting E. coli genome pgl ; S2, replacing the codon encoding aspartic acid at position 128 of phosphoribosylpyrophosphokinase in the genome of the strain obtained in S1 with an alanine codon; S3, replacing the codon encoding lysine at position 326 of the strain obtained in S2 with a glutamine codon, and replacing the codon encoding proline at position 410 with a tryptophan codon; S4, knockout or silencing of repressor protein encoding genes in the genome of the strain obtained from S3 purR and 6-phosphofructokinase encoding genes pfq ; S5, the gene encoding phosphoribosyl pyrophosphokinase in the genome of the strain obtained in S4 prs D128A and glutamate phosphoribosylpyrophosphate amidotransferase encoding genes purF K326Q / P410W The promoter was replaced by P tac promoter; S6. Knockout or silencing of the UMP phosphatase encoding gene in the genome of the strain obtained in S5 nagD and 5'-inosinic acid dehydrogenase encoding genes guaB ; S7. In the genome of the strain obtained in S6, the gene encoding serine hydroxymethyltransferase was inserted into the genome of the strain obtained in S6. glyA The promoter was replaced by P tac Promoter.

3. The construction method according to claim 2, wherein: Gene editing was performed using the CRISPR / Cas9 system. A microbial preparation comprising the Escherichia coli according to claim 1 .

5. A method for producing 5'-inosinic acid, characterized in that: The Escherichia coli according to claim 1 or the microbial preparation according to claim 4 is added to the fermentation system.

6. The method according to claim 5, characterized in that: The fermentation substrate is glucose.

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