A genetically engineered bacterium for producing uridine diphosphate glucose and its construction method and application

Abstract

The invention discloses a genetic engineering bacterium for producing uridine diphosphate glucose and a construction method and application thereof, belonging to the field of genetic engineering technology. The following genes are overexpressed in the genetic engineering bacterium: pyrimidine nucleoside operon gene, carbamoyl phosphate synthetase gene, aspartate carbamoyltransferase gene, orotate phosphoribosyltransferase gene, phosphoribosyl pyrophosphate synthase gene, uridylate kinase gene, glucose phosphomutase gene, uridine triphosphate glucose-1-phosphouridiyltransferase gene; the following genes are not expressed in the genetic engineering bacterium: purine nucleotide repressor protein gene, deoxyribonucleoside triphosphate reductase gene, nucleoside triphosphate pyrophosphohydrolase gene, cytidine triphosphate synthase gene, pyrimidine nucleotidase gene, nucleotidase gene, ornithine carbamoyltransferase gene, UDPG dehydrogenase gene, lacI repressor protein gene. The genetic engineering bacterium can efficiently synthesize UDPG using glucose as a substrate.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a genetic engineering bacterium for producing UDP-glucose, and a construction method and application thereof. Background Art

[0002] Thanks to the rapid development of enzyme engineering, glycosyltransferases have become powerful tools for synthesizing natural and non-natural bioconjugates. However, the substrates catalyzed by eukaryotic glycosyltransferases are limited to a limited number of nucleoside diphosphate sugars, among which uridine diphosphate glucose (UDPG) is one of the most important sugar donors for the biosynthesis of glycosides, oligosaccharides, polysaccharides, glycoproteins and other glycosyl compounds.

[0003] Hu Meirong et al. used uridine monophosphate, sucrose, ATP and other raw materials to generate uridine diphosphate glucose under the action of polyphosphate kinase, uridine monophosphate kinase and sucrose synthase. The enzymes were expressed in Escherichia coli BL21 using plasmids as vectors. However, this method did not construct the metabolic pathway of UDPG in the engineered strain. It required fermentation of the engineered strain to produce enzymes, and then cell crushing to obtain the supernatant to catalyze the generation of uridine diphosphate. The process was complicated and the cost of industrial application was not low enough.

[0004] Wu Xuri et al. designed the UDPG biosynthesis and metabolic pathway of Escherichia coli using the "open source-throttling" scheme and constructed a UDPG engineering strain. The "open source" strategy was to strengthen the nucleoside pathway from orotate pyrophosphorylase pyrE, orotate nucleotide decarboxylase pyrF, and uracil nucleotide kinase pyrH, and knock out the 6-phosphoglucose isomerase gene pgi. The "throttling" strategy was to knock out the UDPG dehydrogenase gene. However, this metabolic modification has the following shortcomings: First, the supply of the precursor uridylic acid is limited, and the key rate-limiting enzyme in the uridylic acid synthesis pathway is not strengthened and the feedback inhibition of uridylic acid is not relieved. Uridylic acid is catalyzed to generate uridine by nucleotidase. Secondly, the supply of UDP is limited. When uridylic acid kinase catalyzes UMP to generate UDP, it is subject to feedback inhibition by UDP, and UDP generates UTP under the catalysis of uridine diphosphate kinase. Finally, the strain must be transformed with a plasmid connected to sucrose synthase before it can convert UDP into UDPG in a substrate containing sucrose.

[0005] Based on this, there is an urgent need in this field to construct more efficient, safe, low-cost, plasmid-free genetically engineered bacteria that can directly produce UDP-glucose from cheap carbon sources such as glucose to meet the needs of industrial production of UDP-glucose. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a genetically engineered bacterium for producing UDP-glucose and a construction method and application thereof, so as to overcome the problem that the production process of UDP-glucose in the prior art is complicated and cannot meet the needs of industrial production of UDP-glucose.

[0007] In a first aspect, the present invention provides a genetically engineered bacterium for producing UDP-glucose, wherein the following genes are overexpressed in the genetically engineered bacterium:

[0008] Pyrimidine nucleoside operon gene, carbamyl phosphate synthetase gene, aspartate carbamyl transaminase gene, orotate phosphoribosyltransferase gene, phosphoribosyl pyrophosphate synthase gene, uridylate kinase gene, glucose phosphomutase gene, uridine triphosphate glucose-1-phospho-uridyltransferase gene;

[0009] The following genes are not expressed in the genetically engineered bacteria:

[0010] Purine nucleotide repressor protein gene, deoxyribonucleoside triphosphate reductase gene, nucleoside triphosphate pyrophosphohydrolase gene, cytidine triphosphate synthase gene, pyrimidine nucleotidase gene, nucleotidase gene, ornithine carbamoyltransferase gene, UDPG dehydrogenase gene, lacI repressor protein gene.

[0011] Compared with the prior art, the genetically engineered bacteria producing UDPG in the present invention strengthens or weakens the following metabolic pathways by overexpressing or not expressing specific genes, so that the genetically engineered bacteria can efficiently synthesize UDPG using glucose as a substrate, specifically:

[0012] Strengthened the supply of the important precursor UMP. Genetically engineered bacteria overexpressing pyrimidine nucleoside operon genes can relieve the feedback inhibition of UMP and strengthen the metabolism of glutamine to UMP. Genetically engineered bacteria overexpress carbamoyl phosphate synthetase genes, in which the glutamic acid at position 951 of the large subunit of carbamoyl phosphatase is missing, thereby relieving the feedback regulation of uridylic acid and strengthening the metabolism of glutamine to carbamoyl phosphate. Genetically engineered bacteria overexpressing aspartate carbamoyltransferase genes can strengthen the metabolism of carbamoyl phosphate and aspartate to synthesize carbamoyl aspartate. Genetically engineered bacteria overexpressing orotate phosphoribosyltransferase genes can strengthen the metabolism of PRPP (5-phosphoribosyl-1-pyrophosphate) and orotate to produce orotidine. The genetically engineered bacteria overexpressed the phosphoribosyl pyrophosphate synthase gene, and the aspartic acid at position 128 of the enzyme mutated to alanine, thereby relieving the feedback inhibition of ATP and ADP on phosphoribosyl pyrophosphate synthase and strengthening the metabolism of 5-phosphoribose and ATP to generate PRPP. The genetically engineered bacteria did not express the purine nucleotide repressor protein gene, which could relieve the feedback inhibition of the repressor protein and the purine repressor complex on the expression of the prsA gene.

[0013] Block the branch metabolism of the precursor UMP. In Escherichia coli, UMP has two metabolic flows: uridine and uridine triphosphate (UTP). The genetically engineered bacteria of the present invention do not express pyrimidine nucleotidase genes and nucleotidase genes, successfully weakening the metabolism of UMP to uridine and uracil, and then converting the metabolism of UMP to uridine to the metabolism of UMP to UTP.

[0014] Weakening the metabolism of UTP to UMP. Escherichia coli contains multiple nucleoside triphosphate pyrophosphohydrolase genes, which can catalyze UTP to UMP. The genetically engineered bacteria of the present invention do not express nucleoside triphosphate pyrophosphohydrolase genes, thereby weakening the metabolism of UTP to UMP and promoting the accumulation of UTP.

[0015] Blocking the branching metabolism of UTP. Deoxyribonucleoside triphosphate reductase catalyzes the conversion of UTP to dUTP, and cytidine triphosphate synthase catalyzes the conversion of UTP to CTP. The genetically engineered bacteria of the present invention do not express deoxyribonucleoside triphosphate reductase genes and cytidine triphosphate synthase genes, thereby blocking the branching metabolism of UTP and promoting UTP accumulation.

[0016] Optimize the anabolism of UTP. The genetically engineered bacteria of the present invention overexpress the uridylate kinase gene, and the 93rd aspartic acid of the uridylate kinase is replaced by alanine, thereby relieving the feedback inhibition of uridine diphosphate on uridylate kinase and reasonably strengthening the metabolism of UMP to UTP.

[0017] Blocking the branch metabolism of carbamyl phosphate. The genetically engineered bacteria of the present invention do not express the ornithine carbamyl transferase gene to avoid the precursor carbamyl phosphate and ornithine being converted into citrulline by ornithine carbamyl transferase, thereby reducing the loss of the precursor carbamyl phosphate.

[0018] Reduce the consumption of uridine diphosphate glucose (UDPG). The genetically engineered bacteria of the present invention do not express the UDPG dehydrogenase gene, thereby preventing UDPG from being converted into uridine diphosphate gluconic acid by UDPG dehydrogenase in the strain body, thereby reducing the consumption of endogenous UDPG.

[0019] Strengthening the anabolism of UDP-glucose. The genetically engineered bacteria of the present invention overexpress the phosphoglucomutase gene, thereby strengthening the metabolism of 6-phosphate glucose to 1-phosphate glucose; overexpress the UDP-glucose-1-phosphate uridyltransferase gene, thereby strengthening the metabolism of UTP and 1-phosphate glucose to synthesize UDP-glucose.

[0020] The genetically engineered bacteria of the present invention do not express the lacI repressor protein gene, which can relieve the feedback repression of the lacI repressor protein on the Ptrc promoter and avoid the repressor protein inhibiting the normal regulation of the Ptrc promoter on the integrated gene.

[0021] Furthermore, the pyrimidine nucleoside operon gene is pyrBCAKDFE, and the nucleotide sequence of pyrBCAKDFE is shown in SEQ ID NO. 1; the pyrimidine nucleoside operon gene pyrBCAKDFE is derived from Bacillus subtilis.

[0022] The carbamyl phosphate synthetase gene is pyrAA / pyrAB E951* , the pyrAA / pyrAB E951* The nucleotide sequence is shown in SEQ ID NO. 2; the carbamoyl phosphate synthetase gene pyrAA / pyrAB E951* Derived from Bacillus subtilis.

[0023] The aspartate transaminase gene is pyrB, and the nucleotide sequence of pyrB is shown in SEQ ID NO. 3; the aspartate transaminase gene pyrB is derived from Bacillus subtilis.

[0024] The orotate phosphoribosyltransferase gene is pyrE, and the nucleotide sequence of pyrE is shown in SEQ ID NO. 4; the orotate phosphoribosyltransferase gene pyrE is derived from Bacillus subtilis.

[0025] The phosphoribosyl pyrophosphate synthase gene is prs D128A , the prs D128A The nucleotide sequence is shown in SEQ ID NO.5; the phosphoribosyl pyrophosphate synthase gene prs D128A Derived from Bacillus subtilis.

[0026] The uridylate kinase gene is pyrH D93A , the pyrH D93A The nucleotide sequence is shown in SEQ ID NO.6; the uridylate kinase gene pyrH D93A Derived from Bacillus subtilis.

[0027] The phosphoglucomutase genes include PGM1 and PGM2, the nucleotide sequence of PGM1 is shown as SEQ ID NO.7, and the nucleotide sequence of PGM2 is shown as SEQ ID NO.8; the phosphoglucomutase genes PGM1 and PGM2 are derived from Saccharomyces cerevisiae.

[0028] The uridine triphosphate glucose-1-phosphate uridyltransferase gene is UGP1, and the nucleotide sequence of UGP1 is shown in SEQ ID NO.9; the uridine triphosphate glucose-1-phosphate uridyltransferase gene UGP1 is derived from Saccharomyces cerevisiae.

[0029] The purine nucleotide repressor gene is purR, and the nucleotide sequence of purR is shown in SEQ ID NO. 10;

[0030] The deoxyribonucleoside triphosphate reductase gene is nrdD, and the nucleotide sequence of nrdD is shown in SEQ ID NO.11;

[0031] The nucleoside triphosphate pyrophosphohydrolase gene includes mazG and yhdE, the nucleotide sequence of mazG is shown in SEQ ID NO. 12, and the nucleotide sequence of yhdE is shown in SEQ ID NO. 13;

[0032] The cytidine triphosphate synthase gene is pyrG, and the nucleotide sequence of pyrG is shown in SEQ ID NO. 14;

[0033] The pyrimidine nucleotidase gene is ygdH, and the nucleotide sequence of ygdH is shown in SEQ ID NO. 15;

[0034] The nucleotidase gene includes ushA, surE, yjjG and yrfG, the nucleotide sequence of ushA is shown in SEQ ID NO.16, the nucleotide sequence of surE is shown in SEQ ID NO.17, the nucleotide sequence of yjjG is shown in SEQ ID NO.18, and the nucleotide sequence of yrfG is shown in SEQ ID NO.19;

[0035] The ornithine carbamoyltransferase gene is argF, and the nucleotide sequence of argF is shown in SEQ ID NO.20;

[0036] The UDPG dehydrogenase gene is ugd, and the nucleotide sequence of ugd is shown in SEQ ID NO. 21;

[0037] The lacI repressor protein gene is lacI, and the nucleotide sequence of lacI is shown in SEQ ID NO.22.

[0038] Furthermore, the above-mentioned pyrimidine nucleoside operon gene, carbamyl phosphate synthetase gene, aspartate carbamyl transaminase gene, orotic acid phosphoribosyltransferase gene, phosphoribosyl pyrophosphate synthase gene, uridylate kinase gene, glucose phosphomutase gene and uridine triphosphate glucose-1-phosphate uridyltransferase gene are all controlled by the strong promoter Ptrc.

[0039] Furthermore, the nucleotide sequence of the strong promoter Ptrc is shown in SEQ ID NO.23.

[0040] Furthermore, the pyrimidine nucleoside operon gene pyrBCAKDFE is located at the pseudogene locus yghX;

[0041] Carbamoyl phosphate synthetase gene pyrAA / pyrAB E951* Located at the pseudogene locus rph;

[0042] The aspartate transaminase gene pyrB is located at the pseudogene site yjiT;

[0043] The orotate phosphoribosyltransferase gene pyrE is located at the pseudogene locus mbhA;

[0044] Phosphoribosyl-pyrophosphate synthase gene prs D128A Located at the gene loci purR, nrdD, and mazG;

[0045] The uridylate kinase gene is located in the gene pyrH D93A Located at gene loci yhdE, pyrG, and ygdH;

[0046] The phosphoglucomutase gene PGM1 is located at the gene locus ushA; PGM2 is located at the gene locus surE;

[0047] The uridine triphosphate glucose-1-phosphate uridyltransferase gene UGP1 is located at gene loci yjjG and yrfG.

[0048] Furthermore, the starting strain used in the construction of the genetically engineered bacteria is Escherichia coli; the Escherichia coli is E. coli W3110.

[0049] In a second aspect, the present invention provides a method for constructing a genetically engineered bacterium, which is used to construct the above-mentioned genetically engineered bacterium, comprising the following steps:

[0050] Integrating the pyrimidine nucleoside operon gene into the pseudogene site yghX of the genetically engineered bacteria to overexpress the pyrimidine nucleoside operon gene;

[0051] Integrating a carbamyl phosphate synthetase gene into the pseudogene site rph of the genetically engineered bacteria to overexpress the carbamyl phosphate synthetase gene;

[0052] Integrating the aspartate transaminase gene into the pseudogene site yjiT of the genetically engineered bacteria to overexpress the aspartate transaminase gene;

[0053] Integrating the orotate phosphoribosyltransferase gene into the pseudogene site mbhA of the genetically engineered bacteria to overexpress the orotate phosphoribosyltransferase gene;

[0054] Integrating the phosphoribosyl pyrophosphate synthase gene into the gene sites purR, nrdD and mazG of the genetically engineered bacteria to overexpress the phosphoribosyl pyrophosphate synthase gene;

[0055] The uridylate kinase gene is located at the gene sites yhdE, pyrG and ygdH of the genetically engineered bacteria, so that the uridylate kinase gene is overexpressed;

[0056] Integrating the phosphoglucomutase gene PGM1 into the gene site ushA of the genetically engineered bacteria to overexpress the phosphoglucomutase gene PGM1;

[0057] Integrating the phosphoglucomutase gene PGM2 into the gene site surE of the genetically engineered bacteria to overexpress the phosphoglucomutase gene PGM2;

[0058] Integrating a UDP-glucose-1-phosphate uridyltransferase gene into the gene sites yjjG and yrfG of the genetically engineered bacteria to overexpress the UDP-glucose-1-phosphate uridyltransferase gene;

[0059] knocking out the ornithine carbamoyl transferase gene so that the ornithine carbamoyl transferase gene is not expressed;

[0060] Knocking out the UDPG dehydrogenase gene so that the UDPG dehydrogenase gene is not expressed;

[0061] The lacI repressor protein gene is knocked out so that the lacI repressor protein gene is not expressed.

[0062] Compared with the prior art, when constructing a genetically engineered bacterium for producing UDP-glucose, the present invention performs all gene operations on the genome of the strain, and no plasmid remains in the strain, and no antibiotics or inducers are used. The genetic traits of the UDP-glucose genetically engineered bacterium finally obtained are clear and stable, the production performance is stable, and the fermentation process is simple.

[0063] In a third aspect, the present invention provides the use of the above-mentioned genetically engineered bacteria in the production of UDP-glucose.

[0064] In a fourth aspect, the present invention provides a method for producing UDP-glucose, comprising the following steps:

[0065] Take the bacterial liquid of the genetically engineered bacteria, apply it to LB medium, culture it at 36.5℃~37.5℃ for 8~10 hours, inoculate it into seed medium, culture it at 36.5℃~37.5℃ for 5~8 hours, maintain the pH value at 6.8~7.1 during the culture process, and obtain seed liquid;

[0066] The seed liquid is inoculated into a fermentation medium for fermentation culture. During the fermentation culture, the pH value is controlled to be 6.8-7.1 and the temperature is controlled to be 36.5°C-37.5°C. After 10-12 hours of fermentation culture, glucose is added using a feedback pulse feeding strategy, and the dissolved oxygen value is controlled to be 25%-35%. The fermentation culture is continued for 36-40 hours to obtain uridine diphosphate glucose.

[0067] Compared with the prior art, the genetically engineered bacteria for producing UDP-glucose constructed in the present invention can produce 35 g / L of UDP-glucose after fermentation under the above-mentioned suitable fermentation conditions, and the conversion rate of glucose to UDP-glucose reaches 30%. Compared with the recombinant strains reported previously, the production and conversion rate of UDP-glucose are improved, and the invention has good industrial application value.

[0068] Furthermore, the feedback pulse feeding strategy is:

[0069] The pulse rate of feeding on the feeding pump is set to 1 / 18~1 / 15s. When the dissolved oxygen value is higher than 35%, the feeding pump automatically starts to add 700~800g / L of glucose solution. When the dissolved oxygen value is lower than 25%, the feeding pump automatically shuts down. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the agarose gel electrophoresis verification diagram in Example 1.

[0071] Figure 2 This is the agarose gel electrophoresis verification diagram in Example 1.

[0072] Figure 3 This is the agarose gel electrophoresis verification diagram in Example 1.

[0073] Figure 4 This is the agarose gel electrophoresis verification diagram in Example 2.

[0074] Figure 5 This is the agarose gel electrophoresis verification diagram in Example 3.

[0075] Figure 6 This is the agarose gel electrophoresis verification diagram in Example 4.

[0076] Figure 7 This is the agarose gel electrophoresis verification diagram in Example 5.

[0077] Figure 8 This is the agarose gel electrophoresis verification diagram in Example 6.

[0078] Fig. 9 This is the agarose gel electrophoresis verification diagram in Example 7.

[0079] Fig.10This is the agarose gel electrophoresis verification diagram in Example 8.

[0080] Fig.11 This is the agarose gel electrophoresis verification diagram in Example 9.

[0081] Fig.12 This is the agarose gel electrophoresis verification diagram in Example 10.

[0082] Fig.13 This is the agarose gel electrophoresis verification diagram in Example 11.

[0083] Fig.14 This is the agarose gel electrophoresis verification diagram in Example 12.

[0084] Fig.15 This is the detection diagram of UDP-glucose in the supernatant in Example 13.

[0085] Fig.16 This is the detection diagram of the UDP-glucose standard in Example 13. DETAILED DESCRIPTION

[0086] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, 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.

[0087] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0088] The embodiment of the present invention uses a CRISPR / Cas9-mediated gene editing method to edit the gene of E. coli W3110 (refer to the literature Metabolic Engineering, 2015, 31: 13-21.). CRISPR / Cas9 is a new type of precise and efficient gene targeting modification technology. The two plasmids used in this method are pGRB and pREDCas9. The pREDCas9 plasmid is a temperature-sensitive plasmid, carrying a gRNA plasmid elimination system, a lambda phage Red recombination system and a Cas9 protein expression system, and has spectinomycin resistance (working concentration: 100 mg / L), and an optimal culture temperature: 32°C; the pGRB plasmid uses pUC18 as a skeleton, contains a promoter J23100, a gRNA-Cas9 binding region sequence and a terminator sequence, and has ampicillin resistance (working concentration: 100 mg / L), and an optimal culture temperature: 37°C. Example 1

[0089] Overexpression of pyrBCAKDFE at the pseudogene locus yghX

[0090] Since the fragment of the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis is relatively large, with the number of bases reaching 9492 bp, the gene pyrBCAKDFE was integrated in segments at the pseudogene site yghX.

[0091] 1. Integration of pyr1

[0092] Using the E. coli W3110 genome as a template, upstream homology arm primers UP-yghX-S (SEQ ID NO.24) and UP-yghX-Ptrc-A (SEQ ID NO.25), and downstream homology arm primers DN-yghX-S1 (SEQ ID NO.26) and DN-yghX-A (SEQ ID NO.27) were designed according to the upstream and downstream gene sequences of its pseudogene yghX, and PCR was used to amplify the upstream and downstream homology arm fragments.

[0093] Using the B. subtilis A260 genome as a template, primers Ptrc-pyr1-S (SEQ ID NO.28) and pyr1-A (SEQ ID NO.29) were designed based on bases 1 to 3260 (pyr1) of the pyrBCAKDFE nucleotide sequence (SEQ ID NO.1) to amplify the pyr1 gene fragment by PCR.

[0094] The promoter Ptrc is designed in the downstream primer of the upstream homology arm of the pseudogene yghX and the upstream primer of the pyr1 gene; the plasmid gene No. 4 is designed in the upstream primer of the downstream homology arm of the pseudogene yghX and the downstream primer of the pyr1 gene. The nucleotide sequence of the plasmid gene No. 4 is shown in SEQ ID NO.155.

[0095] By overlapping PCR, the upstream and downstream homologous arm fragments of the pseudogene yghX obtained by the above PCR and the pyr1 gene fragment were used as templates, and UP-yghX-S (SEQ ID NO.24) and DN-yghX-A (SEQ ID NO.27) were used as primers to obtain the integrated fragment of the pyr1 gene: upstream homologous arm-Ptrc-pyr1-downstream homologous arm.

[0096] A DNA fragment containing the pseudogene yghX target sequence was prepared by annealing primers gRNA-yghX-S (SEQ ID NO.30) and gRNA-yghX-A (SEQ ID NO.31), and the recombinant plasmid pGRB-yghX was obtained after recombination with the linearized pGRB vector.

[0097] The above integration fragment (upstream homology arm-Ptrc-pyr1-downstream homology arm) and the recombinant plasmid pGRB-yghX were electroporated into competent cells of E. coli W3110 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Udg1-1.

[0098] Agarose gel electrophoresis verification of pyr1 integration fragment and positive strains is shown in the figure Figure 1 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the pyr1 gene fragment; Lane 3 is the downstream homology arm; Lane 4 is the integrated fragment of the pyr1 gene; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain.

[0099] 2. Integration of pyr2

[0100] Using the B. subtilis A260 genome as a template, primers pyr2-S (SEQ ID NO.32) and pyr2-A (SEQ ID NO.33) were designed according to positions 2510-6509 (pyr2) of the pyrBCAKDFE nucleotide sequence (SEQ ID NO.1) to PCR amplify the pyr2 fragment.

[0101] Using the E. coli W3110 genome as a template, PCR was performed to amplify the downstream homology arm fragment of its pseudogene yghX using the downstream homology arm primers DN-yghX-S2 (SEQ ID NO.34) and DN-yghX-A (SEQ ID NO.27).

[0102] Plasmid gene No. 5 is designed in the upstream primer of the downstream homology arm of pseudogene yghX and the downstream primer of pyr2 gene. The nucleotide sequence of plasmid gene No. 5 is shown in SEQ ID NO.156.

[0103] By overlapping PCR, the pyr2 fragment obtained by the above PCR and the downstream homology arm fragment of the pseudogene yghX were used as templates, and pyr2-S (SEQ ID NO.32) and DN-yghX-A (SEQ ID NO.27) were used as primers to obtain the integration fragment of pyr2: pyr2-downstream homology arm.

[0104] A DNA fragment containing the target sequence of plasmid gene No. 4 was prepared by annealing primers gRNA-S1 (SEQ ID NO.35) and gRNA-A1 (SEQ ID NO.36), and the recombinant plasmid pGRB-pyr2 was obtained after recombination with the linearized pGRB vector.

[0105] The above integration fragment (pyr2-downstream homology arm) and recombinant plasmid pGRB-pyr2 were electroporated into competent cells of E. coli Udg1-1 containing pREDCas9 plasmid. After screening positive strains, the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg1-2.

[0106] Agarose gel electrophoresis verification of pyr2 integration fragment and positive strains is shown in the figure Figure 2 As shown: Lane M is DNA Marker; Lane 1 is the pyr2 fragment; Lane 2 is the downstream homology arm; Lane 3 is the integrated fragment of the pyr2 gene; Lane 4 is the PCR fragment of E. coli W3110; Lane 5 is the PCR fragment of the positive strain.

[0107] 3. Integration of pyr3

[0108] Using the B. subtilis A260 genome as a template, primers pyr3-S (SEQ ID NO.37) and pyr3-Ptrc-A (SEQ ID NO.38) were designed according to positions 5671-9492 (pyr3) of the pyrBCAKDFE nucleotide sequence (SEQ ID NO.1) to PCR amplify the pyr3 fragment.

[0109] Using the E. coli W3110 genome as a template, PCR was performed to amplify the downstream homology arm fragment of its pseudogene yghX using the downstream homology arm primers DN-Ptrc-yghX-S (SEQ ID NO.39) and DN-yghX-A (SEQ ID NO.27).

[0110] The terminator Ptrc is designed in the upstream primer of the downstream homology arm of the pseudogene yghX and the downstream primer of the pyr3 gene. The gene sequence of the terminator Ptrc is shown in SEQ ID NO.154.

[0111] By overlapping PCR, the pyr3 fragment obtained by the above PCR and the downstream homology arm fragment of the pseudogene yghX were used as templates, and pyr3-S (SEQ ID NO.37) and DN-yghX-A (SEQ ID NO.27) were used as primers to obtain the pyr3 integration fragment: pyr3-downstream homology arm.

[0112] The DNA fragment containing the target sequence of plasmid gene No. 5 was prepared by annealing primers gRNA-S2 (SEQ ID NO.40) and gRNA-A2 (SEQ ID NO.41), and the recombinant plasmid pGRB-pyr3 was obtained after recombination with the linearized pGRB vector.

[0113] The above integration fragment (pyr3-downstream homology arm) and recombinant plasmid pGRB-pyr3 were electroporated into competent cells of E. coli Udg1-2 containing pREDCas9 plasmid. After screening positive strains, the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg1-3.

[0114] Agarose gel electrophoresis verification of pyr3 integration fragment and positive strains is shown in the figure Figure 3 As shown: Lane M is DNA Marker; Lane 1 is the pyr3 fragment; Lane 2 is the downstream homology arm; Lane 3 is the integrated fragment of the pyr3 gene; Lane 4 is the PCR fragment of E. coli W3110; Lane 5 is the PCR fragment of the positive strain. Example 2

[0115] Overexpression of pyrAA / pyrAB at the pseudogene locus rph E951*

[0116] Using the E. coli W3110 genome as a template, upstream homology arm primers UP-rph-S (SEQ ID NO.42) and UP-rph-Ptrc-A (SEQ ID NO.43) were designed according to the upstream and downstream gene sequences of its pseudogene rph; downstream homology arm primers DN-Ptrc-rph-S (SEQ ID NO.44) and DN-rph-A (SEQ ID NO.45) were designed to PCR amplify its upstream and downstream homology arm fragments.

[0117] According to the pyrAA / pyrAB carrying mutation points synthesized by Tianjin Anshengda Biological Company E951* Design primers Ptrc-pyrAA / pyrAB based on the nucleotide sequence E951* -S (SEQ ID NO. 46) and pyrAA / pyrAB E951* -Ptrc-A (SEQ IDNO.47), to pyrAA / pyrAB E951* As a template, pyrAA / pyrAB was obtained by PCR E951* Fragment.

[0118] Ptrc promoter design is located in the antisense strand primer and pyrAA / pyrAB of the upstream homology arm of pseudogene rph E951* The Ptrc terminator was designed in the pyrAA / pyrAB E951* The antisense primer of the gene and the sense primer of the homology arm downstream of the rph pseudogene site.

[0119] By overlapping PCR, the upstream and downstream homology arm fragments of the pseudogene rph obtained above and pyrAA / pyrABE951* The fragment was used as a template, and UP-rph-S (SEQ ID NO.42) and DN-rph-A (SEQ ID NO.45) were used as primers to obtain the integration fragment: upstream homology arm-Ptrc-pyrAA / pyrAB E951* - Downstream homology arm.

[0120] A DNA fragment containing the pseudogene rph target sequence was prepared by annealing primers gRNA-rph-S (SEQ ID NO.48) and gRNA-rph-A (SEQ ID NO.49), and recombined with a linearized pGRB vector to obtain recombinant pGRB-rph.

[0121] The above integration fragment (upstream homology arm-Ptrc-pyrAA / pyrAB E951* - downstream homology arm) and the recombinant plasmid pGRB-rph were electroporated into competent cells of E. coli Udg1-3 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Udg2.

[0122] pyrAA / pyrAB E951* Agarose gel electrophoresis verification of integration fragments and positive strains is shown in the figure below. Figure 4 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is pyrAA / pyrAB E951* Fragment; Lane 3 is the downstream homology arm; Lane 4 is pyrAA / pyrAB E951* Lane 5 is the integrated fragment of the gene; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain. Example 3

[0123] Overexpression of pyrB at the pseudogene locus yjiT

[0124] Using the E. coli W3110 genome as a template, upstream homology arm primers UP-yjiT-S (SEQ ID NO.50) and UP-yjiT-Ptrc-A (SEQ ID NO.51) were designed according to the upstream and downstream gene sequences of its pseudogene yjiT; downstream homology arm primers DN-Ptrc-yjiT-S (SEQ ID NO.52) and DN-yjiT-A (SEQ ID NO.53) were designed to PCR amplify its upstream and downstream homology arm fragments.

[0125] Primers Ptrc-pyrB-S (SEQ ID NO.54) and pyrB-Ptrc-A (SEQ ID NO.55) were designed according to the nucleotide sequence of pyrB, and the pyrB fragment was obtained by PCR amplification using the B. subtilisA260 genome as a template.

[0126] The Ptrc promoter was designed in the antisense primer of the upstream homology arm of the pseudogene yjiT and the sense primer of the pyrB gene; the Ptrc terminator was designed in the antisense primer of the pyrB gene and the sense primer of the downstream homology arm of the pseudogene yjiT.

[0127] By overlapping PCR, the upstream and downstream homologous arm fragments of the pseudogene yjiT obtained by the above PCR and the pyrB fragment were used as templates, and UP-yjiT-S (SEQ ID NO.50) and DN-yjiT-A (SEQ ID NO.53) were used as primers to obtain the integration fragment of the pyrB gene: upstream homologous arm-Ptrc-pyrB-downstream homologous arm.

[0128] A DNA fragment containing the pseudogene yjiT target sequence was obtained by annealing primers gRNA-yjiT-S (SEQ ID NO.56) and gRNA-yjiT-A (SEQ ID NO.57), and the recombinant plasmid pGRB-yjiT was obtained after recombination with the linearized pGRB vector.

[0129] The above integration fragment (upstream homology arm-Ptrc-pyrB-downstream homology arm) and recombinant plasmid pGRB-yjiT were electroporated into competent cells of E. coli Udg2 containing pREDCas9 plasmid. After screening positive strains, the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg3.

[0130] Agarose gel electrophoresis verification of pyrB integration fragment and positive strains is shown in the figure below. Figure 5 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the pyrB fragment; Lane 3 is the downstream homology arm; Lane 4 is the integrated fragment of the pyrB gene; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain. Example 4

[0131] Overexpression of pyrE at the pseudogene locus mbhA

[0132] Using the E. coli W3110 genome as a template, upstream homology arm primers UP-mbhA-S (SEQ ID NO.58) and UP-mbhA-Ptrc-A (SEQ ID NO.59) were designed according to the upstream and downstream gene sequences of its pseudogene yjiT; downstream homology arm primers DN-Ptrc-mbhA-S (SEQ ID NO.60) and DN-mbhA-A (SEQ ID NO.61) were designed to PCR amplify its upstream and downstream homology arm fragments.

[0133] Primers Ptrc-pyrE-S (SEQ ID NO.62) and pyrE-Ptrc-A (SEQ ID NO.63) were designed according to the pyrE nucleotide sequence, and the pyrE fragment was obtained by PCR using the B. subtilisA260 genome as a template.

[0134] The Ptrc promoter was designed in the antisense primer of the upstream homology arm of the pseudogene mbhA and the sense primer of the pyrE gene; the Ptrc terminator was designed in the antisense primer of the pyrE gene and the sense primer of the downstream homology arm of the pseudogene mbhA.

[0135] By overlapping PCR, the upstream and downstream homology arm fragments of the pseudogene yjiT obtained above and the pyrE fragment were used as templates, and UP-mbhA-S (SEQ ID NO.58) and DN-mbhA-A (SEQ ID NO.61) were used as primers to obtain the integration fragment of the pyrE gene: upstream homology arm-Ptrc-pyrE-downstream homology arm.

[0136] A DNA fragment containing the pseudogene mbhA target sequence was prepared by annealing primers gRNA-mbhA-S (SEQ ID NO.64) and gRNA-mbhA-A (SEQ ID NO.65), and the recombinant plasmid pGRB-mbhA was obtained after recombination with the linearized pGRB vector.

[0137] The above integration fragment (upstream homology arm-Ptrc-pyrE-downstream homology arm) and recombinant plasmid pGRB-mbhA were electroporated into competent cells of E. coli Udg3 containing pREDCas9 plasmid. After screening positive strains, the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg4.

[0138] Agarose gel electrophoresis verification of pyrE integration fragment and positive strains is shown in the figure below. Figure 6As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the pyrE fragment; Lane 3 is the downstream homology arm; Lane 4 is the integrated fragment of the pyrE gene; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain. Example 5

[0139] Overexpression of prs at the gene loci purR, nrdD and mazG D128A

[0140] The upstream homology arm primers UP-purR-S (SEQ ID NO.66) and UP-purR-Ptrc-A (SEQ ID NO.67); UP-nrdD-S (SEQ ID NO.68) and UP-nrdD-Ptrc-A (SEQ ID NO.69); UP-mazG-S (SEQ ID NO.70) and UP-mazG-Ptrc-A (SEQ IDNO.71) were designed according to the upstream and downstream sequences of genes purR, nrdD and mazG, respectively; the downstream homology arm primers DN-Ptrc-purR-S (SEQ ID NO.72) and DN-purR-A (SEQ ID NO.73); DN-Ptrc-nrdD-S (SEQ ID NO.74) and DN-nrdD-A (SEQ ID NO.75); DN-Ptrc-mazG-S (SEQID NO.76) and DN-mazG-A (SEQ ID NO.77). Using the E. coli W3110 genome as a template, PCR technology was used to amplify the upstream and downstream homologous arms of genes purR, nrdD and mazG, respectively.

[0141] According to the target gene prs carrying mutation points synthesized by Tianjin Anshengda Biological Company D128A Design primers Ptrc-prs D128A -S (SEQ ID NO.78), prs D128A -Ptrc-A (SEQ ID NO.79), with the target gene prs D128A As a template, prs was obtained by PCR amplification D128A Fragment.

[0142] The Ptrc promoter was designed to be located in the upstream homology arms of genes purR, nrdD and mazG respectively. D128A The Ptrc terminator was designed in the prs D128A The antisense strand primer of the gene and the positive strand primer of the downstream homology arm of genes purR, nrdD and mazG.

[0143] By recombinant PCR, the upstream and downstream homologous arms of the gene purR obtained by PCR and prs D128A The fragment was used as a template, and UP-purR-S (SEQ ID NO.66) and DN-purR-A (SEQ ID NO.73) were used as primers to obtain prs D128A Integration fragment of the gene: purR upstream homology arm-Ptrc-prs D128A -purR downstream homology arm.

[0144] By recombinant PCR, the upstream and downstream homologous arms of the gene nrdD obtained by PCR and prs D128A The fragment was used as a template, and UP-nrdD-S (SEQ ID NO.68) and DN-nrdD-A (SEQ ID NO.75) were used as primers to obtain prs D128A Integration fragment of the gene: nrdD upstream homology arm-Ptrc-prs D128A -nrdD downstream homology arm.

[0145] By recombinant PCR, the upstream and downstream homologous arms of the gene mazG obtained by PCR and prs D128A The fragment was used as a template, and UP-mazG-S (SEQ ID NO.70) and DN-mazG-A (SEQ ID NO.77) were used as templates to obtain prs D128A Integration fragment of the gene: mazG upstream homology arm-Ptrc-prs D128A -mazG downstream homology arm.

[0146] The DNA fragments containing the target sequences of purR gene, nrdD gene and mazG gene were prepared by annealing primers gRNA-purR-S (SEQ ID NO.80) and gRNA-purR-A (SEQ ID NO.81); gRNA-nrdD-S (SEQ ID NO.82) and gRNA-nrdD-A (SEQ ID NO.83); gRNA-mazG-S (SEQ ID NO.84) and gRNA-mazG-A (SEQ ID NO.85), and recombined with the linearized pGRB vector to obtain recombinant plasmids pGRB-purR, pGRB-nrdD and pGRB-mazG, respectively.

[0147] The above integration fragment purR upstream homology arm -Ptrc-prs D128A-purR downstream homology arm and recombinant plasmid pGRB-purR were electroporated into E. coli Udg4 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive strains were obtained by PCR colony verification, and the two plasmids used for gene editing were eliminated to obtain strain E. coliUdg5.

[0148] The integration fragment nrdD upstream homology arm-Ptrc-prs D128A The downstream homology arm of -nrdD and the recombinant plasmid pGRB-nrdD were electroporated into E. coli Udg5 competent cells containing the pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. The positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain the strain E. coliUdg6.

[0149] Integration fragment mazG upstream homology arm-Ptrc-prs D128A -mazG downstream homology arm and recombinant plasmid pGRB-mazG were electrotransformed into E. coli Udg6 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg7. Agarose gel electrophoresis verification is shown in the figure below Figure 7 As shown: Lane M is a DNA Marker; Lane 1 is the upstream homology arm of the mazG gene; Lane 2 is the prs D128A Fragment; Lane 3 is the downstream homology arm of the mazG gene; Lane 4 is the upstream homology arm of the integrated fragment mazG - Ptrc-prs D128A -mazG downstream homology arm; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain. Example 6

[0150] Overexpression of pyrH at the gene loci yhdE, pyrG and ygdH D93A

[0151] According to the upstream and downstream sequences of genes yhdE, pyrG and ygdH, upstream homology arm primers UP-yhdE-S (SEQ ID NO.86) and UP-yhdE-Ptrc-A (SEQ ID NO.87); UP-pyrG-S (SEQ ID NO.88) and UP-pyrG-Ptrc-A (SEQ ID NO.89); UP-ygdH-S (SEQ ID NO.90) and UP-ygdH-Ptrc-A (SEQ IDNO.91), and downstream homology arm primers DN-Ptrc-yhdE-S (SEQ ID NO.92) and DN-yhdE-A (SEQ ID NO.93); DN-Ptrc-pyrG-S (SEQ ID NO.94) and DN-pyrG-A (SEQ ID NO.95); DN-Ptrc-ygdH-S (SEQ ID NO.96); Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the genes yhdE, pyrG and ygdH were amplified by PCR technology.

[0152] According to the target gene pyrH carrying the mutation point synthesized by Tianjin Anshengda Biological Company D93A Design primers Ptrc-pyrH D93A -S (SEQ ID NO.98), pyrH D93A -Ptrc-A (SEQ ID NO.99), with the target gene pyrH D93A As a template, pyrH was obtained by PCR D93A Fragment.

[0153] The Ptrc promoter was designed to be located in the upstream homology arms of genes yhdE, pyrG and ygdH respectively, and the antisense strand primer and pyrH D93A The Ptrc terminator was designed in the positive strand primer of the pyrH D93A The antisense strand primer of the gene and the positive strand primer of the downstream homology arm of genes yhdE, pyrG and ygdH.

[0154] By recombinant PCR, the upstream and downstream homologous arms of the gene yhdE obtained by PCR and pyrH D93A The fragment was used as a template, and UP-yhdE-S (SEQ ID NO.86) and DN-yhdE-A (SEQ ID NO.93) were used as primers to obtain pyrH D93A Integration fragment of the gene: yhdE upstream homology arm - Ptrc-pyrH D93A -yhdE downstream homology arm.

[0155] By recombinant PCR, the upstream and downstream homology arms of the gene pyrG and pyrH obtained by the above PCR were used to generate D93A The fragment was used as a template, and UP-pyrG-S (SEQ ID NO.88) and DN-pyrG-A (SEQ ID NO.95) were used as primers to obtain pyrH D93A Integration fragment of the gene: pyrG upstream homology arm - Ptrc-pyrH D93A -pyrG downstream homology arm.

[0156] By recombinant PCR, the upstream and downstream homology arms of the gene ygdH and pyrH obtained by the above PCR were used to generate D93A The fragment was used as a template, and UP-ygdH-S (SEQ ID NO.90) and DN-ygdH-A (SEQ ID NO.97) were used as primers to obtain pyrH D93A Integration fragment of the gene: ygdH upstream homology arm - Ptrc-pyrH D93A -ygdH downstream homology arm.

[0157] DNA fragments containing target sequences of genes yhdE, pyrG and ygdH were prepared by annealing primers gRNA-yhdE-S (SEQ ID NO.100) and gRNA-yhdE-A (SEQ ID NO.101); gRNA-pyrG-S (SEQ ID NO.102) and gRNA-pyrG-A (SEQ ID NO.103); gRNA-ygdH-S (SEQ IDNO.104) and gRNA-ygdH-A (SEQ ID NO.105), and recombined with the linearized pGRB vector to obtain recombinant plasmids pGRB-yhdE, pGRB-pyrG and pGRB-ygdH.

[0158] The upstream homology arm of the integration fragment yhdE-Ptrc-pyrH D93A The downstream homology arm of -yhdE and pGRB-yhdE were electroporated into E. coli Udg7 competent cells containing the pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg8.

[0159] The upstream homology arm of the integration fragment pyrG - Ptrc-pyrH D93AThe downstream homology arm of -pyrG and pGRB-pyrG were electroporated into E. coli Udg8 competent cells containing the pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg9.

[0160] Integration fragment ygdH upstream homology arm - Ptrc-pyrH D93A -ygdH downstream homology arm and pGRB-ygdH were electrotransformed into E. coli Udg9 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated, that is, strain E. coli Udg10 was obtained. The agarose gel electrophoresis verification figure is shown in the figure Figure 8 As shown: Lane M is a DNA Marker; Lane 1 is the upstream homology arm of the ygdH gene; Lane 2 is the upstream homology arm of the pyrH gene D93A Fragment; Lane 3 is the downstream homology arm of the ygdH gene; Lane 4 is the upstream homology arm of the integrated fragment ygdH - Ptrc-pyrH D93A -ygdH downstream homology arm; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain. Example 7

[0161] Overexpression of PGM1 gene at the ushA gene locus

[0162] According to the upstream and downstream sequences of the ushA gene, upstream homology arm primers UP-ushA-S (SEQ ID NO.106) and UP-ushA-Ptrc-A (SEQ ID NO.107) and downstream homology arm primers DN-Ptrc-ushA-S (SEQ ID NO.108) and DN-ushA-A (SEQ ID NO.109) were designed. The upstream and downstream homology arms of the ushA gene were amplified using PCR technology using the E. coli W3110 genome as a template.

[0163] According to the target gene PGM1, primers Ptrc-PGM1-S (SEQ ID NO.110) and PGM1-Ptrc-A (SEQID NO.111) were designed, and the PGM1 gene was amplified by PCR technology using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template.

[0164] The Ptrc promoter was designed in the antisense primer of the upstream homology arm of the ushA gene and the sense primer of the PGM1 gene; the Ptrc terminator was designed in the antisense primer of the PGM1 gene and the sense primer of the downstream homology arm of the ushA gene.

[0165] By recombinant PCR, the upstream and downstream homologous arms of the ushA gene obtained by the above PCR and the PGM1 gene were used as templates, and UP-ushA-S (SEQ ID NO.106) and DN-ushA-A (SEQ ID NO.109) were used as primers to obtain the integrated fragment of the PGM1 gene: upstream homologous arm-Ptrc-PGM1-downstream homologous arm.

[0166] A DNA fragment containing the ushA gene target sequence was prepared by annealing primers gRNA-ushA-S (SEQ ID NO.112) and gRNA-ushA-A (SEQ ID NO.113), and the recombinant plasmid pGRB-ushA was obtained after recombination with the linearized pGRB vector.

[0167] The above integration fragment (upstream homology arm-Ptrc-PGM1-downstream homology arm) and pGRB-ushA were electroporated into E. coli Udg10 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. The positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg11. The agarose gel electrophoresis verification is shown in the figure below. Fig. 9 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the PGM1 fragment; Lane 3 is the downstream homology arm; Lane 4 is the integration fragment; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain. Example 8

[0168] Overexpression of PGM2 gene at the surE gene locus

[0169] According to the upstream and downstream sequences of the surE gene, upstream homology arm primers UP-surE-S (SEQ ID NO.114) and UP-surE-Ptrc-A (SEQ ID NO.115) and downstream homology arm primers DN-Ptrc-surE-S (SEQ ID NO.116) and DN-surE-A (SEQ ID NO.117) were designed. The upstream and downstream homology arms of the surE gene were amplified using PCR technology using the E. coli W3110 genome as a template.

[0170] According to the target gene PGM2, primers Ptrc-PGM2-S (SEQ ID NO.118) and PGM2-Ptrc-A (SEQID NO.119) were designed, and the PGM2 gene was amplified by PCR technology using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template.

[0171] The Ptrc promoter was designed in the antisense primer of the upstream homology arm of the surE gene and the sense primer of the PGM2 gene; the Ptrc terminator was designed in the antisense primer of the PGM2 gene and the sense primer of the downstream homology arm of the surE gene.

[0172] By recombinant PCR, the upstream and downstream homologous arms of the surE gene obtained by the above PCR and the PGM2 gene were used as templates, and UP-surE-S (SEQ ID NO.114) and DN-surE-A (SEQ ID NO.117) were used as primers to obtain the integrated fragment of the PGM2 gene: upstream homologous arm-Ptrc-PGM2-downstream homologous arm.

[0173] A DNA fragment containing the surE gene target sequence was prepared by annealing primers gRNA-surE-S (SEQ ID NO.120) and gRNA-surE-A (SEQ ID NO.121), and the recombinant plasmid pGRB-surE was obtained after recombination with the linearized pGRB vector.

[0174] The integration fragment (upstream homology arm-Ptrc-PGM2-downstream homology arm) and pGRB-surE were electroporated into E. coli Udg11 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. The positive strains were verified by PCR colonies, and then the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg12. The agarose gel electrophoresis verification is shown in the figure below. Fig.10 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the PGM2 fragment; Lane 3 is the downstream homology arm; Lane 4 is the integration fragment; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain. Example 9

[0175] Overexpression of UGP1 at the yjjG and yrfG gene loci

[0176] According to the upstream and downstream sequences of yjjG and yrfG genes, upstream homology arm primers UP-yjjG-S (SEQ ID NO.122) and UP-yjjG-Ptrc-A (SEQ ID NO.123); UP-yrfG-S (SEQ ID NO.124) and UP-yrfG-Ptrc-A (SEQ ID NO.125); downstream homology arm primers DN-Ptrc-yjjG-S (SEQ ID NO.126) and DN-yjjG-A (SEQ ID NO.127); DN-Ptrc-yrfG-S (SEQ ID NO.128) and DN-yrfG-A (SEQ ID NO.129) were designed respectively. The upstream and downstream homology arms of yjjG and yrfG genes were amplified by PCR technology using the E. coli W3110 genome as a template.

[0177] Primers Ptrc-UGP1-S (SEQ ID NO.130) and UGP1-Ptrc-A (SEQ ID NO.131) were designed according to the nucleotide sequence of the UGP1 gene, and the UGP1 gene was amplified by PCR technology using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template.

[0178] The Ptrc promoter was designed in the antisense primer of the upstream homologous arms of the yjjG and yrfG genes and the sense primer of the UGP1 gene; the Ptrc terminator was designed in the antisense primer of the UGP1 gene and the sense primer of the downstream homologous arms of the yjjG and yrfG genes.

[0179] By recombinant PCR, the upstream and downstream homologous arms of the yjjG gene obtained by the above PCR and the UGP1 gene were used as templates, and UP-yjjG-S (SEQ ID NO.123) and DN-yjjG-A (SEQ ID NO.128) were used as primers to obtain the integrated fragment of the UGP1 gene: yjjG upstream homologous arm-Ptrc-UGP1-yjjG downstream homologous arm.

[0180] By recombinant PCR, the upstream and downstream homologous arms of the yrfG gene obtained by the above PCR and the UGP1 gene were used as templates, and UP-yrfG-S (SEQ ID NO.125) and DN-yrfG-A (SEQ ID NO.130) were used as primers to obtain the integrated fragment of the UGP1 gene: yrfG upstream homologous arm-Ptrc-UGP1-yrfG downstream homologous arm.

[0181] DNA fragments containing yjjG and yrfG gene target sequences were prepared by annealing primers gRNA-yjjG-S (SEQ ID NO.132) and gRNA-yjjG-A (SEQ ID NO.133); gRNA-yrfG-S (SEQ ID NO.134) and gRNA-yrfG-A (SEQ ID NO.135), and recombined with the linearized pGRB vector to obtain recombinant vectors pGRB-yjjG and pGRB-yrfG.

[0182] The integrated fragments yjjG upstream homology arm-Ptrc-UGP1-yjjG downstream homology arm and pGRB-yjjG were electroporated into E. coli Udg12 competent cells containing the pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg13.

[0183] The integration fragment yrfG upstream homology arm-Ptrc-UGP1-yrfG downstream homology arm and pGRB-yghE were electrotransformed into E. coli Udg13 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. The positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated, and the strain E. coli Udg14 was obtained. The agarose gel electrophoresis verification figure is shown in the figure below. Fig.11 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm of yrfG; Lane 2 is the UGP1 fragment; Lane 3 is the downstream homology arm of yrfG; Lane 4 is the integrated fragment yrfG upstream homology arm-Ptrc-UGP1-yrfG downstream homology arm; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain. Example 10

[0184] Knockout of the ornithine transcarbamylase argF gene

[0185] According to the upstream and downstream sequences of the argF gene, upstream homology arm primers UP-argF-S (SEQ ID NO.136) and UP-argF-A (SEQ ID NO.137) and downstream homology arm primers DN-argF-S (SEQ ID NO.138) and DN-argF-A (SEQ ID NO.139) were designed. The upstream and downstream homology arms of the argF gene were amplified by PCR technology using the genome of the uridine-producing bacterium E. coli W3110 as a template.

[0186] By recombinant PCR, the upstream and downstream homologous arms of the argF gene obtained above were used as templates, and UP-argF-S (SEQ ID NO.136) and DN-argF-A (SEQ ID NO.139) were used as primers to obtain the knockout fragment of the argF gene: upstream homologous arm-downstream homologous arm.

[0187] A DNA fragment containing the argF gene target sequence was prepared by annealing primers gRNA-argF-S (SEQ ID NO.140) and gRNA-argF-A (SEQ ID NO.141), and the recombinant plasmid pGRB-argF was obtained after recombination with the linearized pGRB vector.

[0188] The knockout fragment (upstream homology arm-downstream homology arm) and pGRB-argF were electrotransformed into E. coli Udg14 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg15. Agarose gel electrophoresis verification is shown in the figure below. Fig.12 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the downstream homology arm; Lane 3 is the upstream homology arm-downstream homology arm of the knockout fragment; Lane 4 is the PCR fragment of E. coli W3110; Lane 5 is the PCR fragment of the positive strain. Embodiment 11

[0189] Knockout of UDPG dehydrogenase gene ugd

[0190] According to the upstream and downstream sequences of the ugd gene, upstream homology arm primers UP-ugd-S (SEQ ID NO.142) and UP-ugd-A (SEQ ID NO.143) and downstream homology arm primers DN-ugd-S (SEQ ID NO.144) and DN-ugd-A (SEQID NO.145) were designed. The upstream and downstream homology arms of the ugd gene were amplified by PCR technology using the genome of the uridine-producing bacterium E. coli W3110 as a template.

[0191] By recombinant PCR, the upstream and downstream homologous arms of the ugd gene obtained by the above PCR were used as templates, and UP-ugd-S (SEQ ID NO.142) and DN-ugd-A (SEQ ID NO.145) were used as primers to obtain the knockout fragment of the ugd gene: upstream homologous arm-downstream homologous arm.

[0192] The DNA fragment containing the ugd gene target sequence was prepared by annealing primers gRNA-ugd-S (SEQ ID NO.146) and gRNA-ugd-A (SEQ ID NO.147), and the recombinant plasmid pGRB-ugd was obtained after recombination with the linearized pGRB vector.

[0193] The knockout fragment (upstream homology arm-downstream homology arm) and pGRB-ugd were electroporated into E. coli Udg15 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. The positive strains were verified by PCR colonies, and then the two plasmids used for gene editing were eliminated to obtain strain E. coli Udg16. The agarose gel electrophoresis verification is shown in the figure below. Fig.13 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the downstream homology arm; Lane 3 is the upstream homology arm-downstream homology arm of the knockout fragment; Lane 4 is the PCR fragment of E. coli W3110; Lane 5 is the PCR fragment of the positive strain. Example 12

[0194] Knockout of lacI repressor gene lacI

[0195] According to the upstream and downstream sequences of the lacI gene, upstream homology arm primers UP-lacI-S (SEQ ID NO.148) and UP-lacI-A (SEQ ID NO.149) and downstream homology arm primers DN-lacI-S (SEQ ID NO.150) and DN-lacI-A (SEQ ID NO.151) were designed. The upstream and downstream homology arms of the lacI gene were amplified by PCR technology using the genome of the uridine-producing bacterium E. coli W3110 as a template.

[0196] By recombinant PCR, the upstream and downstream homologous arms of the lacI gene obtained by the above PCR were used as templates, and UP-lacI-S (SEQ ID NO.148) and DN-lacI-A (SEQ ID NO.151) were used as primers to obtain the knockout fragment of the lacI gene: upstream homologous arm-downstream homologous arm.

[0197] The DNA fragment containing the lacI gene target sequence was prepared by annealing primers gRNA-lacI-S (SEQ ID NO.152) and gRNA-lacI-A (SEQ ID NO.153), and the recombinant plasmid pGRB-lacI was obtained after recombination with the linearized pGRB vector.

[0198] The knockout fragment (upstream homology arm-downstream homology arm) and pGRB-lacI were electroporated into E. coli LacI16 competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive strains were obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain strain E. coliUdg17. Agarose gel electrophoresis verification is shown in the figure below. Fig.14 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the downstream homology arm; Lane 3 is the upstream homology arm-downstream homology arm of the knockout fragment; Lane 4 is the PCR fragment of E. coli W3110; Lane 5 is the PCR fragment of the positive strain. Example 13

[0199] LB medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar powder 20 g / L.

[0200] Seed culture medium: glucose 30 g / L, yeast powder 5 g / L, peptone 3 g / L, KH2PO4 1.2 g / L, MgSO4 0.5 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·H2O 0.01 g / L, V B1 、V B3 、V B5 、V B12 0.0013 g / L each, V H 0.001 g / L; LD-XP3020 defoamer 100μL / L, adjust the pH to 7 with NaOH solution.

[0201] Fermentation medium: initial glucose 20 g / L, anhydrous sodium citrate 2 g / L, peptone 5 g / L, L-methionine 0.3 g / L, KH2PO4 2 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 0.02 g / L, yeast powder 4 g / L, MnSO40.01 g / L, V B1 、V B3 、V B5 、V B12 0.001 g / L each, V H 0.0005 g / L; LD-XP3020 defoamer 100 μL / L, adjust the pH to 7 with NaOH solution.

[0202] The strain E. coli Udg17 obtained in Example 12 was evenly spread on LB solid culture medium to obtain a bacterial solution of genetically engineered bacteria. The bacterial solution was evenly spread on LB culture medium and cultured at 37°C for 9 hours. A single colony was picked into physiological saline and inoculated into 50 mL of seed culture medium and cultured at 37°C for 6.5 hours. During the culture process, ammonia water was added to maintain the pH value at 7 to obtain a seed solution.

[0203] The above seed solution was inoculated into the fermentation medium at a volume ratio of 2%, and fermentation culture was started. During fermentation culture, the pH value was controlled at 7 and the temperature was 37°C. After 12 hours of fermentation, glucose was added using a feedback pulse feeding strategy: the pulse rate of feeding on the feeding pump was set to 1 / 15s. When the dissolved oxygen value was higher than 35%, the feeding pump automatically started to add 800g / L of glucose solution. When the dissolved oxygen value was lower than 25%, the feeding pump automatically closed. Fermentation culture was continued for 40 hours.

[0204] After the fermentation is completed, a high-pressure homogenizer is used to break the bacteria at a pressure of 800 bar for 5 minutes; the cell disruption liquid is filtered through a ceramic membrane (20 nm) to obtain a UDPG ceramic membrane filtrate, and the UDPG ceramic membrane filtrate is filtered through an ultrafiltration membrane (10000 Da) to collect the UDPG ultrafiltrate; the UDPG ultrafiltrate is desalted by a cation exchange resin (desalting standard: the conductivity of the UDPG clear liquid is <5000 us / cm), and then decolorized by macroporous resin adsorption to obtain a UDPG decolorized liquid; the UDPG decolorized liquid is filtered through a nanofiltration membrane (pore size of 200 Da) to collect a pure UDPG solution.

[0205] The pure UDPG solution obtained above was diluted 50 to obtain a clear solution, which was measured by HPLC. The results were as follows: Fig.15 shown. Fig.16 This is the detection chart of the UDP-glucose standard. Fig.15 and Fig.16 By comparison, it is proved that the embodiment of the present invention produces UDP-glucose by fermentation. Comparative Example 1

[0206] Using E. coli W3110 as the starting strain, a genetically engineered strain was constructed according to the methods of Examples 2 to 12, that is, the step of Example 1 to overexpress pyrBCAKDFE was not performed. In Example 2, the integration fragment (upstream homology arm-Ptrc-pyrAA / pyrABE951*-downstream homology arm) and the recombinant plasmid pGRB-rph were electroporated into competent cells of E. coli W3110 containing the pREDCas9 plasmid, and after screening the positive strains, the plasmid was eliminated to obtain the strain E. coli Udg2′. The strain E. coliUdg2′ was constructed into a genetically engineered strain according to the methods of Examples 3 to 12, and the genetically engineered strain was fermented according to the method of Example 13. Comparative Example 2

[0207] Using E. coli W3110 as the starting strain, a genetically engineered strain was constructed according to the methods of Examples 1, 3 to 12, except that the step of Example 2 was not performed to overexpress pyrAA / pyrAB. E951* In Example 3, the integration fragment (upstream homology arm-Ptrc-pyrB-downstream homology arm) and the recombinant plasmid pGRB-yjiT were electroporated into competent cells of E. coli Udg1-3 containing the pREDCas9 plasmid, and the positive strains were screened and then the plasmid was eliminated to obtain the strain E. coli Udg3′, and the strain E. coli Udg3′ was constructed into a genetically engineered strain according to the methods of Examples 4 to 12, and the genetically engineered strain was fermented according to the method of Example 13. Comparative Example 3

[0208] Using E. coli W3110 as the starting strain, a genetically engineered strain was constructed according to the methods of Examples 1 to 2 and 4 to 12, except that the step of Example 3 to overexpress pyrB was not performed, and in Example 4, the integration fragment (upstream homology arm-Ptrc-pyrE-downstream homology arm) and the recombinant plasmid pGRB-mbhA were electroporated into competent cells of E. coli Udg2 containing the pREDCas9 plasmid, and after screening positive strains, the plasmid was eliminated to obtain strain E. coli Udg4′, and strain E. coli Udg4′ was constructed into a genetically engineered strain according to the methods of Examples 5 to 12, and the genetically engineered strain was fermented according to the method of Example 13. Comparative Example 4

[0209] Using E. coli W3110 as the starting strain, genetically engineered strains were constructed according to the methods of Examples 1 to 4, 6 to 12, except that the step of Example 5 was not performed to overexpress prs at the gene loci purR, nrdD and mazG, respectively. D128AIn Example 6, the upstream homology arm of the integration fragment yhdE - Ptrc-pyrHD93A-downstream homology arm of yhdE and pGRB-yhdE were electrotransformed into E. coli Udg4 competent cells containing the pREDCas9 plasmid, and single colonies were obtained by recovery culture. Positive recombinants were obtained by PCR colony verification, and then pGRB-yhdE used for gene editing was eliminated, that is, the strain E. coli Udg8′; the upstream homology arm of the integration fragment - Ptrc-pyrHD93A-downstream homology arm and pGRB-pyrG were electrotransformed into E. coliUdg8′ competent cells containing the pREDCas9 plasmid, and single colonies were obtained by recovery culture. Positive recombinants were obtained by PCR colony verification, and then pGRB-pyrG used for gene editing was eliminated, that is, the strain E. coli Udg9′; the upstream homology arm of the integration fragment - Ptrc-pyrHD93A-downstream homology arm and pGRB-ygdH were electrotransformed into E. coli Udg9′ competent cells containing pREDCas9 plasmid, and single colonies were obtained by resuscitation culture. Positive recombinants were obtained by PCR colony verification, and then pGRB-ygdH used for gene editing was eliminated, that is, strain E. coli Udg10′, and then strain E. coli Udg10′ was constructed into a genetically engineered strain according to the methods of Examples 7 to 12, and the genetically engineered strain was fermented according to the method of Example 13. Comparative Example 5

[0210] Using E. coli W3110 as the starting strain, a genetically engineered strain was constructed according to the methods of Examples 1 to 9 and 11 to 12, except that the step of Example 10 was not performed to knock out the ornithine carbamoyltransferase argF gene, and in Example 11, the knockout fragment (upstream homology arm-downstream homology arm) and pGRB-ugd were electroporated into E. coli Udg14 competent cells containing the pREDCas9 plasmid, and single colonies were obtained by resuscitation culture, and positive recombinants were obtained by PCR colony verification, and then pGRB-ugd used for gene editing was eliminated, that is, strain E. coli Udg16′, and strain E. coli Udg16′ was constructed into a genetically engineered strain according to the method of Example 12, and the genetically engineered strain was fermented according to the method of Example 13. Comparative Example 6

[0211] Using E. coli W3110 as the starting strain, a genetically engineered strain, namely, strain E. coli Udg16, was constructed according to the methods of Examples 1 to 11, and the strain E. coli Udg16 was fermented according to the method of Example 13.

[0212] Test Case

[0213] The pure uridine diphosphate glucose (UDPG) solutions collected in Example 12 and Comparative Examples 1 to 6 were measured by HPLC. The results are shown in Table 1.

[0214] Table 1

[0215]

[0216] From the above results, it can be seen that compared with Examples 1 to 12, Comparative Example 1 did not overexpress the pyrBCAKDFE gene, and Comparative Example 2 did not overexpress pyrAA / pyrAB E951* After the pyrB gene was not overexpressed in comparative example 3, the yield and conversion rate of UDPG decreased, indicating that the overexpression of pyrBCAKDFE gene, pyrAA / pyrAB E951* Gene or pyrB gene can improve the yield and conversion rate of UDPG. Comparative Example 4 does not overexpress prs at gene sites purR, nrdD and mazG D128A The production and conversion rate of UDPG decreased, indicating that purR, nrdD and mazG genes were not expressed and prs D128A Gene can improve the yield and conversion rate of UDPG. After the ornithine carbamoyl transferase argF gene was not knocked out in comparative example 5, the yield and conversion rate of UDPG both decreased, indicating that not expressing the ornithine carbamoyl transferase argF gene can improve the yield and conversion rate of UDPG. After the lacI repressor protein gene lacI was not knocked out in comparative example 6, the yield and conversion rate of UDPG decreased significantly, indicating that the expression of this gene would inhibit the regulation of other integrated genes by the Ptrc promoter, thereby significantly reducing the yield and conversion rate of UDPG.

[0217] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium for producing UDP-glucose, characterized in that: The following genes are overexpressed in the genetically engineered bacteria: Pyrimidine nucleoside operon gene, carbamyl phosphate synthetase gene, aspartate carbamyl transaminase gene, orotate phosphoribosyltransferase gene, phosphoribosyl pyrophosphate synthase gene, uridylate kinase gene, glucose phosphomutase gene, uridine triphosphate glucose-1-phospho-uridyltransferase gene; The pyrimidine nucleoside operon gene is the pyrBCAKDFE gene, and the nucleotide sequence of the pyrBCAKDFE gene is shown in SEQ ID NO. 1; the pyrBCAKDFE gene is located at the pseudogene site yghX; The carbamyl phosphate synthetase gene is pyrAA / pyrAB E951* Gene, the pyrAA / pyrAB E951* The nucleotide sequence of the gene is shown in SEQ ID NO. 2; the pyrAA / pyrAB E951* The gene is located at the pseudogene locus rph; The aspartate transaminase gene is the pyrB gene, and the nucleotide sequence of the pyrB gene is shown in SEQ ID NO.3; the pyrB gene is located at the pseudogene site yjiT; The orotate phosphoribosyltransferase gene is the pyrE gene, and the nucleotide sequence of the pyrE gene is shown in SEQ ID NO.4; the pyrE gene is located at the pseudogene site mbhA; The phosphoribosyl pyrophosphate synthase gene is prs D128A Gene, the prs D128A The nucleotide sequence of the gene is shown in SEQ ID NO.5; the prs D128A The genes are located at the gene loci purR, nrdD, and mazG; The uridylate kinase gene is pyrH D93A gene, the pyrH D93A The nucleotide sequence of the gene is shown in SEQ ID NO.6; the pyrH D93A The genes are located at loci yhdE, pyrG, and ygdH; The glucose phosphomutase gene includes a PGM1 gene and a PGM2 gene, the nucleotide sequence of the PGM1 gene is shown in SEQ ID NO.7, and the nucleotide sequence of the PGM2 gene is shown in SEQ ID NO.8; the PGM1 gene is located at the gene site ushA; the PGM2 gene is located at the gene site surE; The uridine triphosphate glucose-1-phosphate uridyl transferase gene is the UGP1 gene, and the nucleotide sequence of the UGP1 gene is shown in SEQ ID NO. 9; the UGP1 gene is located at gene sites yjjG and yrfG; The following genes are not expressed in the genetically engineered bacteria: Purine nucleotide repressor gene, deoxyribonucleoside triphosphate reductase gene, nucleoside triphosphate pyrophosphohydrolase gene, cytidine triphosphate synthase gene, pyrimidine nucleotidase gene, nucleotidase gene, ornithine carbamoyltransferase gene, UDPG dehydrogenase gene, lacI repressor gene; The purine nucleotide repressor protein gene is the purR gene, and the nucleotide sequence of the purR gene is shown in SEQ ID NO.10; The deoxyribonucleoside triphosphate reductase gene is the nrdD gene, and the nucleotide sequence of the nrdD gene is shown in SEQ ID NO. 11; The nucleoside triphosphate pyrophosphohydrolase gene includes a mazG gene and a yhdE gene, the nucleotide sequence of the mazG gene is shown in SEQ ID NO. 12, and the nucleotide sequence of the yhdE gene is shown in SEQ ID NO. 13; The cytidine triphosphate synthase gene is a pyrG gene, and the nucleotide sequence of the pyrG gene is shown in SEQ ID NO. 14; The pyrimidine nucleotidase gene is the ygdH gene, and the nucleotide sequence of the ygdH gene is shown in SEQ ID NO. 15; The nucleotidase gene includes ushA gene, surE gene, yjjG gene and yrfG gene, the nucleotide sequence of the ushA gene is shown in SEQ ID NO.16, the nucleotide sequence of the surE gene is shown in SEQ ID NO.17, the nucleotide sequence of the yjjG gene is shown in SEQ ID NO.18, and the nucleotide sequence of the yrfG gene is shown in SEQ ID NO.19; The ornithine carbamoyltransferase gene is an argF gene, and the nucleotide sequence of the argF gene is shown in SEQ ID NO.20; The UDPG dehydrogenase gene is the ugd gene, and the nucleotide sequence of the ugd gene is shown in SEQ ID NO. 21; The lacI repressor protein gene is a lacI gene, and the nucleotide sequence of the lacI gene is shown in SEQ ID NO. 22; The starting strain used in the construction of the genetically engineered bacteria is Escherichia coli; the Escherichia coli is E. coliW3110.

2. The genetically engineered bacterium according to claim 1, characterized in that: The pyrimidine nucleoside operon gene, carbamyl phosphate synthetase gene, aspartate carbamyl transaminase gene, orotate phosphoribosyl transferase gene, phosphoribosyl pyrophosphate synthase gene, uridylate kinase gene, glucose phosphomutase gene and uridine triphosphate glucose-1-phosphate uridyl transferase gene are all controlled by the strong promoter Ptrc.

3. The genetically engineered bacterium according to claim 1, characterized in that The nucleotide sequence of the strong promoter Ptrc is shown in SEQ ID NO.

23.

4. A method for constructing a genetically engineered bacterium, used to construct the genetically engineered bacterium according to any one of claims 1 to 3, characterized in that: The following steps are involved: Integrating the pyrimidine nucleoside operon gene into the pseudogene site yghX of the genetically engineered bacteria to overexpress the pyrimidine nucleoside operon gene; Integrating a carbamyl phosphate synthetase gene into the pseudogene site rph of the genetically engineered bacteria to overexpress the carbamyl phosphate synthetase gene; Integrating the aspartate transaminase gene into the pseudogene site yjiT of the genetically engineered bacteria to overexpress the aspartate transaminase gene; Integrating the orotate phosphoribosyltransferase gene into the pseudogene site mbhA of the genetically engineered bacteria to overexpress the orotate phosphoribosyltransferase gene; Integrating the phosphoribosyl pyrophosphate synthase gene into the gene sites purR, nrdD and mazG of the genetically engineered bacteria to overexpress the phosphoribosyl pyrophosphate synthase gene; The uridylate kinase gene is located at the gene sites yhdE, pyrG and ygdH of the genetically engineered bacteria, so that the uridylate kinase gene is overexpressed; Integrating the phosphoglucomutase gene PGM1 into the gene site ushA of the genetically engineered bacteria to overexpress the phosphoglucomutase gene PGM1; Integrating the phosphoglucomutase gene PGM2 into the gene site surE of the genetically engineered bacteria to overexpress the phosphoglucomutase gene PGM2; Integrating a UDP-glucose-1-phosphate uridyltransferase gene into the gene sites yjjG and yrfG of the genetically engineered bacteria to overexpress the UDP-glucose-1-phosphate uridyltransferase gene; knocking out the ornithine carbamoyltransferase gene so that the ornithine carbamoyltransferase gene is not expressed; Knocking out the UDPG dehydrogenase gene so that the UDPG dehydrogenase gene is not expressed; The lacI repressor protein gene is knocked out so that the lacI repressor protein gene is not expressed.

5. Use of the genetically engineered bacteria according to any one of claims 1 to 3 in the production of UDP-glucose.

6. A method for producing UDP-glucose, characterized in that: The following steps are involved: Take the bacterial liquid of any one of the genetically engineered bacteria of claims 1 to 3, apply it to LB medium, culture it at 36.5°C to 37.5°C for 8 to 10 hours, inoculate it into seed medium, culture it at 36.5°C to 37.5°C for 5 to 8 hours, maintain the pH value at 6.8 to 7.1 during the culture process, and obtain seed liquid; The seed liquid is inoculated into a fermentation medium for fermentation culture. During the fermentation culture, the pH value is controlled to be 6.8-7.1 and the temperature is controlled to be 36.5°C-37.5°C. After 10-12 hours of fermentation culture, glucose is added using a feedback pulse feeding strategy, the dissolved oxygen value is controlled to be 25%-35%, and the fermentation culture is continued for 36-40 hours to obtain uridine diphosphate glucose.

7. The method for producing UDP-glucose according to claim 6, characterized in that: The feedback pulse feeding strategy is: The pulse rate of feeding on the feeding pump is set to 1 / 18~1 / 15s. When the dissolved oxygen value is higher than 35%, the feeding pump automatically starts to add 700~800g / L of glucose solution. When the dissolved oxygen value is lower than 25%, the feeding pump automatically shuts down.

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