An engineered strain for producing uridine diphosphate, a construction method thereof, and a method for synthesizing rebaudioside M

Abstract

The present invention discloses an engineered strain for producing uridine diphosphate, a construction method and a method for synthesizing rebaudioside M, belonging to the technical field of genetic engineering. The engineered strain integrates genes such as the pyrimidine nucleoside operon genes pyrBCAKDFE, the carbonic anhydrase gene cynT, and the carbamoyl phosphate synthetase gene pyrAA / AB<supgt;E951*< / supgt; on the genome, and deletes genes such as the aspartate carbamoyltransferase gene pyrB, the orotate phosphoribosyltransferase gene pyrE, and the phosphoribosyl pyrophosphate synthase gene prs<supgt;D128A< / supgt>. The engineered strain can efficiently synthesize uridine diphosphate and can act together with a recombinant strain expressing functional proteins to catalyze the synthesis of rebaudioside M using sucrose and rebaudioside A as substrates.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to an engineering strain for producing uridine diphosphate, a construction method thereof, and a method for synthesizing rebaudioside M. Background Art

[0002] Rebaudioside M is a natural sweetener extracted from Stevia rebaudiana, and its sweetness is about 300-400 times that of sucrose, and it has no bitterness and aftertaste, and is widely used in various fields such as food, health products, and medicine. In the prior art, methods for obtaining rebaudioside M include plant extraction method, fermentation method, enzyme conversion method, etc. Among them, the plant extraction method mainly extracts from Stevia rebaudiana by water or ethanol, but the content of natural rebaudioside M in the leaves of Stevia rebaudiana is low, only accounting for 0.4-0.5% of the dry weight of the Stevia rebaudiana leaves, which limits industrial production. When producing rebaudioside M by fermentation method, traditional engineering strains are used for fermentation. For example, Saccharomyces cerevisiae fermentation method is used to synthesize rebaudioside M. The fermentation of Saccharomyces cerevisiae requires 144 h, and the yield of rebaudioside M is only 67 mg / L, which has the problems of long fermentation time and low yield.

[0003] In addition, in the prior art, there are two conversion methods for producing rebaudioside M by enzyme conversion method. One is to use a crude enzyme solution containing sucrose synthase and glycosyltransferase to generate rebaudioside M in one pot with rebaudioside D, UDP, MgCl2, and phosphate buffer. This method uses UDP and rebaudioside D as raw materials, and has the problem of relatively high production cost. The other uses steviol glycoside and sucrose as raw materials and generates rebaudioside M through whole-cell catalysis of recombinant cells for 20 h. In this method, the yield of uridine diphosphate produced by the engineering strain is only 15.137 mmol / L, and the yield of uridine diphosphate is low, resulting in a low yield of rebaudioside M. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an engineering strain for producing uridine diphosphate, a construction method thereof, and a method for synthesizing rebaudioside M, so as to overcome the technical problems of low yield of uridine diphosphate and insufficient high yield of rebaudioside M in the prior art.

[0005] In the first aspect, the present invention provides an engineering strain for producing uridine diphosphate, and the engineering strain for producing uridine diphosphate integrates the following genes on the genome:

[0006] Pyrimidine nucleoside operon genes pyrBCAKDFE, carbonic anhydrase gene cynT, carbamoyl phosphate synthetase genes pyrAA / AB E951* , aspartate carbamoyltransferase gene pyrB, orotate phosphoribosyltransferase gene pyrE, phosphoribosyl pyrophosphate synthase gene prs D128A , uridylate kinase gene pyrHD93A 。

[0007] The engineered strain for producing uridine diphosphate has the following genes deleted from its genome:

[0008] The lactose operon regulatory gene lacI, the repressor protein genes purR, pepA and argR, the homoserine dehydrogenase gene thrA, the ornithine carbamoyltransferase gene argF, the uridine diphosphate kinase gene udk, the nucleotide hydrolase genes ushA, surE, yjjG and yrfG, the pyrimidine nucleotide hydrolase gene ygdH, and the global transcriptional regulatory gene arcA.

[0009] Compared with the prior art, the engineered strain for producing uridine diphosphate in the present invention has specific genes integrated into and deleted from its genome, thereby regulating the metabolism of the engineered strain, enabling it to efficiently produce uridine diphosphate (UDP), and further increasing the yield of UDP, specifically as follows:

[0010] Enhancing the supply of the important precursor UMP: The pyrimidine nucleoside operon genes PyrBCAKDFE that can relieve the feedback inhibition of UMP are integrated into the genome of the engineered strain, strengthening the metabolism from glutamine to UMP. The carbonic anhydrase gene cynT is integrated into the genome of the engineered strain, strengthening the metabolism of the reaction of CO2 and H2O to generate HCO3 - The carbamoyl phosphate synthetase gene pyrAA / AB is integrated into the genome of the engineered strain E951* , in which the glutamic acid at position 951 of the large subunit of carbamoyl phosphate synthetase is deleted, thereby relieving the feedback regulation of uridylic acid and strengthening the metabolism of glutamine to synthesize carbamoyl phosphate. The aspartate carbamoyltransferase gene pyrB is integrated into the genome of the engineered strain, strengthening the metabolism of aspartate and carbamoyl phosphate to synthesize carbamoyl aspartic acid. The orotate phosphoribosyltransferase gene pyrE is integrated into the genome of the engineered strain, strengthening the metabolism of PRPP (5-phosphoribosyl-1-pyrophosphate) and orotate to generate orotidine monophosphate. The phosphoribosyl pyrophosphate synthase gene prs is integrated into the genome of the engineered strain D128A , and the aspartic acid at position 128 of this enzyme is 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 purine nucleotide repressor gene purR is deleted from the genome of the engineered strain, and further relieves the binding of the repressor protein and the purine repressor to prs D128AFeedback inhibition of gene expression. The pepA gene and argR gene are deleted from the genome of the engineered strain, thereby relieving the inhibition of the carbamoyl phosphate-encoding gene operon by the repressor proteins encoded by the pepA gene and the argR gene. The homoserine dehydrogenase gene thrA is deleted from the genome of the engineered strain, blocking the metabolism of L-aspartic acid dehydrogenation to homoserine. The ornithine carbamoyltransferase gene argF is deleted from the genome of the engineered strain, blocking the metabolism of the carbamoyl group of carbamoyl phosphate transferring to ornithine to produce citrulline.

[0011] Blocking the branched metabolism of the precursor UMP: In Escherichia coli, UMP has two metabolic pathways, namely uridine and uridine diphosphate. The engineered strain for producing uridine diphosphate in the present invention lacks the pyrimidine nucleotidase gene ygdH, and lacks the nucleotide hydrolase genes ushA, surE, yjjG, and yrfG, successfully weakening the metabolism of UMP to uridine and uracil, and then converting the metabolism of UMP to uridine into the metabolism of UMP to UDP.

[0012] Blocking the phosphorylation metabolism of UDP: Uridine diphosphate kinase in Escherichia coli affects the accumulation of uridine diphosphate. Uridine diphosphate is phosphorylated by uridine diphosphate kinase to produce uridine triphosphate. The engineered strain of uridine diphosphate in the present invention lacks the uridine diphosphate kinase gene udk, thereby blocking the phosphorylation reaction of the product UDP and promoting the accumulation of UDP.

[0013] Optimizing the synthetic metabolism of UDP: On the basis of relieving the feedback inhibition of UDP, the uridylate kinase gene pyrH is integrated into the genome of the engineered strain for producing uridine diphosphate in the present invention D93A and reasonably strengthening the metabolism of UMP to UDP.

[0014] Accelerating the TCA cycle and weakening the coupling flux of PRdnBP (de novo biosynthesis pathway of pyrimidine ribonucleoside) and acetate metabolism: The global transcriptional regulatory gene arcA is deleted from the genome of the engineered strain, weakening the coupling flux of PRdnBP and acetate metabolism, reducing acetate production, relieving the inhibition of arcA on TCA, and accelerating the TCA cycle, reducing carbon metabolic loss.

[0015] The lactose operon regulatory gene lacI is deleted from the genetically engineered strain of the present invention, which can relieve the feedback repression of the Ptrc promoter by the lacI repressor protein and avoid the normal regulation of the integrated gene by the repressor protein inhibiting the Ptrc promoter.

[0016] Furthermore, the gene sequences of pyrBCAKDFE are as shown in SEQ ID NO. 1.

[0017] The gene sequence of cynT is as shown in SEQ ID NO. 2.

[0018] pyrAA / AB E951* The gene sequence of

[0019] pyrB is shown in SEQ ID NO. 4.

[0020] The gene sequence of pyrE is shown in SEQ ID NO. 5.

[0021] prsD 128A The gene sequence of

[0022] pyrH D93A is shown in SEQ ID NO. 7.

[0023] The gene sequence of lacI is shown in SEQ ID NO. 8.

[0024] The gene sequence of purR is shown in SEQ ID NO. 9.

[0025] The gene sequence of pepA is shown in SEQ ID NO. 10.

[0026] The gene sequence of argR is shown in SEQ ID NO. 11.

[0027] The gene sequence of thrA is shown in SEQ ID NO. 12.

[0028] The gene sequence of argF is shown in SEQ ID NO. 13.

[0029] The gene sequence of udk is shown in SEQ ID NO. 14.

[0030] The gene sequence of ushA is shown in SEQ ID NO. 15.

[0031] The gene sequence of surE is shown in SEQ ID NO. 16.

[0032] The gene sequence of yjjG is shown in SEQ ID NO. 17.

[0033] The gene sequence of yrfG is shown in SEQ ID NO. 18.

[0034] The gene sequence of ygdH is shown in SEQ ID NO. 19.

[0035] The gene sequence of arcA is shown in SEQ ID NO. 20.

[0036] Second aspect, the present invention provides a method for constructing an engineered strain for constructing the above-mentioned engineered strain, comprising the following steps:

[0037] Integrate the pyrimidine nucleoside operon genes pyrBCAKDFE from Bacillus subtilis at the pseudogene locus yghX.

[0038] Integrate the carbonic anhydrase gene cynT at the gene locus purR.

[0039] Integrate the carbamoyl phosphate synthase genes pyrAA / AB from Bacillus subtilis at the gene locus lacI E951* .

[0040] Integrate the aspartate carbamoyltransferase gene pyrB from Bacillus subtilis at the gene locus pepA.

[0041] Integrate the orotate phosphoribosyltransferase gene pyrE from Bacillus subtilis at the gene locus argR.

[0042] Integrate the phosphoribosyl pyrophosphate synthase gene prs from Bacillus subtilis at the gene loci thrA, argF, and udk respectively D128A .

[0043] Integrate the uridine monophosphate kinase gene pyrH from Bacillus subtilis at the gene loci ushA, surE, yjjG, and yrfG respectively D93A .

[0044] Knock out the pyrimidine nucleotidase gene ygdH.

[0045] Knock out the global transcriptional regulatory gene arcA.

[0046] Compared with the prior art, the gene operations involved in constructing the engineered strain for producing uridine diphosphate in the present invention are all carried out on the genome, without plasmid residues, without the use of antibiotics and inducers. The finally obtained engineered strain for producing uridine diphosphate has clear and stable genetic traits, stable production performance, and a simple fermentation process.

[0047] Furthermore, the integrated genes are all controlled by the strong promoter Ptrc, and the gene sequence of the strong promoter Ptrc is as shown in SEQ ID NO. 21.

[0048] Third aspect, the present invention provides a method for synthesizing rebaudioside M, comprising the following steps:

[0049] Ferment and culture the above-mentioned engineered strain for producing uridine diphosphate to obtain the fermented engineered strain for producing uridine diphosphate.

[0050] Ferment and culture the functional protein expression recombinant strain to obtain the fermented functional protein expression recombinant strain.

[0051] The engineered strain producing uridine diphosphate and the recombinant strain expressing functional protein after fermentation are configured into the following reaction system: potassium dihydrogen phosphate 2.0 - 2.5 g / L, dipotassium hydrogen phosphate 25 - 35 g / L, ammonium sulfate 3.0 - 3.5 g / L, magnesium chloride 2.0 - 2.5 g / L, glycerol 8 - 12 g / L, CTAB 2.0 - 2.5 g / L, the engineered strain producing uridine diphosphate after fermentation 25 - 40 g / L, the recombinant strain expressing functional protein after fermentation 100 - 130 g / L, sucrose 100 - 200 g / L, rebaudioside A 50 - 80 g / L.

[0052] React the above reaction system under the conditions of pH value 6.5 - 7.5 and temperature 36.5 - 37.5 for 18 - 24 h. After the reaction, dilute the reaction solution by 100 - 200 times and centrifuge to obtain the supernatant containing rebaudioside M.

[0053] Compared with the prior art, the reaction system configured with the engineered strain producing uridine diphosphate and the recombinant strain expressing functional protein after fermentation of the present invention can efficiently convert rebaudioside A into rebaudioside M, further improving the yield and conversion rate of rebaudioside M.

[0054] Furthermore, the preparation method of the recombinant strain expressing functional protein after fermentation includes the following steps:

[0055] Connect the sucrose synthase gene susy, the uridine diphosphate glycosyltransferase gene EUGT11, and the uridine diphosphate glycosyltransferase gene SrUGT76G4 to the plasmid to construct a recombinant plasmid.

[0056] Transfer the recombinant plasmid into the host strain to obtain the recombinant strain expressing functional protein.

[0057] Ferment and culture the recombinant strain expressing functional protein to obtain the recombinant strain expressing functional protein after fermentation.

[0058] Among them, the host strain is Escherichia coli; the Escherichia coli is Escherichia coli BL21(DE3).

[0059] In the above technical solution, the susy gene can encode sucrose synthase, and sucrose synthase can use sucrose and UDP as substrates to produce fructose and UDPG; the EUGT11 gene can encode a glycosyltransferase, and this glycosyltransferase can transfer the glucose group in UDPG to rebaudioside A to generate rebaudioside D and UDP; the SrUGT76G4 gene can encode another glycosyltransferase, and this glycosyltransferase has a preference for the C19 position of steviol glycosides, forming a β-1,3-glycosidic bond at the C19 position, so that this glycosyltransferase can use UDPG and rebaudioside D as substrates to catalyze the production of rebaudioside M and UDP.

[0060] In addition, all gene operations involved in the recombinant strain expressing the functional protein constructed in the present invention are carried out on the pET28A plasmid, which carries kanamycin resistance. The finally obtained recombinant strain of the functional protein has clear and stable genetic traits, stable production performance, and a simple fermentation process.

[0061] Furthermore, the sucrose synthase gene susy is derived from Nitrosomonas polymorphis, tobacco or Arabidopsis thaliana.

[0062] The uridine diphosphate glycosyltransferase gene EUGT11 is derived from Stevia rebaudiana.

[0063] The uridine diphosphate glycosyltransferase gene SrUGT76G4 is derived from Stevia rebaudiana.

[0064] Furthermore, the recombinant strain expressing the functional protein is fermented and cultured, including the following steps:

[0065] Take the bacterial liquid of the recombinant strain expressing the functional protein, evenly coat it on the LB medium containing kanamycin, culture it at 36.5 - 37.5 °C for 8 - 10 h, pick a single colony and inoculate it into the seed medium containing kanamycin, and culture it at a temperature of 36.5 - 37.5 °C and a pH value of 6.5 - 7.5 for 5 - 8 h to obtain a seed liquid.

[0066] Inoculate the seed liquid into the fermentation medium containing kanamycin, ferment and culture it at a temperature of 36.5 - 37.5 °C and a pH value of 6.5 - 7.5. When the OD value reaches 10 - 20, lower the temperature to 25 - 30 °C, supplement and flow the medium to control the glucose concentration within 1 g / L, and continue to ferment and culture for 24 - 30 h.

[0067] After the fermentation is completed, centrifuge the obtained fermentation broth, discard the supernatant, and obtain the recombinant strain expressing the functional protein after fermentation.

[0068] Furthermore, the engineering strain producing uridine diphosphate is fermented and cultured, including the following steps:

[0069] Take the bacterial solution of the engineered strain for producing uridine diphosphate, coat it on the LB medium, culture it at 36.5 - 37.5 °C for 8 - 10 h, pick a single bacterium and inoculate it into the seed medium, and culture it at a temperature of 36.5 - 37.5 °C and a pH value of 6.5 - 7.5 for 5 - 8 h to obtain the seed solution.

[0070] Inoculate the seed solution into the fermentation medium, and after fermenting and culturing at a temperature of 36.5 - 37.5 °C and a pH value of 6.5 - 7.5 for 10 - 12 h, add glucose using a feedback pulse feeding strategy, and continue fermenting and culturing for 36 - 40 h.

[0071] After the fermentation is completed, centrifuge the obtained fermentation broth, discard the supernatant, and obtain the engineered strain for producing uridine diphosphate after fermentation.

[0072] Furthermore, the feedback pulse feeding strategy for adding glucose is as follows:

[0073] Set the pulse rate of feeding on the feeding pump to 1 / 18 - 1 / 15 s. When the dissolved oxygen value is higher than 35%, the feeding pump automatically starts to add a glucose solution of 700 - 800 g / L. When the dissolved oxygen value is lower than 25%, the feeding pump automatically shuts off. Description of the Drawings

[0074] Figure 1 It is the verification diagram of agarose gel electrophoresis for integrating the pyr1 gene in Example 1.

[0075] Figure 2 It is the verification diagram of agarose gel electrophoresis for integrating the pyr2 gene in Example 1.

[0076] Figure 3 It is the verification diagram of agarose gel electrophoresis for integrating the pyr3 gene in Example 1.

[0077] Figure 4 It is the verification diagram of agarose gel electrophoresis for integrating the cynT gene in Example 1.

[0078] Figure 5 It is the verification diagram of agarose gel electrophoresis for integrating the pyrAA / AB E951* gene in Example 1.

[0079] Figure 6 It is the verification diagram of agarose gel electrophoresis for integrating the pyrB gene in Example 1.

[0080] Figure 7 It is the verification diagram of agarose gel electrophoresis for integrating the pyrE gene in Example 1.

[0081] Figure 8 It is the verification diagram of agarose gel electrophoresis for integrating the prs D128AAgarose gel electrophoresis verification diagram of the gene.

[0082] Figure 9 For the integration of pyrH in Example 1 D90A Agarose gel electrophoresis verification diagram of the gene.

[0083] Figure 10 Agarose gel electrophoresis verification diagram for the knockout of the ygdH gene in Example 1.

[0084] Figure 11 Agarose gel electrophoresis verification diagram for the knockout of the ArcA gene in Example 1. Detailed implementation manners

[0085] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be 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.

[0086] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified. Example 1

[0087] Construction of an engineering strain for producing uridine diphosphate

[0088] In the embodiment of the present invention, the gene editing of E. coli W3110 (ATCC27325) is carried out by using the CRISPR / Cas9-mediated gene editing method (reference can be made to the literature Metabolic Engineering, 2015, 31: 13-21.). CRISPR / Cas9 is a precise and efficient new gene targeting modification technology. The two plasmids used in this method are pGRB and pREDCas9 respectively. The pREDCas9 plasmid is a temperature-sensitive plasmid, carrying the elimination system of the gRNA plasmid, the Red recombination system of λ phage and the Cas9 protein expression system, with spectinomycin resistance (working concentration: 100 mg / L), and the culture temperature is 32 °C; the pGRB plasmid, with pUC18 as the backbone, contains the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence, with ampicillin resistance (working concentration: 100 mg / L), and the culture temperature is 37 °C.

[0089] 1. Integrate the pyrimidine nucleoside operon genes pyrBCAKDFE from Bacillus subtilis at the pseudogene locus yghX:

[0090] Due to the large fragment of the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis, with a base number as high as 9492 bp, the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis was integrated in segments at the pseudogene locus yghX.

[0091] (1)Integration of pyr1

[0092] Using the E. coli W3110 genome as a template, upstream homologous arm primers UP-yghX-S (SEQ ID NO.28) and UP-yghX-Ptrc-A (SEQ ID NO.29), and downstream homologous arm primers DN-yghX-S1 (SEQ ID NO.30) and DN-yghX-A (SEQ ID NO.31) were designed according to the upstream and downstream gene sequences of its pseudogene yghX, and the upstream and downstream homologous arm fragments were amplified by PCR.

[0093] Using the B.subtilis A260 genome as a template, primers Ptrc-pyr1-S (SEQ ID NO.32) and pyr1-A (SEQ ID NO.33) were designed at the 1st to 3260th bases (pyr1) of the pyrBCAKDFE nucleotide sequence (SEQ ID NO.1), and the pyr1 gene fragment was amplified by PCR.

[0094] The promoter Ptrc was designed in the downstream primer of the upstream homologous arm of the pseudogene yghX and the upstream primer of the pyr1 gene. The 4th plasmid gene was designed in the upstream primer of the downstream homologous arm of the pseudogene yghX and the downstream primer of the pyr1 gene. The nucleotide sequence of the 4th plasmid gene is shown in SEQ ID NO.136.

[0095] By the method of overlap PCR, using the upstream and downstream homologous arm fragments of the pseudogene yghX and the pyr1 gene fragment obtained above as templates, and UP-yghX-S (SEQ ID NO.28) and DN-yghX-A (SEQ ID NO.31) as primers, the integration fragment of the pyr1 gene was obtained: upstream homologous arm - Ptrc - pyr1 - downstream homologous arm.

[0096] A DNA fragment containing the target sequence of the pseudogene yghX was prepared by annealing primers gRNA-yghX-S (SEQ ID NO.34) and gRNA-yghX-A (SEQ ID NO.35), and after recombination with the linearized pGRB vector, the recombinant plasmid pGRB-yghX was obtained.

[0097] The integration fragment of the pyr1 gene (upstream homologous arm - Ptrc - pyr1 - downstream homologous arm) and the recombinant plasmid pGRB - yghX were electrotransformed into the competent cells of E. coli W3110 containing the pREDCas9 plasmid. After screening for positive strains, the two plasmids pGRB - yghX and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud1 - 1.

[0098] The agarose gel electrophoresis verification diagrams of the integration fragment of the pyr1 gene and the positive strain are as Figure 1 shown: Lane M is the DNA Marker; Lane 1 is the upstream homologous arm; Lane 2 is the pyr1 gene fragment; Lane 3 is the downstream homologous arm; Lane 4 is the integration 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.36) and pyr2 - A (SEQ ID NO.37) 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, primers DN - yghX - S2 (SEQ ID NO.38) and DN - yghX - A (SEQ ID NO.35) based on the downstream homologous arm of the pseudogene yghX were used to PCR - amplify its downstream homologous arm fragment.

[0102] The 5 - plasmid gene was designed in the upstream primer of the downstream homologous arm of the pseudogene yghX and the downstream primer of the pyr2 gene. The nucleotide sequence of the 5 - plasmid gene is as shown in SEQ ID NO.137.

[0103] By the method of overlap PCR, using the obtained pyr2 fragment and the downstream homologous arm fragment of the pseudogene yghX as templates, and pyr2 - S (SEQ ID NO.36) and DN - yghX - A (SEQ ID NO.35) as primers, the integration fragment of pyr2: pyr2 - downstream homologous arm was obtained.

[0104] A DNA fragment containing the target sequence of the 4 - plasmid gene was prepared by annealing primers gRNA - S1 (SEQ ID NO.39) and gRNA - A1 (SEQ ID NO.40), and after recombination with the linearized pGRB vector, the recombinant plasmid pGRB - pyr2 was obtained.

[0105] The integration fragment of pyr2 (pyr2-downstream homologous arm) and the recombinant plasmid pGRB-pyr2 were electrotransformed into the competent cells of E. coli Ud1-1 containing the plasmid pREDCas9. After screening for positive strains, the two plasmids pGRB-pyr2 and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud1-2.

[0106] The agarose gel electrophoresis verification diagrams of the pyr2 integration fragment and the positive strain are as Figure 2 shown: Lane M is the DNA Marker; Lane 1 is the pyr2 gene fragment; Lane 2 is the downstream homologous arm; Lane 3 is the integration 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.41) and pyr3-Ptrc-A (SEQ ID NO.42) were designed based on 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, primers DN-Ptrc-yghX-S (SEQ ID NO.43) and DN-yghX-A (SEQ ID NO.35) for the downstream homologous arm of the pseudogene yghX were used to PCR amplify its downstream homologous arm fragment.

[0110] The terminator Ptrc was designed in the upstream primer of the downstream homologous arm of the pseudogene yghX and the downstream primer of the pyr3 gene. The gene sequence of the terminator Ptrc is as shown in SEQ ID NO.22.

[0111] By the method of overlap PCR, using the pyr3 fragment and the downstream homologous arm fragment of the pseudogene yghX obtained by the above PCR as templates, and using pyr3-S (SEQ ID NO.41) and DN-yghX-A (SEQ ID NO.35), the integration fragment of the pyr3 gene: pyr3-downstream homologous arm was obtained.

[0112] The DNA fragment containing the gene target sequence of plasmid No. 5 was prepared by annealing primers gRNA-S2 (SEQ ID NO. 44) and gRNA-A2 (SEQ ID NO. 45), and the recombinant plasmid pGRB-pyr3 was obtained after recombination with the linearized pGRB vector.

[0113] The integration fragment of the pyr3 gene (pyr3-downstream homologous arm) and the recombinant plasmid pGRB-pyr3 were electrotransformed into the competent cells of E. coli Ud1-2 containing the pREDCas9 plasmid. After screening positive strains, the two plasmids pGRB-pyr3 and pREDCas9 for gene editing were eliminated to obtain the strain E. coli Ud1-3.

[0114] The agarose gel electrophoresis verification diagrams of the pyr3 integration fragment and the positive strain are as Figure 3 shown: Lane M is the DNA Marker; Lane 1 is the pyr3 fragment; Lane 2 is the downstream homologous arm; Lane 3 is the integration 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.

[0115] 2. Integrate the carbonic anhydrase gene cynT at the gene locus purR:

[0116] Using the E. coli W3110 genome as a template, upstream homologous arm primers UP-purR-S (SEQ ID NO. 46) and UP-purR-Ptrc-A (SEQ ID NO. 47) were designed according to the upstream and downstream gene sequences of purR; downstream homologous arm primers DN-Ptrc-purR-S (SEQ ID NO. 48) and DN-purR-A (SEQ ID NO. 49); the upstream and downstream homologous arm fragments of purR were amplified by PCR.

[0117] Primers Ptrc-cynT-S (SEQ ID NO. 50) and cynT-Ptrc-A (SEQ ID NO. 51) were designed according to the cynT gene sequence, and the cynT fragment was amplified by PCR.

[0118] The Ptrc promoter was designed in the antisense strand primer of the upstream homologous arm of the purR gene and the sense strand primer of the cynT gene; the Ptrc terminator was designed in the antisense strand primer of the cynT gene and the sense strand primer of the downstream homologous arm of the purR gene.

[0119] By the method of overlapping PCR, using the cynT fragment obtained by the above PCR and the upstream and downstream homologous arm fragments of purR as templates, and using UP-purR-S (SEQ ID NO.46) and DN-purR-A (SEQ ID NO.49) as primers, the integration fragment of cynT was obtained: upstream homologous arm - Ptrc - cynT - downstream homologous arm.

[0120] The DNA fragment containing the target sequence of the purR gene was prepared by annealing the primers gRNA-purR-S (SEQ ID NO.52) and gRNA-purR-A (SEQ ID NO.53), and the recombinant plasmid pGRB-purR was obtained after recombination with the linearized pGRB vector.

[0121] The integration fragment of cynT (upstream homologous arm - Ptrc - cynT - downstream homologous arm) and the recombinant plasmid pGRB-purR were electrotransformed into the competent cells of E. coli Ud1-3 containing the pREDCas9 plasmid. After screening positive strains, the two plasmids pGRB-purR and pREDCas9 for gene editing were eliminated to obtain the strain E. coli Ud2.

[0122] The agarose gel electrophoresis verification diagrams of the cynT integration fragment and the positive strain are as Figure 4 shown: Lane M is DNA Marker; Lane 1 is the upstream homologous arm; Lane 2 is the cynT gene fragment; Lane 3 is the downstream homologous arm; Lane 4 is the integration fragment of the cynT gene; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain.

[0123] 3. Integrate the carbamoyl phosphate synthetase gene pyrAA / AB from Bacillus subtilis at the gene locus lacI E951* :

[0124] Using the E. coli W3110 genome as a template, the upstream homologous arm primers UP-lacI-S (SEQ ID NO.54) and UP-lacI-Ptrc-A (SEQ ID NO.55) were designed according to the upstream and downstream gene sequences of the gene lacI; the downstream homologous arm primers DN-Ptrc-lacI-S (SEQ ID NO.56) and DN-lacI-A (SEQ ID NO.57) were designed, and their upstream and downstream homologous arm fragments were amplified by PCR.

[0125] According to the nucleotide sequence of pyrAA / AB carrying mutation sites synthesized by Tianjin Anshengda Biotechnology Co., Ltd. E951* the primer Ptrc-pyrAA / AB was designed E951*-S (SEQ ID NO.58) and pyrAA / AB E951* -Ptrc-A (SEQ ID NO.59); using pyrAA / AB E951* as a template, pyrAA / AB was obtained by PCR E951* fragment.

[0126] The Ptrc promoter was designed in the antisense strand primer of the upstream homologous arm of the lacI gene and the sense strand primer of the pyrAA / AB E951* gene; the Ptrc terminator was designed in the antisense strand primer of the pyrAA / AB E951* gene and the sense strand primer of the downstream homologous arm of the lacI gene locus.

[0127] By the method of overlap PCR, using the pyrAA / AB E951* fragment obtained by the above PCR and the upstream and downstream homologous arm fragments of the lacI gene as templates, and using UP-lacI-S (SEQ ID NO.54) and DN-lacI-A (SEQ ID NO.57) as primers, the integration fragment of the pyrAA / AB E951* gene was obtained: upstream homologous arm - Ptrc - pyrAA / AB E951* - downstream homologous arm.

[0128] A DNA fragment containing the target sequence of the lacI gene was prepared by annealing the primers gRNA-lacI-S (SEQ ID NO.60) and gRNA-lacI-A (SEQ ID NO.61), and after recombination with the linearized pGRB vector, the recombinant plasmid pGRB-lacI was obtained.

[0129] The integration fragment of the pyrAA / AB E951* gene (upstream homologous arm - Ptrc - pyrAA / AB E951* - downstream homologous arm) and the recombinant plasmid pGRB-lacI were electrotransformed into the competent cells of E. coli Ud2 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids pGRB-lacI and pREDCas9 for gene editing were eliminated, and the strain E. coli Ud3 was obtained.

[0130] pyrAA / AB E951* The agarose gel electrophoresis verification diagram of the integration fragment of the gene and the positive strain is as Figure 5 shown: Lane M is the DNA Marker; Lane 1 is the upstream homologous arm; Lane 2 is the pyrAA / AB E951* gene fragment; Lane 3 is the downstream homologous arm; Lane 4 is the pyrAA / AB E951*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.

[0131] 4. Integrate the aspartate carbamoyltransferase gene pyrB from Bacillus subtilis at the gene locus pepA:

[0132] Using the E. coli W3110 genome as a template, upstream homologous arm primers UP-pepA-S (SEQ ID NO.62) and UP-pepA-Ptrc-A (SEQ ID NO.63) were designed according to the upstream and downstream gene sequences of the pepA gene; downstream homologous arm primers DN-Ptrc-pepA-S (SEQ ID NO.64) and DN-pepA-A (SEQ ID NO.65) were designed, and their upstream and downstream homologous arm fragments were amplified by PCR.

[0133] Primers Ptrc-pyrB(bsu)-S (SEQ ID NO.66) and pyrB(bsu)-Ptrc-A (SEQ ID NO.67) were designed according to the pyrB nucleotide sequence, and using the B.subtilis A260 genome as a template, the pyrB fragment was amplified by PCR.

[0134] The Ptrc promoter was designed in the antisense strand primer of the upstream homologous arm of the pepA gene and the sense strand primer of the pyrB gene; the Ptrc terminator was designed in the antisense strand primer of the pyrB gene and the sense strand primer of the downstream homologous arm of the pepA gene locus.

[0135] By the method of overlap PCR, using the upstream and downstream homologous arm fragments of the pepA gene and the pyrB fragment obtained by the above PCR as templates, and using UP-pepA-S (SEQ ID NO.62) and DN-pepA-A (SEQ ID NO.65) as primers, the integrated fragment of the pyrB gene was obtained: upstream homologous arm - Ptrc - pyrB - downstream homologous arm.

[0136] A DNA fragment containing the target sequence of the pepA gene was prepared by annealing primers gRNA-pepA-S (SEQ ID NO.68) and gRNA-pepA-A (SEQ ID NO.69), and after recombination with the linearized pGRB vector, the recombinant plasmid pGRB-pepA was obtained.

[0137] The integration fragment of the pyrB gene (upstream homology arm - Ptrc - pyrB - downstream homology arm) and the recombinant plasmid pGRB - pepA were electrotransformed into the competent cells of E. coli Ud3 containing the pREDCas9 plasmid. After screening for positive strains, the two plasmids pGRB - pepA and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud4.

[0138] The agarose gel electrophoresis verification diagrams of the integration fragment of pyrB and the positive strain are as Figure 6 shown: Lane M is the DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the pyrB gene fragment; Lane 3 is the downstream homology arm; Lane 4 is the integration 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.

[0139] 5. Integrate the orotate phosphoribosyltransferase gene pyrE from Bacillus subtilis at the gene locus argR:

[0140] Using the E. coli W3110 genome as a template, upstream homology arm primers UP - argR - S (SEQ ID NO.70) and UP - argR - Ptrc - A (SEQ ID NO.71) were designed according to the gene sequences upstream and downstream of the argR gene; downstream homology arm primers DN - Ptrc - argR - S (SEQ ID NO.72) and DN - argR - A (SEQ ID NO.73) were designed, and their upstream and downstream homology arm fragments were amplified by PCR.

[0141] Primers Ptrc - pyrE(bsu) - S (SEQ ID NO.74) and pyrE(bsu) - Ptrc - A (SEQ ID NO.75) were designed according to the pyrB nucleotide sequence, and the pyrE fragment was obtained by PCR using the B. subtilis A260 genome as a template.

[0142] The Ptrc promoter was designed in the antisense strand primer of the upstream homology arm of the argR gene and the sense strand primer of the pyrE gene; the Ptrc terminator was designed in the antisense strand primer of the pyrE gene and the sense strand primer of the downstream homology arm of the argR gene locus.

[0143] By the method of overlap PCR, using the upstream and downstream homology arm fragments of the gene argR and the pyrE fragment obtained by the above PCR as templates, and using UP - argR - S (SEQ ID NO.70) and DN - argR - A (SEQ ID NO.73) as primers, the integration fragment of the pyrE gene: upstream homology arm - Ptrc - pyrE - downstream homology arm was obtained.

[0144] The DNA fragment containing the target sequence of the argR gene was prepared by annealing the primers gRNA-argR-S (SEQ ID NO.76) and gRNA-argR-A (SEQ ID NO.77), and the recombinant plasmid pGRB-argR was obtained after recombination with the linearized pGRB vector.

[0145] The integration fragment of the pyrE gene (upstream homologous arm - Ptrc - pyrE - downstream homologous arm) and the recombinant plasmid pGRB-argR were electrotransformed into the competent cells of E. coli Ud4 containing the pREDCas9 plasmid. After screening positive strains, the two plasmids pGRB-argR and pREDCas9 for gene editing were eliminated to obtain the strain E. coli Ud5.

[0146] The agarose gel electrophoresis verification diagrams of the integration fragment of pyrE and the positive strain are shown as Figure 7 follows: Lane M is DNA Marker; Lane 1 is the upstream homologous arm; Lane 2 is the pyrE gene fragment; Lane 3 is the downstream homologous arm; Lane 4 is the integration 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.

[0147] 6. Integrate the phosphoribosyl pyrophosphate synthase gene prs from Bacillus subtilis at the gene loci thrA, argF, and udk respectively D128A :

[0148] The upstream homologous arm primers UP-thrA-S (SEQ ID NO.78) and UP-thrA-Ptrc-A (SEQ ID NO.79); UP-argF-S (SEQ ID NO.80) and UP-argF-Ptrc-A (SEQ ID NO.81); UP-udk-S (SEQ ID NO.82) and UP-udk-Ptrc-A (SEQ ID NO.83); the downstream homologous arm primers DN-Ptrc-thrA-S (SEQ ID NO.84) and DN-thrA-A (SEQ ID NO.85); DN-Ptrc-argF-S (SEQ ID NO.86) and DN-argF-A (SEQ ID NO.87); DN-Ptrc-udk-S (SEQ ID NO.88) and DN-udk-A (SEQ ID NO.89) were designed respectively according to the upstream and downstream sequences of the genes thrA, argF, and udk. Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the genes thrA, argF, and udk were amplified by PCR technology respectively.

[0149] According to Tianjin Anshengda Biotechnology Company, the target gene prs carrying mutation sites was synthesized. D128A Primers Ptrc-prs D128A -S (SEQ ID NO.90) and prs D128A -Ptrc-A (SEQ ID NO.91) were designed based on the gene sequence of the target gene prs D128A As a template, the prs D128A fragment was obtained by PCR amplification.

[0150] The Ptrc promoter was respectively designed in the antisense strand primers of the upstream homologous arms of genes thrA, argF, and udk and the sense strand primer of the prs D128A gene; the Ptrc terminator was respectively designed in the antisense strand primer of the prs D128A gene and the sense strand primers of the downstream homologous arms of the purR, argF, and udk gene loci.

[0151] By the method of recombinant PCR, using the upstream and downstream homologous arms of the gene thrA and the prs D128A fragment obtained by the above PCR as templates, and using UP-thrA-S (SEQ ID NO.78) and DN-thrA-A (SEQ ID NO.85) as primers, the integration fragment of the prs D128A gene was obtained: thrA upstream homologous arm - Ptrc-prs D128A -thrA downstream homologous arm.

[0152] By the method of recombinant PCR, using the upstream and downstream homologous arms of the gene argF and the prs D128A fragment obtained by the above PCR as templates, and using UP-argF-S (SEQ ID NO.80) and DN-argF-A (SEQ ID NO.87) as primers, the integration fragment of the prs D128A gene was obtained: argF upstream homologous arm - Ptrc-prs D128A -argF downstream homologous arm.

[0153] By the method of recombinant PCR, using the upstream and downstream homologous arms of the gene udk and the prs D128A fragment obtained by the above PCR as templates, and using UP-udk-S (SEQ ID NO.82) and DN-udk-A (SEQ ID NO.89) as primers, the integration fragment of the prs D128A gene was obtained: udk upstream homologous arm - Ptrc-prs D128A -udk downstream homologous arm.

[0154] The DNA fragments containing the target sequences of thrA, argF, and udk genes were respectively prepared by annealing the primers gRNA-thrA-S (SEQ ID NO.92) and gRNA-thrA-A (SEQ ID NO.93); gRNA-argF-S (SEQ ID NO.94) and gRNA-argF-A (SEQ ID NO.95); gRNA-udk-S (SEQ ID NO.96) and gRNA-udk-A (SEQ ID NO.97). After recombination with the linearized pGRB vector respectively, the recombinant plasmids pGRB-thrA, pGRB-argF, and pGRB-udk were obtained respectively.

[0155] The integration fragment of the prs D128A gene, the upstream homologous arm of thrA - Ptrc - prs D128A - the downstream homologous arm of thrA and pGRB-thrA were electrotransformed into the competent cells of E. coli Ud5 containing the pREDCas9 plasmid. After resuscitation and culture, single colonies were obtained. Positive strains were obtained by PCR colony verification, and then the two plasmids pGRB-thrA and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud6.

[0156] The integration fragment of the prs D128A gene, the upstream homologous arm of argF - Ptrc - prs D128A - the downstream homologous arm of argF and pGRB-argF were electrotransformed into the competent cells of E. coli Ud6 containing the pREDCas9 plasmid. After resuscitation and culture, single colonies were obtained. Positive strains were obtained by PCR colony verification, and then the two plasmids pGRB-argF and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud7.

[0157] The integration fragment of the prs D128A gene, the upstream homologous arm of udk - Ptrc - prs D128A - the downstream homologous arm of udk and pGRB-udk were electrotransformed into the competent cells of E. coli Ud7 containing the pREDCas9 plasmid. After resuscitation and culture, single colonies were obtained. Positive strains were obtained by PCR colony verification, and then the two plasmids pGRB-udk and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud8. The verification diagram of agarose gel electrophoresis is as Figure 8 shown: The M lane is the DNA Marker; the 1 lane is the upstream homologous arm; the 2 lane is the prs D128A gene fragment; the 3 lane is the downstream homologous arm; the 4 lane is the prs D128AIntegrated fragments of the gene; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain.

[0158] 7. Integrate the uridine kinase gene pyrH from Bacillus subtilis at the gene loci ushA, surE, yjjG, and yrfG respectively D90A :

[0159] Design upstream homologous arm primers UP-ushA-S (SEQ ID NO.98) and UP-ushA-Ptrc-A (SEQ ID NO.99); UP-surE-S (SEQ ID NO.100) and UP-surE-Ptrc-A (SEQ ID NO.101); UP-yjjG-S (SEQ ID NO.102) and UP-yjjG-Ptrc-A (SEQ ID NO.103); UP-yrfG-S (SEQ ID NO.104) and UP-yrfG-Ptrc-A (SEQ ID NO.105); downstream homologous arm primers DN-Ptrc-ushA-S (SEQ ID NO.106) and DN-ushA-A (SEQ ID NO.107); DN-Ptrc-surE-S (SEQ ID NO.108) and DN-surE-A (SEQ ID NO.109); DN-Ptrc-yjjG-S (SEQ ID NO.110) and DN-yjjG-A (SEQ ID NO.111); DN-Ptrc-yrfG-S (SEQ ID NO.112) and DN-yrfG-A (SEQ ID NO.113) according to the upstream and downstream sequences of the gene. Use the E. coli W3110 genome as a template and amplify the upstream and downstream homologous arms of the genes ushA, surE, yjjG, and yrfG respectively by PCR technology.

[0160] Synthesize the target gene pyrH with mutation sites according to Tianjin Anshengda Biotechnology Co., Ltd. D90A Design primers Ptrc-pyrH D90A -S (SEQ ID NO.114), pyrH D90A -Ptrc-A (SEQ ID NO.115). Use the target gene pyrH D90A as a template and obtain the pyrH D90A fragment by PCR.

[0161] Design antisense strand primers of the Ptrc promoter at the upstream homologous arms of the genes ushA, surE, yjjG, and yrfG and pyrH D90Ain the sense strand primer of the gene; the Ptrc terminator was designed in pyrH D90A in the antisense strand primer of the gene and the sense strand primers of the homologous arms downstream of the ushA, surE, yjjG, and yrfG gene loci.

[0162] By recombinant PCR, using the upstream and downstream homologous arms of the ushA gene and the pyrH D90A fragment obtained by the above PCR as templates, and UP-ushA-S (SEQ ID NO.98) and DN-ushA-A (SEQ ID NO.107) as primers, the integration fragment of the pyrH D90A gene was obtained: ushA upstream homologous arm - Ptrc - pyrH D90A - ushA downstream homologous arm.

[0163] By recombinant PCR, using the upstream and downstream homologous arms of the surE gene and the pyrH D90A fragment obtained by the above PCR as templates, and UP-surE-S (SEQ ID NO.100) and DN-surE-A (SEQ ID NO.109) as primers, the integration fragment of the pyrH D90A gene was obtained: surE upstream homologous arm - Ptrc - pyrH D90A - surE downstream homologous arm.

[0164] By recombinant PCR, using the upstream and downstream homologous arms of the yjjG gene and the pyrH D90A fragment obtained by the above PCR as templates, and UP-yjjG-S (SEQ ID NO.102) and DN-yjjG-A (SEQ ID NO.111) as primers, the integration fragment of the pyrH D90A gene was obtained: yjjG upstream homologous arm - Ptrc - pyrH D90A - yjjG downstream homologous arm.

[0165] By recombinant PCR, using the upstream and downstream homologous arms of the yrfG gene and the pyrH D90A fragment obtained by the above PCR as templates, and UP-yrfG-S (SEQ ID NO.104) and DN-yrfG-A (SEQ ID NO.113) as primers, the integration fragment of the pyrH D90A gene was obtained: yrfG upstream homologous arm - Ptrc - pyrH D90A - yrfG downstream homologous arm.

[0166] Separate DNA fragments containing the target sequences of the ushA, surE, yjjG, and yrfG genes were prepared by annealing with primers gRNA-ushA-S (SEQ ID NO.116) and gRNA-ushA-A (SEQ ID NO.117); gRNA-surE-S (SEQ ID NO.118) and gRNA-surE-A (SEQ ID NO.119); gRNA-yjjG-S (SEQ ID NO.120) and gRNA-yjjG-A (SEQ ID NO.121); gRNA-yrfG-S (SEQ ID NO.122) and gRNA-yrfG-A (SEQ ID NO.123), respectively. After recombination with the linearized pGRB vector, recombinant plasmids pGRB-ushA, pGRB-surE, pGRB-yjjG, and pGRB-yrfG were obtained respectively.

[0167] The integration fragment, upstream homologous arm of ushA - Ptrc - pyrH D90A - downstream homologous arm of ushA and pGRB-ushA were electrotransformed into competent E. coli Ud8 cells containing the pREDCas9 plasmid. After recovery and culture, single colonies were obtained. Positive strains were obtained by PCR colony verification, and then the two plasmids pGRB-ushA and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud9.

[0168] The integration fragment, upstream homologous arm of surE - Ptrc - pyrH D90A - downstream homologous arm of surE and pGRB-surE were electrotransformed into competent E. coli Ud9 cells containing the pREDCas9 plasmid. After recovery and culture, single colonies were obtained. Positive strains were obtained by PCR colony verification, and then the two plasmids pGRB-surE and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud10.

[0169] The integration fragment, upstream homologous arm of yjjG - Ptrc - pyrH D90A - downstream homologous arm of yjjG and pGRB-yjjG were electrotransformed into competent E. coli Ud10 cells containing the pREDCas9 plasmid. After recovery and culture, single colonies were obtained. Positive strains were obtained by PCR colony verification, and then the two plasmids pGRB-yjjG and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud11.

[0170] The integration fragment, upstream homologous arm of yrfG - Ptrc - pyrH D90AThe downstream homologous arm of -yrfG and pGRB-yrfG were electrotransformed into competent E. coli Ud11 cells containing the pREDCas9 plasmid. After recovery culture, single colonies were obtained. Positive strains were obtained through PCR colony verification, and then the two plasmids pGRB-yrfG and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud12. The verification diagram of agarose gel electrophoresis is as shown in Figure 9 follows: Lane M is the DNA Marker; Lane 1 is the upstream homologous arm; Lane 2 is the pyrH D90A gene fragment; Lane 3 is the downstream homologous arm; Lane 4 is the integrated fragment of the pyrH D90A gene; Lane 5 is the PCR fragment of E. coli W3110; Lane 6 is the PCR fragment of the positive strain.

[0171] 8. Knockout of the pyrimidine nucleotidase gene ygdH:

[0172] According to the upstream and downstream sequences of the ygdH gene, upstream homologous arm primers UP-ygdH-S (SEQ ID NO.124), UP-ygdH-A (SEQ ID NO.125) and downstream homologous arm primers DN-ygdH-S (SEQ ID NO.126), DN-ygdH-A (SEQ ID NO.127) were designed. Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms were amplified by PCR technology.

[0173] By the method of recombinant PCR, using the upstream and downstream homologous arms of the ygdH gene obtained by the above PCR as templates and UP-ygdH-S (SEQ ID NO.124) and DN-ygdH-A (SEQ ID NO.127) as primers, the knockout fragment of the ygdH gene was obtained: upstream homologous arm - downstream homologous arm.

[0174] A DNA fragment containing the target sequence of the ygdH gene was prepared by annealing primers gRNA-ygdH-S (SEQ ID NO.128) and gRNA-ygdH-A (SEQ ID NO.129), and after recombination with the linearized pGRB vector, the recombinant vector pGRB-ygdH was obtained.

[0175] The knockout fragment upstream homologous arm - downstream homologous arm of the ygdH gene and pGRB-ygdH were electrotransformed into competent E. coli Ud12 cells containing the pREDCas9 plasmid. After recovery culture, single colonies were obtained. Positive strains were obtained through PCR colony verification, and then the two plasmids pGRB-ygdH and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud13.

[0176] The verification diagram of agarose gel electrophoresis is as Figure 10 shown: Lane M is the DNA Marker; Lane 1 is the upstream homologous arm; Lane 2 is the downstream homologous arm; Lane 3 is the upstream homologous arm - downstream homologous arm of the knockout fragment of gene ygdH; Lane 4 is the PCR fragment of E. coli W3110; Lane 5 is the PCR fragment of the positive strain.

[0177] 9. Knockout of the global transcriptional regulatory gene ArcA:

[0178] According to the upstream and downstream sequences of the ArcA gene, the upstream homologous arm primers UP-ArcA-S (SEQ ID NO.130), UP-ArcA-A (SEQ ID NO.131) and the downstream homologous arm primers DN-ArcA-S (SEQ ID NO.132), DN-ArcA-A (SEQ ID NO.133) were designed. Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms were amplified by PCR technology.

[0179] By the method of recombinant PCR, using the upstream and downstream homologous arms of the ArcA gene obtained by the above PCR as templates, and UP-ArcA-S (SEQ ID NO.130) and DN-ArcA-A (SEQ ID NO.133) as primers, the knockout fragment of the ArcA gene: upstream homologous arm - downstream homologous arm was obtained.

[0180] A DNA fragment containing the target sequence of the ArcA gene was prepared by annealing the primers gRNA-ArcA-S (SEQ ID NO.134) and gRNA-ArcA-A (SEQ ID NO.135), and after recombination with the linearized pGRB vector, the recombinant plasmid pGRB-ArcA was obtained.

[0181] The upstream homologous arm - downstream homologous arm of the ArcA gene knockout fragment and pGRB-ArcA were electrotransformed into the competent cells of E. coli Ud13 containing the pREDCas9 plasmid. After resuscitation culture, single colonies were obtained. Positive strains were obtained by PCR colony verification, and then the two plasmids pGRB-ArcA and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud14. The verification diagram of agarose gel electrophoresis is as Figure 11 shown: Lane M is the DNA Marker; Lane 1 is the upstream homologous arm; Lane 2 is the downstream homologous arm; Lane 3 is the upstream homologous arm - downstream homologous arm of the ArcA gene knockout fragment; Lane 4 is the PCR fragment of E. coli W3110; Lane 5 is the PCR fragment of the positive strain. Example 2

[0182] Construction of Recombinant Strains Expressing Functional Proteins

[0183] Using the pET28A plasmid as the expression vector, the promoter Ptrc and the EUGT11 gene were integrated onto the plasmid using XbaⅠ and BamHⅠ enzymes to obtain the recombinant expression plasmid pET28A-Ptrc-EUGT11. Using the recombinant expression plasmid pET28A-Ptrc-EUGT11 as the expression vector, the promoter Ptrc and the SrUGT76G4 gene were integrated onto the plasmid using SacⅠ and SalⅠ enzymes to obtain the recombinant expression plasmid pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4. Using the recombinant expression plasmid pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4 as the expression vector, the promoter Ptrc and the susy(nmu) gene, the promoter Ptrc and the susy(nta) gene, and the promoter Ptrc and the susy(nth) gene were integrated onto the plasmid using ApaⅠ and HapⅠ enzymes respectively to obtain the recombinant expression plasmids pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nmu), pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nta), and pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nth).

[0184] Among them, the susy(nmu) gene is the sucrose synthase gene susy derived from Nitrosomonas polymorphis, and its nucleotide sequence is shown in SEQ ID NO.23; the susy(nta) gene is the sucrose synthase gene susy derived from Nicotiana tabacum, and its nucleotide sequence is shown in SEQ ID NO.24; the susy(ath) gene is the sucrose synthase susy derived from Arabidopsis thaliana, and its nucleotide sequence is shown in SEQ ID NO.25; the EUGT11 gene is derived from Stevia rebaudiana, and its nucleotide sequence is shown in SEQ ID NO.26; the SrUGT76G4 gene is derived from Stevia rebaudiana, and its nucleotide sequence is shown in SEQ ID NO.27.

[0185] The recombinant expression plasmids pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nmu), pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nta) and pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nth) were respectively transformed into Escherichia coli BL21(DE3) cells by the method of CaCl2 chemical transformation. After resuscitation and culture, 100 μL / L of the bacterial solution was taken and spread on LB medium containing kanamycin resistance for culture. Single colonies after transformation were picked and verified by PCR to obtain the functional protein-expressing recombinant strain E. coli lep3 containing the recombinant expression plasmid pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nmu), the functional protein-expressing recombinant strain E. coli lep4 containing the recombinant expression plasmid pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nta), and the functional protein-expressing recombinant strain E. coli lep5 containing the recombinant expression plasmid pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4-Ptrc-susy(nth). Example 3

[0186] Fermentation of the engineered strain for producing uridine diphosphate

[0187] Prepare the following media:

[0188] LB medium: Peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar powder 20 g / L.

[0189] Seed medium: Glucose 30 g / L, yeast extract 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 Each 0.0013 g / L, V H 0.001 g / L; LD-XP3020 antifoaming agent 100 μL / L, adjust the pH to 7.0 with NaOH solution.

[0190] Fermentation medium: initial glucose 20 g / L, sodium citrate anhydrous 2 g / L, peptone 5 g / L, L-methionine 0.3 g / L, KH2PO4 6 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 0.02 g / L, glutamic acid 2 g / L, yeast powder 4 g / L, MnSO4 0.01 g / L, V B1 、V B3 、V B5 、V B12 each 0.001 g / L, V H 0.0005 g / L; LD-XP3020 antifoaming agent 100 μL / L, adjust the pH to 7.0 with NaOH solution.

[0191] Take the bacterial liquid of the uridine diphosphate genetic engineering strain E. coli Ud14 in Example 1, evenly coat it on the LB medium, culture it at 37 °C for 9 h, pick a single colony into physiological saline, and inoculate it into 50 mL of the seed medium. During the culture process, maintain the pH value at 7 by adding ammonia water, and culture it at 37 °C for 6.5 h to obtain the seed liquid.

[0192] Inoculate the seed liquid into the fermentation medium for fermentation culture according to an inoculation amount of 2% volume ratio. After fermentation culture for 11 h under the conditions of a temperature of 37 °C and a pH value of 7, glucose is supplemented by a feedback pulse feeding strategy, that is, the pulse rate of feeding is set to 1 / 15 s on the feeding pump. When the dissolved oxygen value is higher than 35%, the feeding pump automatically starts to supplement the 800 g / L glucose solution. When the dissolved oxygen value is lower than 25%, the feeding pump automatically closes, and continue fermentation culture for 36 h.

[0193] After the fermentation is completed, take 10 mL of the fermentation bacterial liquid, break the cells with an ultrasonic crusher, centrifuge at 13500 rpm for 10 min, discard the precipitate, and obtain the supernatant containing UDP (uridine diphosphate). The yield of UDP in the supernatant is measured by HPLC to be 38 g / L.

[0194] Centrifuge the remaining fermentation broth at 5000 rpm for 10 min, discard the supernatant, collect the bacterial cells, and obtain the engineering strain E. coli Ud15 for producing uridine diphosphate after fermentation. Example 4

[0195] Fermentation of the functional protein expression recombinant strain

[0196] Prepare the following medium:

[0197] LB medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar powder 20 g / L.

[0198] Seed culture medium: Glucose 30 g / L, yeast powder 5 g / L, peptone 3 g / L, KH2PO4 1.15 g / L, MgSO4 0.6 g / L, kanamycin 50 μg / ml, LD-XP3020 antifoaming agent 100 μL / L.

[0199] Fermentation culture medium: Initial glucose 20 g / L, peptone 12 g / L, yeast powder 5 g / L, KH2PO4 6 g / L, MgSO4·7H2O 2 g / L, K2HPO4 6.3 g / L, kanamycin 50 μg / ml, LD-XP3020 antifoaming agent 100 μL / L.

[0200] Feed culture medium: Glucose 600 g / L, MgSO4·7H2O 2 g / L, yeast powder 75 g / L.

[0201] Take the bacterial liquid of the functional protein-expressing recombinant strain E. coli lep3 in Example 2, evenly coat it on the LB medium containing 50 μg / ml kanamycin, culture it at 37 °C for 8 h, pick a single colony into physiological saline, and inoculate it into 50 mL of the seed culture medium containing 50 μg / ml kanamycin, culture it at 37 °C for 6.5 h, and maintain the pH value at 7 by adding ammonia water during the culture process to obtain the seed liquid.

[0202] Inoculate the seed liquid into the fermentation culture medium containing 50 μg / ml kanamycin for fermentation culture at an inoculation amount of 2% by volume. Control the pH value at 7 by adding ammonia water during the culture process, culture it at 37 °C. When the OD value of the strain reaches 10, control the fermentation temperature at 25 °C. When the dissolved oxygen soars, start to feed the feed culture medium to control the residual sugar (glucose) in the fermentation broth within 1 g / L, and continue fermentation culture for 26 h.

[0203] After fermentation, centrifuge the fermentation broth at 5000 rpm for 10 min, discard the supernatant, collect the bacterial cells, and obtain the fermented functional protein-expressing recombinant strain E. coli lep3-1.

[0204] Ferment and culture the functional protein-expressing recombinant strains E. coli lep4 and E. coli lep5 in Example 2 respectively according to the above method to obtain the fermented functional protein-expressing recombinant strains E. coli lep4-1 and E. coli lep5-1 respectively. Example 5

[0205] Whole-cell catalyzed synthesis of Rebaudioside M

[0206] 1. Prepare the reaction system with E. coli Ud15 and E. coli lep3-1 as follows: potassium dihydrogen phosphate 2.0 g / L, dipotassium hydrogen phosphate 25 g / L, ammonium sulfate 3.0 g / L, magnesium chloride 2.0 g / L, glycerol 8 g / L, CTAB 2.0 g / L, E. coli Ud15 33 g / L, E. coli lep3-1 115 g / L, sucrose 150 g / L, and rebaudioside A 65 g / L.

[0207] React the above reaction system at a rotation speed of 250 rpm, a temperature of 37 °C, and a pH value of 7 for 20 h. After the reaction, dilute the obtained reaction solution 200 times, shake well, centrifuge at 13,000 r / min for 10 min at room temperature, and collect the supernatant containing rebaudioside M.

[0208] Filter the supernatant through an organic membrane with a pore size of 0.22 μm, and determine the content of rebaudioside M in the filtrate by HPLC. The HPLC uses a Waters C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase is acetonitrile: water (pH 7.0) = 30:70, the flow rate is 1 mL / min, and the column temperature is 40 °C. Detection is carried out using an ultraviolet detector. At a wavelength of 210 nm and an injection volume of 10 μL, the content of rebaudioside M is measured to be 56.55 g / L, and the conversion rate is 87%.

[0209] 2. Prepare the reaction system with E. coli Ud15 and E. coli lep4-1 as follows: potassium dihydrogen phosphate 2.5 g / L, dipotassium hydrogen phosphate 35 g / L, ammonium sulfate 3.5 g / L, magnesium chloride 2.5 g / L, glycerol 12 g / L, CTAB 2.5 g / L, E. coli Ud15 40 g / L, E. coli lep4-1 130 g / L, sucrose 200 g / L, and rebaudioside A 80 g / L.

[0210] React the above reaction system at a rotation speed of 250 rpm, a temperature of 37 °C, and a pH value of 7 for 20 h. After the reaction, dilute the obtained reaction solution 200 times, shake well, centrifuge at 13,000 r / min for 10 min at room temperature, and collect the supernatant.

[0211] The supernatant was filtered through an organic membrane with a pore size of 0.22 μm, and the content of rebaudioside M in the filtrate was determined by HPLC. The HPLC was performed using a Waters C18 column (4.6 mm × 250 mm, 5 μm), with the mobile phase being acetonitrile: water (pH 7.0) = 30:70, a flow rate of 1 mL / min, and a column temperature of 40°C. Detection was carried out using a UV detector at a wavelength of 210 nm and an injection volume of 10 μL. The content of rebaudioside M was measured to be 72 g / L, and the conversion rate was 90%.

[0212] 3. A reaction system was prepared using E. coli Ud15 and E. coli lep5-1 as follows: potassium dihydrogen phosphate 2.2 g / L, dipotassium hydrogen phosphate 30 g / L, ammonium sulfate 3.1 g / L, magnesium chloride 2.1 g / L, glycerol 10 g / L, CTAB 2.2 g / L, E. coli Ud15 25 g / L, E. coli lep5-1 100 g / L, sucrose 100 g / L, and rebaudioside A 50 g / L.

[0213] The above reaction system was reacted under the conditions of a rotation speed of 250 rpm, a temperature of 37°C, and a pH value of 7 for 20 h. After the reaction ended, the resulting reaction solution was diluted 200 times, shaken well, centrifuged at 13,000 r / min for 10 min at room temperature, and the supernatant was collected.

[0214] The supernatant was filtered through a 0.22-μm organic membrane, and the content of rebaudioside M in the filtrate was determined by HPLC. The HPLC was performed using a Waters C18 column (4.6 mm × 250 mm, 5 μm), with the mobile phase being acetonitrile: water (pH 7.0) = 30:70, a flow rate of 1 mL / min, and a column temperature of 40°C. Detection was carried out using a UV detector at a wavelength of 210 nm and an injection volume of 10 μL. The content of rebaudioside M was measured to be 48.5 g / L, and the conversion rate was 97%. Comparative Example 1

[0215] An engineering strain for producing uridine diphosphate was constructed according to the method of Example 1, with the only difference being that pyrBCAKDFE was not integrated at the pseudogene locus yghX of E. coli W3110. The integration fragment of cynT (upstream homologous arm - Ptrc - cynT - downstream homologous arm) and the recombinant plasmid pGRB - purR were electrotransformed into the competent cells of E. coli W3110 containing the pREDCas9 plasmid. After screening positive strains, the two plasmids used for gene editing were eliminated to obtain strain E. coli Ud2′, and subsequent gene editing was performed on strain E. coli Udg2′. Comparative Example 2

[0216] Construct an engineered strain for producing uridine diphosphate according to the method of Example 1, with the only difference being that pyrAA / AB is not integrated at the gene locus lacI. E951* , Transform the integration fragment of the pyrB gene (upstream homologous arm - Ptrc - pyrB - downstream homologous arm) and the recombinant plasmid pGRB - pepA into the competent cells of E. coli Ud2 containing the pREDCas9 plasmid. After screening for positive strains, eliminate the two plasmids used for gene editing to obtain the strain E. coli Ud4′, and perform subsequent gene editing on the strain E. coli Udg4′. Comparative Example 3

[0217] Construct an engineered strain for producing uridine diphosphate according to the method of Example 1, with the only difference being that pyrB is not integrated at the gene locus pepA. Transform the integration fragment of the pyrE gene (upstream homologous arm - Ptrc - pyrE - downstream homologous arm) and the recombinant plasmid pGRB - argR into the competent cells of E. coli Ud3 containing the pREDCas9 plasmid. After screening for positive strains, eliminate the two plasmids used for gene editing to obtain the strain E. coli Ud5′, and perform subsequent gene editing on the strain E. coli Udg5′. Comparative Example 4

[0218] Construct an engineered strain for producing uridine diphosphate according to the method of Example 1, with the only difference being that pyrE is not integrated at the gene locus argR. D128A The integration fragment of the prs gene, the upstream homologous arm of thrA - Ptrc - prs D128A - the downstream homologous arm of thrA and pGRB - thrA were electrotransformed into the competent cells of E. coli Ud4 containing the pREDCas9 plasmid. After resuscitation culture to obtain single colonies, positive strains were obtained by PCR colony verification. Then, eliminate the two plasmids used for gene editing to obtain the strain E. coli Ud6′, and perform subsequent gene editing on the strain E. coli Udg6′. Comparative Example 5

[0219] Construct an engineered strain for producing uridine diphosphate according to the method of Example 1, with the only difference being that the pyrimidine nucleotidase gene ygdH is not knocked out. Transform the knockout fragment of the ArcA gene, the upstream homologous arm - downstream homologous arm and pGRB - ArcA into the competent cells of E. coli Ud12 containing the pREDCas9 plasmid. After resuscitation culture to obtain single colonies, positive strains were obtained by PCR colony verification. Then, eliminate the two plasmids used for gene editing to obtain the strain E. coli Ud14′. Comparative Example 6

[0220] Construct an engineered strain for producing uridine diphosphate according to the method of Example 1, except that the global transcriptional regulatory gene ArcA is not knocked out.

[0221] Test Example

[0222] Ferment and culture the engineered strains for producing uridine diphosphate constructed in Example 1 and Comparative Examples 1 - 6 according to the method of Example 3, and detect the yield of UDP. The results are shown in Table 1.

[0223] Table 1

[0224]

[0225] Ferment and culture the engineered strains for producing uridine diphosphate constructed in Example 1 and Comparative Examples 1 - 6 according to the method of Example 3 respectively, to obtain the strains E. coli Ud15, E. coli Ud15 - 1, E. coli Ud15 - 2, E. coli Ud15 - 3, E. coli Ud15 - 4, E. coli Ud15 - 5, E. coli Ud15 - 6 for producing uridine diphosphate after fermentation respectively. Then synthesize rebaudioside M according to the method of Example 5 respectively, and detect the yield and conversion rate of rebaudioside M (RM). The results are shown in Table 2 and Table 3.

[0226] Table 2

[0227]

[0228] Table 3

[0229]

[0230] From the results in Table 1, Table 2, and Table 3 above, it can be seen that in Comparative Example 1, the pyrBCAKDFE genes were not integrated, in Comparative Example 2, the pyrAA / AB E951* genes were not integrated at the gene locus lacI, in Comparative Example 3, the pyrB gene was not integrated at the gene locus pepA, in Comparative Example 4, the pyrE gene was not integrated at the gene locus argR. After that, the yield of UDP, the yield and conversion rate of RM all decreased, indicating that integrating the above - mentioned specific genes at the above - mentioned specific gene loci can increase the yield of UDP and thus increase the yield and conversion rate of RM. In Comparative Example 5, after the pyrimidine nucleotidase gene ygdH was not knocked out, the yield of UDP, the yield and conversion rate of RM all decreased, indicating that not expressing the pyrimidine nucleotidase gene ygdH can increase the yield of UDP and thus increase the yield and conversion rate of RM. In Comparative Example 6, after the global transcriptional regulatory gene ArcA was not knocked out, the yield of UDP, the yield and conversion rate of RM all decreased, indicating that not expressing the global transcriptional regulatory gene ArcA can increase the yield of UDP and thus increase the yield and conversion rate of RM.

[0231] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An engineered strain for producing uridine diphosphate, characterized in that: The engineered strain producing uridine diphosphate has the following genes integrated into its genome: Pyrimidine nucleoside operon gene pyrBCAKDFE, carbonic anhydrase gene cynT, carbamoyl phosphate synthetase gene pyrAA / AB E951* , aspartate transaminase gene pyrB, orotate phosphoribosyltransferase gene pyrE, phosphoribosyl pyrophosphate synthase gene prs D128A 、Uridylate kinase gene pyrH D93A ; The gene sequence of the pyrBCAKDFE is shown in SEQ ID NO. 1; The gene sequence of cynT is shown in SEQ ID NO. 2; The pyrAA / AB E951* The gene sequence is shown in SEQ ID NO. 3; The gene sequence of pyrB is shown in SEQ ID NO. 4; The gene sequence of pyrE is shown in SEQ ID NO.5; The prs D128A The gene sequence is shown in SEQ ID NO.6; The pyrH D93A The gene sequence is shown in SEQ ID NO.7; The engineered strain producing uridine diphosphate has the following genes missing from its genome: The lactose operon regulatory gene lacI, the repressor protein genes purR, pepA and argR, the homoserine dehydrogenase gene thrA, the ornithine carbamoyltransferase gene argF, the uridine diphosphate kinase gene udk, the nucleotidase genes ushA, surE, yjjG and yrfG, the pyrimidine nucleotidase gene ygdH, and the global transcription regulatory gene arcA; The gene sequence of lacI is shown in SEQ ID NO.8; The gene sequence of purR is shown in SEQ ID NO.9; The gene sequence of pepA is shown in SEQ ID NO. 10; The gene sequence of argR is shown in SEQ ID NO. 11; The gene sequence of thrA is shown in SEQ ID NO. 12; The gene sequence of argF is shown in SEQ ID NO. 13; The gene sequence of udk is shown in SEQ ID NO. 14; The gene sequence of ushA is shown in SEQ ID NO. 15; The gene sequence of surE is shown in SEQ ID NO. 16; The gene sequence of yjjG is shown in SEQ ID NO. 17; The gene sequence of yrfG is shown in SEQ ID NO. 18; The gene sequence of ygdH is shown in SEQ ID NO. 19; The gene sequence of arcA is shown in SEQ ID NO. 20; The method for constructing the engineered strain comprises the following steps: The pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis was integrated into the pseudogene site yghX; The carbonic anhydrase gene cynT was integrated at the gene locus purR; Integration of the carbamoyl phosphate synthetase gene pyrAA / AB from Bacillus subtilis at the gene locus lacI E951* ; The aspartate transaminase gene pyrB from Bacillus subtilis was integrated into the gene locus pepA; The orotate phosphoribosyltransferase gene pyrE from Bacillus subtilis was integrated into the gene locus argR; The phosphoribosyl-pyrophosphate synthase gene prs from Bacillus subtilis was integrated into the gene loci thrA, argF and udk respectively. D128A ; The uridine kinase gene pyrH from Bacillus subtilis was integrated into the gene loci ushA, surE, yjjG and yrfG respectively. D93A ; Knockout of the pyrimidine nucleotidase gene ygdH; Knockout of the global transcriptional regulator gene arcA; The starting strain used in the construction of the engineered strain is E. coli W3110.

2. The engineered strain according to claim 1, characterized in that The integrated genes are all controlled by the strong promoter Ptrc, and the gene sequence of the strong promoter Ptrc is shown in SEQ ID NO.

21.

3. A method for synthesizing rebaudioside M, characterized in that: The following steps are involved: Fermenting the engineered strain for producing uridine diphosphate according to claim 1 to obtain the engineered strain for producing uridine diphosphate after fermentation; Fermenting and culturing the functional protein-expressing recombinant strain to obtain a fermented functional protein-expressing recombinant strain; The method for preparing the recombinant strain expressing the functional protein after fermentation comprises the following steps: The sucrose synthase gene susy, the UDP-glycosyltransferase gene EUGT11 and the UDP-glycosyltransferase gene SrUGT76G4 were connected to the plasmid to construct a recombinant plasmid; The nucleotide sequence of the sucrose synthase gene susy is shown in SEQ ID NO.23, SEQ ID NO.24 or SEQ ID NO.25; the nucleotide sequence of the uridine diphosphate glycosyltransferase gene EUGT11 is shown in SEQ ID NO.26; the nucleotide sequence of the uridine diphosphate glycosyltransferase gene SrUGT76G4 is shown in SEQ ID NO.27; The recombinant plasmid is transferred into a host strain to obtain a functional protein-expressing recombinant strain; Fermenting and culturing the functional protein-expressing recombinant strain to obtain a fermented functional protein-expressing recombinant strain; Wherein, the host strain is Escherichia coli; the Escherichia coli is Escherichia coli BL21 (DE3); The fermented engineering strain for producing UDP-glucose and the functional protein expression recombinant strain are configured into the following reaction system: 2.0-2.5 g / L potassium dihydrogen phosphate, 25-35 g / L potassium hydrogen phosphate, 3.0-3.5 g / L ammonium sulfate, 2.0-2.5 g / L magnesium chloride, 8-12 g / L glycerol, 2.0-2.5 g / L CTAB, 25-40 g / L of the fermented engineering strain for producing UDP-glucose, 100-130 g / L of the fermented functional protein expression recombinant strain, 100-200 g / L sucrose, and 50-80 g / L rebaudioside A; The reaction system is reacted for 18 to 24 hours at a pH value of 6.5 to 7.5 and a temperature of 36.5 to 37.

5. After the reaction is completed, the reaction solution is diluted 100 to 200 times and centrifuged to obtain a supernatant containing rebaudioside M.

4. The method according to claim 3, characterized in that The sucrose synthase gene susy is derived from polymorphic nitrosobacter, tobacco or Arabidopsis; The UDP-glycosyltransferase gene EUGT11 is derived from Stevia rebaudiana; The UDP-glycosyltransferase gene SrUGT76G4 is derived from Stevia rebaudiana.

5. The method according to claim 3, characterized in that: The functional protein expression recombinant strain is subjected to fermentation culture, comprising the following steps: Take the bacterial liquid of the recombinant strain expressing the functional protein, evenly spread it in the LB medium containing kanamycin, and culture it at 36.5-37.5℃ for 8-10h, pick a single colony and inoculate it into the seed medium containing kanamycin, and culture it at the temperature of 36.5-37.5℃ and pH of 6.5-7.5 for 5-8h to obtain the seed liquid; The seed liquid was inoculated into a fermentation medium containing kanamycin, and fermented at a temperature of 36.5-37.5°C and a pH of 6.5-7.

5. When the OD value reached 10-20, the temperature was lowered to 25-30°C, and the concentration of glucose was controlled within 1 g / L by feeding the medium, and the fermentation was continued for 24-30 hours. After the fermentation is finished, the obtained fermentation liquid is centrifuged, and the supernatant is discarded to obtain the fermented functional protein expression recombinant strain.

6. The method according to claim 3, characterized in that The engineered strain producing uridine diphosphate is fermented and cultured. The following steps are involved: Take the bacterial liquid of the engineering strain producing uridine diphosphate, apply it to LB medium, culture it at 36.5-37.5°C for 8-10 hours, pick a single bacterium and inoculate it into the seed medium, culture it at a temperature of 36.5-37.5°C and a pH of 6.5-7.5 for 5-8 hours to obtain the seed liquid; The seed liquid is inoculated into a fermentation medium, and after fermentation culture for 10 to 12 hours at a temperature of 36.5 to 37.5° C. and a pH value of 6.5 to 7.5, glucose is added by a feedback pulse feeding strategy, and the fermentation culture is continued for 36 to 40 hours; After the fermentation is finished, the obtained fermentation liquid is centrifuged, and the supernatant is discarded to obtain the fermented engineered strain for producing uridine diphosphate.

7. The method according to claim 6, characterized in that The feedback pulse feeding strategy is used to add glucose: 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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