Engineering strain for producing uridine diphosphate, construction method and method for synthesizing rebaudioside M

Abstract

The invention discloses an engineering strain for producing uridine diphosphate, a construction method and a method for synthesizing rebaudioside M, and belongs to the technical field of gene engineering. According to the engineering strain, genes such as a pyrimidine nucleoside operon gene pyrBCAKDFE, a carbonic anhydrase gene cynT and a carbamyl phosphate synthetase gene pyrAA / ABE951 * are integrated on a genome, and genes such as an aspartate carbamyl transaminase gene pyrB, an orotic acid phosphoribose transferase gene pyrE and a phosphoribose pyrophosphate synthase gene prsD128A are deleted. The engineering strain can efficiently synthesize uridine diphosphate, and can act together with a functional protein expression recombinant strain to catalytically synthesize rebaudioside M by taking sucrose and rebaudioside A as substrates.

Description

Technical Field

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

[0002] Rebaudioside M is a natural sweetener extracted from stevia, which is about 300-400 times sweeter than sucrose, and has no bitterness and aftertaste. It is widely used in various fields such as food, health products, and medicine. In the prior art, the methods for obtaining rebaudioside M include plant extraction, fermentation, enzyme conversion, etc. Among them, the plant extraction method mainly extracts from stevia through water or ethanol, but the content of natural rebaudioside M in stevia leaves is low, accounting for only 0.4-0.5% of the dry weight of stevia leaves, which limits industrial production. When producing rebaudioside M by fermentation, traditional engineering strain fermentation is used, for example, brewer's yeast fermentation method is used to synthesize rebaudioside M, brewer's yeast fermentation requires 144h, 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. One is to use crude enzyme solution containing sucrose synthase and glycosyltransferase with rebaudioside D, UDP, MgCl2, and phosphate buffer in a one-pot method to produce rebaudioside M. This method uses UDP and rebaudioside D as raw materials, and there is a problem of relatively high production cost. The other method uses stevioside and sucrose as raw materials and generates rebaudioside M through recombinant cell whole cell catalysis for 20 h. In this method, the yield of uridine diphosphate produced by the engineered 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 object of the present invention is to provide an engineered strain for producing uridine diphosphate, a construction method and a method for synthesizing rebaudioside M, so as to overcome the technical problems of low uridine diphosphate production and insufficient rebaudioside M production in the prior art.

[0005] In a first aspect, the present invention provides an engineered strain for producing uridine diphosphate, wherein the engineered strain for 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 .

[0006] The engineered strain producing uridine diphosphate lacks the following genes in 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.

[0007] Compared with the prior art, the engineered strain for producing uridine diphosphate in the present invention integrates and deletes specific genes in the genome, thereby regulating the metabolism of the engineered strain, enabling it to efficiently produce uridine diphosphate (UDP), thereby further increasing the yield of UDP, as follows: Strengthen the supply of the important precursor UMP: The genome of the engineered strain is integrated with the pyrimidine nucleoside operon gene PyrBCAKDFE, which can relieve the feedback inhibition of UMP, and strengthen the metabolism of glutamine to UMP. The genome of the engineered strain is integrated with the carbonic anhydrase gene cynT, which strengthens the reaction of CO2 and H2O to produce HCO3 - The carbamoyl phosphate synthetase gene pyrAA / AB was integrated into the genome of the engineered strain. E951* , where the glutamate 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 carbamoyl phosphate. The aspartate transaminase gene pyrB is integrated into the genome of the engineered strain, strengthening the metabolism of aspartate and carbamoyl phosphate to synthesize carbamoyl aspartate. 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 produce orotidine. The phosphoribosyl pyrophosphate synthase gene prs is integrated into the genome of the engineered strain D128A The aspartic acid at position 128 of this enzyme mutated to alanine, thereby relieving the feedback inhibition of ATP and ADP on phosphoribosyl pyrophosphate synthase and strengthening the metabolism of 5-phosphoribosyl and ATP to generate PRPP. The purine nucleotide repressor protein gene purR is missing from the genome of this engineered strain, thereby relieving the repressor protein and purine repressor complex from inhibiting prs D128AFeedback inhibition of gene expression. The pepA gene and argR gene are missing from the genome of the engineered strain, thereby relieving the inhibition of the repressor protein encoded by the pepA gene and the repressor protein encoded by the argR gene on the carbamoyl phosphate encoding gene operon. The homoserine dehydrogenase gene thrA is missing from the genome of the engineered strain, blocking the metabolism of L-aspartate dehydrogenation to homoserine. The ornithine carbamoyltransferase gene argF is missing from the genome of the engineered strain, blocking the transfer of the carbamoyl group of carbamoyl phosphate to ornithine to produce citrulline.

[0008] Blocking the branched metabolism of the precursor UMP: UMP in Escherichia coli has two metabolic flows, uridine and uridine diphosphate. The engineered strain for producing uridine diphosphate in the present invention lacks the pyrimidine nucleotidase gene ygdH and the nucleotidase genes ushA, surE, yjjG and yrfG, which successfully weakens the metabolism of UMP to uridine and uracil, and then converts the metabolism of UMP to uridine to the metabolism of UMP to UDP.

[0009] Blocking the phosphorylation metabolism of UDP: UDP in Escherichia coli will affect the accumulation of UDP. UDP is phosphorylated to generate UDP by UDP. The UDP engineered bacteria in the present invention lack the UDP kinase gene udk, thereby blocking the phosphorylation reaction of the product UDP and promoting the accumulation of UDP.

[0010] Optimizing UDP anabolism: Based on the release of UDP feedback inhibition, the present invention integrates the uridylate kinase gene pyrH into the genome of the uridine diphosphate production engineering strain. D93A , rationally strengthening the metabolism of UMP to UDP.

[0011] Accelerate the TCA cycle and weaken the coupling flux of PRdnBP (pyrimidine ribonucleoside de novo biosynthesis pathway) and acetic acid metabolism: The genome of this engineered strain lacks the global transcriptional regulatory gene arcA, which weakens the coupling flux of PRdnBP and acetic acid metabolism, reduces acetic acid production, relieves the inhibition of arcA on TCA, accelerates the TCA cycle, and reduces carbon metabolic losses.

[0012] The genetically engineered strain of the invention lacks the lactose operon regulatory gene lacI, 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.

[0013] Furthermore, the gene sequence of pyrBCAKDFE is shown in SEQ ID NO.1.

[0014] The gene sequence of cynT is shown in SEQ ID NO.2.

[0015] pyrAA / AB E951* The gene sequence is shown in SEQ ID NO.3.

[0016] The gene sequence of pyrB is shown in SEQ ID NO.4.

[0017] The gene sequence of pyrE is shown in SEQ ID NO.5.

[0018] pR 128A The gene sequence is shown in SEQ ID NO.6.

[0019] pyr D93A The gene sequence is shown in SEQ ID NO.7.

[0020] The gene sequence of lacI is shown in SEQ ID NO.8.

[0021] The gene sequence of purR is shown in SEQ ID NO.9.

[0022] The gene sequence of pepA is shown in SEQ ID NO.10.

[0023] The gene sequence of argR is shown in SEQ ID NO.11.

[0024] The gene sequence of thrA is shown in SEQ ID NO.12.

[0025] The gene sequence of argF is shown in SEQ ID NO.13.

[0026] The gene sequence of udk is shown in SEQ ID NO.14.

[0027] The gene sequence of ushA is shown in SEQ ID NO.15.

[0028] The gene sequence of surE is shown in SEQ ID NO.16.

[0029] The gene sequence of yjjG is shown in SEQ ID NO.17.

[0030] The gene sequence of yrfG is shown in SEQ ID NO.18.

[0031] The gene sequence of ygdH is shown in SEQ ID NO.19.

[0032] The gene sequence of arcA is shown in SEQ ID NO. 20.

[0033] In a second aspect, the present invention provides a method for constructing an engineered strain, which is used to construct the above-mentioned engineered strain, comprising the following steps: The pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis was integrated into the pseudogene site yghX.

[0034] The carbonic anhydrase gene cynT was integrated at the gene locus purR.

[0035] Integration of the carbamoyl phosphate synthetase gene pyrAA / AB from Bacillus subtilis at the gene locus lacI E951* .

[0036] The aspartate transaminase gene pyrB from Bacillus subtilis was integrated into the gene site pepA.

[0037] The orotate phosphoribosyltransferase gene pyrE from Bacillus subtilis was integrated into the gene site argR.

[0038] The phosphoribosyl-pyrophosphate synthase gene prs from Bacillus subtilis was integrated into the gene loci thrA, argF and udk respectively. D128A .

[0039] The uridine kinase gene pyrH from Bacillus subtilis was integrated into the gene loci ushA, surE, yjjG and yrfG respectively. D93A .

[0040] Knockout of the pyrimidine nucleotidase gene ygdH.

[0041] Knockout of the global transcriptional regulator gene arcA.

[0042] Compared with the prior art, the genetic operations involved in constructing the engineering strain for producing uridine diphosphate in the present invention are all performed on the genome, no plasmid residues, no antibiotics and inducers are used, and the finally obtained engineering strain for producing uridine diphosphate has clear and stable genetic traits, stable production performance, and a simple fermentation process.

[0043] Furthermore, 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.

[0044] In a third aspect, the present invention provides a method for synthesizing rebaudioside M, comprising the following steps: The engineered strain for producing uridine diphosphate is fermented and cultured to obtain the engineered strain for producing uridine diphosphate after fermentation.

[0045] The functional protein expression recombinant strain is fermented and cultured to obtain the fermented functional protein expression recombinant strain.

[0046] The fermented engineering strain for producing uridine diphosphate and the recombinant strain for expressing functional protein were configured into the following reaction system: 2.0~2.5 g / L of potassium dihydrogen phosphate, 25~35 g / L of potassium hydrogen phosphate, 3.0~3.5 g / L of ammonium sulfate, 2.0~2.5 g / L of magnesium chloride, 8~12 g / L of glycerol, 2.0~2.5 g / L of CTAB, 25~40 g / L of the fermented engineering strain for producing uridine diphosphate, 100~130 g / L of the fermented recombinant strain for expressing functional protein, 100~200 g / L of sucrose, and 50~80 g / L of rebaudioside A.

[0047] The reaction system is reacted at a pH value of 6.5-7.5 and a temperature of 36.5-37.5 for 18-24 hours. After the reaction is completed, the reaction solution is diluted 100-200 times and centrifuged to obtain a supernatant containing rebaudioside M.

[0048] Compared with the prior art, the reaction system configured by the fermented engineering strain for producing uridine diphosphate and the recombinant strain for expressing functional protein can efficiently convert rebaudioside A into rebaudioside M, thereby further improving the yield and conversion rate of rebaudioside M.

[0049] Furthermore, the method for preparing the functional protein expression recombinant strain after fermentation comprises the following steps: The sucrose synthase gene susy, the uridine diphosphate glycosyltransferase gene EUGT11 and the uridine diphosphate glycosyltransferase gene SrUGT76G4 were connected to the plasmid to construct a recombinant plasmid.

[0050] The recombinant plasmid is transferred into the host strain to obtain a recombinant strain expressing a functional protein.

[0051] The functional protein expression recombinant strain is fermented and cultured to obtain the fermented functional protein expression recombinant strain.

[0052] Wherein, the host strain is Escherichia coli; the Escherichia coli is Escherichia coli BL21 (DE3).

[0053] In the above technical scheme, the susy gene can encode sucrose synthase, which can generate fructose and UDPG using sucrose and UDP as substrates; the EUGT11 gene can encode a glycosyltransferase, which can transfer the glucose group in UDPG to rebaudioside A to generate rebaudioside D and UDP; the SrUGT76G4 gene can encode another glycosyltransferase, which has a preference for the C19 position of stevioside and forms a β-1,3-glycosidic bond at the C19 position, so that the glycosyltransferase can catalyze the generation of rebaudioside M and UDP using UDPG and rebaudioside D as substrates.

[0054] In addition, the gene operations involved in the functional protein expression recombinant strain constructed by the present invention are all performed on the pET28A plasmid, which carries kanamycin resistance. The functional protein engineering strain finally obtained has clear and stable genetic traits, stable production performance, and a simple fermentation process.

[0055] Furthermore, the sucrose synthase gene susy is derived from polymorphic nitrosobacter, tobacco or Arabidopsis thaliana.

[0056] The UDP-glycosyltransferase gene EUGT11 is derived from Stevia rebaudiana.

[0057] The UDP-glycosyltransferase gene SrUGT76G4 is derived from Stevia rebaudiana.

[0058] Furthermore, the functional protein expression recombinant strain is fermented and cultured, 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 a temperature of 36.5-37.5℃ and a pH of 6.5-7.5 for 5-8h to obtain the seed liquid.

[0059] 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 adding the medium, and the fermentation was continued for 24-30 hours.

[0060] After the fermentation is completed, the obtained fermentation liquid is centrifuged, and the supernatant is discarded to obtain the fermented functional protein expression recombinant strain.

[0061] Further, fermentation culture of the engineered strain producing uridine diphosphate comprises the following steps: Take the bacterial liquid of the engineered strain producing uridine diphosphate, spread it on LB medium, culture it at 36.5-37.5℃ for 8-10h, pick out a single bacterium and inoculate it into the seed medium, culture it at 36.5-37.5℃ and pH 6.5-7.5 for 5-8h to obtain seed liquid.

[0062] The seed liquid was inoculated into the fermentation medium, and fermented for 10 to 12 hours at a temperature of 36.5 to 37.5°C and a pH of 6.5 to 7.5. Then, glucose was added using a feedback pulse feeding strategy and the fermentation was continued for 36 to 40 hours.

[0063] 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.

[0064] Furthermore, a feedback pulse feeding strategy is used to add glucose, as follows: 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

[0065] Figure 1 This is an agarose gel electrophoresis verification diagram of the integrated pyr1 gene in Example 1.

[0066] Figure 2 This is an agarose gel electrophoresis verification diagram of the integrated pyr2 gene in Example 1.

[0067] Figure 3 This is an agarose gel electrophoresis verification diagram of the integrated pyr3 gene in Example 1.

[0068] Figure 4 This is an agarose gel electrophoresis verification diagram of the integrated cynT gene in Example 1.

[0069] Figure 5 For the integration of pyrAA / AB in Example 1 E951* Agarose gel electrophoresis verification of the gene.

[0070] Figure 6 This is an agarose gel electrophoresis verification diagram of the integrated pyrB gene in Example 1.

[0071] Figure 7 This is an agarose gel electrophoresis verification diagram of the integrated pyrE gene in Example 1.

[0072] Figure 8 Integrate prs in Example 1 D128A Agarose gel electrophoresis verification of the gene.

[0073] Fig. 9 For the integration of pyrH in Example 1 D90A Agarose gel electrophoresis verification of the gene.

[0074] Fig.10 This is an agarose gel electrophoresis verification diagram of knocking out the ygdH gene in Example 1.

[0075] Fig.11 This is an agarose gel electrophoresis verification diagram of the ArcA gene knockout in Example 1. DETAILED DESCRIPTION

[0076] 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.

[0077] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified. Example 1

[0078] Construction of an engineered strain producing uridine diphosphate In the embodiment of the present invention, a CRISPR / Cas9-mediated gene editing method is used to edit the gene of E. coli W3110 (ATCC27325) (refer to 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 that carries a gRNA plasmid elimination system, a lambda phage Red recombination system, and a Cas9 protein expression system. It has spectinomycin resistance (working concentration: 100 mg / L) and a culture temperature of 32°C. The pGRB plasmid uses pUC18 as a backbone, contains a promoter J23100, a gRNA-Cas9 binding region sequence, and a terminator sequence, and has ampicillin resistance (working concentration: 100 mg / L) and a culture temperature of 37°C.

[0079] 1. Integrate the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis at the pseudogene site yghX: Since the fragment of the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis is relatively large, with the number of bases as high as 9492 bp, the pyrimidine nucleoside operon gene pyrBCAKDFE from Bacillus subtilis is segmentally integrated into the pseudogene site yghX.

[0080] (1) Integration of pyr1 Using the E. coli W3110 genome as a template, upstream homology arm primers UP-yghX-S (SEQ ID NO.28) and UP-yghX-Ptrc-A (SEQ ID NO.29), and downstream homology 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 PCR was used to amplify the upstream and downstream homology arm fragments.

[0081] 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 bases 1 to 3260 (pyr1) of the pyrBCAKDFE nucleotide sequence (SEQ ID NO.1), and the pyr1 gene fragment was obtained by PCR amplification.

[0082] 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.136.

[0083] By overlapping PCR, the upstream and downstream homologous arm fragments of the pseudogene yghX obtained above and the pyr1 gene fragment were used as templates, and UP-yghX-S (SEQ ID NO.28) and DN-yghX-A (SEQ ID NO.31) were used as primers to obtain the integrated fragment of the pyr1 gene: upstream homologous arm-Ptrc-pyr1-downstream homologous arm.

[0084] A DNA fragment containing the pseudogene yghX target sequence was prepared by annealing primers gRNA-yghX-S (SEQ ID NO.34) and gRNA-yghX-A (SEQ ID NO.35), and the recombinant plasmid pGRB-yghX was obtained after recombination with the linearized pGRB vector.

[0085] The integrated fragment of the pyr1 gene (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 pGRB-yghX and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud1-1.

[0086] The agarose gel electrophoresis verification of the integrated fragment of the pyr1 gene and the positive strain 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.

[0087] (2) Integration of pyr2 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 the 2510-6509 (pyr2) nucleotide sequence of pyrBCAKDFE (SEQ ID NO.1), and the pyr2 fragment was amplified by PCR.

[0088] Using the E. coli W3110 genome as a template, PCR was performed to amplify the downstream homology arm fragment of the pseudogene yghX using the downstream homology arm primers DN-yghX-S2 (SEQ ID NO.38) and DN-yghX-A (SEQ ID NO.35).

[0089] 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.137.

[0090] By overlapping PCR, the pyr2 fragment obtained above and the downstream homology arm fragment of the pseudogene yghX were used as templates, and pyr2-S (SEQ ID NO.36) and DN-yghX-A (SEQ ID NO.35) were used as primers to obtain the integration fragment of pyr2: pyr2-downstream homology arm.

[0091] The DNA fragment containing the target sequence of plasmid gene No. 4 was prepared by annealing primers gRNA-S1 (SEQ ID NO.39) and gRNA-A1 (SEQ ID NO.40), and the recombinant plasmid pGRB-pyr2 was obtained after recombination with the linearized pGRB vector.

[0092] The integrated fragment of pyr2 (pyr2-downstream homology arm) and the recombinant plasmid pGRB-pyr2 were electroporated into competent cells of E. coli Ud1-1 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids pGRB-pyr2 and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud1-2.

[0093] 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 gene 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.

[0094] (3) Integration of pyr3 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 according to the 5671-9492 (pyr3) nucleotide sequence of pyrBCAKDFE (SEQ ID NO.1), and the pyr3 fragment was amplified by PCR.

[0095] Using the E. coli W3110 genome as a template, PCR was performed to amplify the downstream homology arm fragment of the pseudogene yghX using the downstream homology arm primers DN-Ptrc-yghX-S (SEQ ID NO.43) and DN-yghX-A (SEQ ID NO.35).

[0096] 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.22.

[0097] By overlapping PCR, the pyr3 fragment obtained by the above PCR and the downstream homologous arm fragment of the pseudogene yghX were used as templates, and pyr3-S (SEQ ID NO.41) and DN-yghX-A (SEQ ID NO.35) were used to obtain the integrated fragment of the pyr3 gene: pyr3-downstream homologous arm.

[0098] The DNA fragment containing the target sequence of plasmid gene 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.

[0099] The integration fragment of the pyr3 gene (pyr3-downstream homology arm) and the recombinant plasmid pGRB-pyr3 were electroporated into competent cells of E. coli Ud1-2 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids pGRB-pyr3 and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud1-3.

[0100] 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.

[0101] 2. Integrate the carbonic anhydrase gene cynT at the gene locus purR: Using the E. coli W3110 genome as a template, upstream homology arm primers UP-purR-S (SEQ ID NO.46) and UP-purR-Ptrc-A (SEQ ID NO.47) and downstream homology arm primers DN-Ptrc-purR-S (SEQ ID NO.48) and DN-purR-A (SEQ ID NO.49) were designed according to the upstream and downstream gene sequences of purR; PCR was used to amplify the upstream and downstream homology arm fragments of purR.

[0102] 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 obtained by PCR amplification.

[0103] The Ptrc promoter was designed in the antisense primer of the upstream homology arm of the purR gene and the sense primer of the cynT gene; the Ptrc terminator was designed in the antisense primer of the cynT gene and the sense primer of the downstream homology arm of the purR gene.

[0104] By overlapping PCR, the cynT fragment obtained by the above PCR and the upstream and downstream homology arm fragments of purR were used as templates, and UP-purR-S (SEQ ID NO.46) and DN-purR-A (SEQ ID NO.49) were used as primers to obtain the integration fragment of cynT: upstream homology arm-Ptrc-cynT-downstream homology arm.

[0105] A DNA fragment containing the purR gene target sequence was prepared by annealing 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.

[0106] The integration fragment of cynT (upstream homology arm-Ptrc-cynT-downstream homology arm) and the recombinant plasmid pGRB-purR were electroporated into competent cells of E. coli Ud1-3 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids pGRB-purR and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud2.

[0107] Agarose gel electrophoresis verification of cynT integration fragment and positive strains is shown in the figure below. Figure 4As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the cynT gene fragment; Lane 3 is the downstream homology arm; Lane 4 is the integrated 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.

[0108] 3. Integrate the carbamoyl phosphate synthetase gene pyrAA / AB from Bacillus subtilis at the gene locus lacI E951* : Using the E. coli W3110 genome as a template, upstream homology 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 gene lacI; downstream homology arm primers DN-Ptrc-lacI-S (SEQ ID NO.56) and DN-lacI-A (SEQ ID NO.57) were designed to PCR amplify the upstream and downstream homology arm fragments.

[0109] According to the synthesis of pyrAA / AB carrying mutation points by Tianjin Anshengda Biological Company E951* Design primer Ptrc-pyrAA / AB based on the nucleotide sequence E951* -S (SEQ ID NO.58) and pyrAA / AB E951* -Ptrc-A (SEQ ID NO.59); with pyrAA / AB E951* As a template, pyrAA / AB was obtained by PCR E951* Fragment.

[0110] Ptrc promoter design in the lacI gene upstream homology arm of the antisense strand primer and pyrAA / AB E951* The positive strand primer of the gene; the Ptrc terminator is designed in pyrAA / AB E951* The antisense primer of the gene and the sense primer of the homology arm downstream of the lacI gene site.

[0111] By overlapping PCR, the pyrAA / AB obtained by the above PCR E951* The fragment and the upstream and downstream homology arm fragments of gene lacI were used as templates, UP-lacI-S (SEQ ID NO.54) and DN-lacI-A (SEQ ID NO.57) were used as primers to obtain pyrAA / AB E951* Integration fragment of the gene: upstream homology arm-Ptrc-pyrAA / AB E951* - Downstream homology arm.

[0112] A DNA fragment containing the lacI gene target sequence was prepared by annealing primers gRNA-lacI-S (SEQ ID NO.60) and gRNA-lacI-A (SEQ ID NO.61), and the recombinant plasmid pGRB-lacI was obtained after recombination with the linearized pGRB vector.

[0113] pyrAA / AB E951* Integration fragment of the gene (upstream homology arm-Ptrc-pyrAA / AB E951* - downstream homology arm) and the recombinant plasmid pGRB-lacI were electroporated into competent cells of E. coli Ud2 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids pGRB-lacI and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud3.

[0114] pyrAA / AB E951* The agarose gel electrophoresis verification of the gene integration fragment and the positive strain is shown in the figure Figure 5 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is pyrAA / AB E951* Gene fragment; Lane 3 is the downstream homology arm; Lane 4 is pyrAA / AB 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.

[0115] 4. Integrate the aspartate transaminase gene pyrB from Bacillus subtilis at the gene locus pepA: Using the E. coli W3110 genome as a template, upstream homology 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 homology arm primers DN-Ptrc-pepA-S (SEQ ID NO.64) and DN-pepA-A (SEQ ID NO.65) were designed to PCR amplify the upstream and downstream homology arm fragments.

[0116] 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 the pyrB fragment was amplified by PCR using the B. subtilisA260 genome as a template.

[0117] The Ptrc promoter was designed in the antisense primer of the upstream homology arm of the pepA gene 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 pepA gene site.

[0118] By overlapping PCR, the upstream and downstream homologous arm fragments of the gene pepA and the pyrB fragment obtained by the above PCR were used as templates, and UP-pepA-S (SEQ ID NO.62) and DN-pepA-A (SEQ ID NO.65) were used as primers to obtain the integration fragment of the pyrB gene: upstream homologous arm-Ptrc-pyrB-downstream homologous arm.

[0119] A DNA fragment containing the pepA gene target sequence was prepared by annealing primers gRNA-pepA-S (SEQ ID NO.68) and gRNA-pepA-A (SEQ ID NO.69), and the recombinant plasmid pGRB-pepA was obtained after recombination with the linearized pGRB vector.

[0120] The integrated fragment of the pyrB gene (upstream homology arm-Ptrc-pyrB-downstream homology arm) and the recombinant plasmid pGRB-pepA were electroporated into the competent cells of E. coli Ud3 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids pGRB-pepA and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud4.

[0121] The agarose gel electrophoresis verification of the integrated fragment of pyrB and the positive strain is shown in the figure Figure 6 As shown: Lane M is 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 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.

[0122] 5. Integrate the orotate phosphoribosyltransferase gene pyrE from Bacillus subtilis at the gene locus argR: 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 upstream and downstream gene sequences of the gene argR; downstream homology arm primers DN-Ptrc-argR-S (SEQ ID NO.72) and DN-argR-A (SEQ ID NO.73) were designed, and the upstream and downstream homology arm fragments were amplified by PCR.

[0123] Primers Ptrc-pyrE(bsu)-S (SEQ ID NO.74) and pyrE(bsu)-Ptrc-A (SEQ ID NO.75) were designed based on the pyrB nucleotide sequence, and the pyrE fragment was obtained by PCR using the B. subtilisA260 genome as a template.

[0124] The Ptrc promoter was designed in the antisense primer of the upstream homology arm of the argR gene 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 argR gene site.

[0125] By overlapping PCR, the upstream and downstream homology arm fragments of the gene argR obtained by the above PCR and the pyrE fragment were used as templates, and UP-argR-S (SEQ ID NO.70) and DN-argR-A (SEQ ID NO.73) were used as primers to obtain the integration fragment of the pyrE gene: upstream homology arm-Ptrc-pyrE-downstream homology arm.

[0126] A DNA fragment containing the argR gene target sequence was prepared by annealing 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.

[0127] The integrated fragment of the pyrE gene (upstream homology arm-Ptrc-pyrE-downstream homology arm) and the recombinant plasmid pGRB-argR were electroporated into competent cells of E. coli Ud4 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids pGRB-argR and pREDCas9 used for gene editing were eliminated to obtain the strain E. coli Ud5.

[0128] The agarose gel electrophoresis verification of the integrated fragment of pyrE and the positive strain is shown in the figure Figure 7 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is the pyrE gene 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.

[0129] 6. Integrate the phosphoribosyl pyrophosphate synthase gene prs from Bacillus subtilis at the gene loci thrA, argF and udk respectively D128A : According to the upstream and downstream sequences of genes thrA, argF and udk, upstream homology 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 IDNO.83); downstream homology 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 IDNO.88) and DN-udk-A (SEQ ID NO.89) were designed respectively. Using the E. coli W3110 genome as a template, PCR technology was used to amplify the upstream and downstream homologous arms of genes thrA, argF and udk, respectively.

[0130] 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.90), prs D128A -Ptrc-A (SEQ ID NO.91), with the target gene prs D128A As a template, prs was obtained by PCR amplification D128A Fragment.

[0131] The Ptrc promoter was designed to be located in the upstream homology arms of genes thrA, argF and udk respectively. D128A The Ptrc terminator was designed in the prs D128A The antisense primer of the gene and the positive primer of the downstream homology arm of the purR, argF and udk gene sites.

[0132] By recombinant PCR, the upstream and downstream homologous arms of the gene thrA obtained by the above PCR and prs D128A The fragment was used as a template, and UP-thrA-S (SEQ ID NO.78) and DN-thrA-A (SEQ ID NO.85) were used as primers to obtain prs D128A Integration fragment of the gene: thrA upstream homology arm-Ptrc-prs D128A -thrA downstream homology arm.

[0133] By recombinant PCR, the upstream and downstream homologous arms of the gene argF obtained by the above PCR and prs D128A The fragment was used as a template, and UP-argF-S (SEQ ID NO.80) and DN-argF-A (SEQ ID NO.87) were used as primers to obtain prs D128A Integration fragment of the gene: argF upstream homology arm-Ptrc-prs D128A -ArgF downstream homology arm.

[0134] By recombinant PCR, the upstream and downstream homology arms of the gene udk obtained by the above PCR and prs D128A The fragment was used as a template, and UP-udk-S (SEQ ID NO.82) and DN-udk-A (SEQ ID NO.89) were used as primers to obtain prs D128A Integration fragment of gene: udk upstream homology arm-ptrc-prs D128A -UDK downstream homology arm.

[0135] The DNA fragments containing the target sequences of thrA, argF and udk genes were prepared by annealing 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), and recombined with the linearized pGRB vector to obtain recombinant plasmids pGRB-thrA, pGRB-argF and pGRB-udk, respectively.

[0136] PRS D128A Integration fragment of gene thrA upstream homology arm - Ptrc-prs D128A -thrA downstream homology arm and pGRB-thrA were electroporated into E. coli Ud5 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 pGRB-thrA and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud6.

[0137] PRS D128A Integration fragment of gene argF upstream homology arm-Ptrc-prs D128A-argF downstream homology arm and pGRB-argF were electroporated into E. coli Ud6 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 pGRB-argF and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud7.

[0138] PRS D128A Integration fragment of gene udk upstream homology arm - Ptrc-prs D128A -udk downstream homology arm and pGRB-udk were electrotransformed into E. coli Ud7 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 pGRB-udk and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud8. Agarose gel electrophoresis verification is shown in the figure below Figure 8 As shown: Lane M is DNA Marker; Lane 1 is the upstream homology arm; Lane 2 is prs D128A Gene fragment; Lane 3 is the downstream homology arm; Lane 4 is the prs D128A 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.

[0139] 7. Integrate the uridylate kinase gene pyrH from Bacillus subtilis at the gene sites ushA, surE, yjjG and yrfG respectively D90A : According to the upstream and downstream sequences of the gene, upstream homology 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 homology 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.110). 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). Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the genes ushA, surE, yjjG and yrfG were amplified by PCR technology.

[0140] According to the target gene pyrH carrying the mutation point synthesized by Tianjin Anshengda Biological Company D90A Design primers Ptrc-pyrH D90A -S (SEQ ID NO.114), pyrH D90A -Ptrc-A (SEQ ID NO.115), with the target gene pyrH D90A As a template, pyrH was obtained by PCR D90A Fragment.

[0141] The Ptrc promoter was designed in the upstream homology arms of genes ushA, surE, yjjG and yrfG respectively. D90A The Ptrc terminator was designed in the pyrH D90A The antisense strand primer of the gene and the positive strand primer of the downstream homologous arm of the ushA, surE, yjjG, and yrfG gene loci.

[0142] By recombinant PCR, the upstream and downstream homologous arms of the gene ushA and pyrH D90AThe fragment was used as a template, and UP-ushA-S (SEQ ID NO.98) and DN-ushA-A (SEQ ID NO.107) were used as primers to obtain pyrH D90A Integration fragment of the gene: ushA upstream homology arm-Ptrc-pyrH D90A -ushA downstream homology arm.

[0143] By recombinant PCR, the upstream and downstream homology arms of the gene surE and pyrH obtained by the above PCR were used to generate D90A The fragment was used as a template, and UP-surE-S (SEQ ID NO.100) and DN-surE-A (SEQ ID NO.109) were used as primers to obtain pyrH D90A Integration fragment of the gene: surE upstream homology arm-Ptrc-pyrH D90A -surE downstream homology arm.

[0144] By recombinant PCR, the upstream and downstream homology arms of gene yjjG and pyrH D90A The fragment was used as a template, and UP-yjjG-S (SEQ ID NO.102) and DN-yjjG-A (SEQ ID NO.111) were used as primers to obtain pyrH D90A Integration fragment of the gene: yjjG upstream homology arm-Ptrc-pyrH D90A -yjjG downstream homology arm.

[0145] By recombinant PCR, the upstream and downstream homologous arms of the gene yrfG and pyrH D90A The fragment was used as a template, and UP-yrfG-S (SEQ ID NO.104) and DN-yrfG-A (SEQ ID NO.113) were used as primers to obtain pyrH D90A Integration fragment of the gene: yrfG upstream homology arm-Ptrc-pyrH D90A -yrfG downstream homology arm.

[0146] DNA fragments containing ushA, surE, yjjG and yrfG gene target sequences were respectively prepared by annealing 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 IDNO.120) and gRNA-yjjG-A (SEQ ID NO.121); gRNA-yrfG-S (SEQ ID NO.122) and gRNA-yrfG-A (SEQ ID NO.123), and recombined with the linearized pGRB vector to obtain recombinant plasmids pGRB-ushA, pGRB-surE, pGRB-yjjG and pGRB-yrfG, respectively.

[0147] The integration fragment ushA upstream homology arm-Ptrc-pyrH D90A -ushA downstream homology arm and pGRB-ushA were electroporated into E. coli Ud8 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 pGRB-ushA and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud9.

[0148] The integration fragment surE upstream homology arm-Ptrc-pyrH D90A -surE downstream homology arm and pGRB-surE were electroporated into E. coli Ud9 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 pGRB-surE and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud10.

[0149] The integration fragment yjjG upstream homology arm-Ptrc-pyrH D90A -yjjG downstream homology arm and pGRB-yjjG were electroporated into E. coli Ud10 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 pGRB-yjjG and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud11.

[0150] The integration fragment yrfG upstream homology arm-Ptrc-pyrH D90A-yrfG downstream homology arm and pGRB-yrfG were electrotransformed into E. coli Ud11 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 pGRB-yrfG and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud12. 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 pyrH D90A gene fragment; Lane 3 is the downstream homology arm; Lane 4 is pyrH D90A 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.

[0151] 8. Knockout of pyrimidine nucleotidase gene ygdH: According to the upstream and downstream sequences of the ygdH gene, upstream homology arm primers UP-ygdH-S (SEQ ID NO.124) and UP-ygdH-A (SEQ ID NO.125) and downstream homology arm primers DN-ygdH-S (SEQ ID NO.126) and DN-ygdH-A (SEQ ID NO.127) were designed, and the upstream and downstream homology arms were amplified by PCR technology using the E. coli W3110 genome as a template.

[0152] By recombinant PCR, the upstream and downstream homologous arms of the ygdH gene obtained by the above PCR were used as templates, and UP-ygdH-S (SEQ ID NO.124) and DN-ygdH-A (SEQ ID NO.127) were used as primers to obtain the knockout fragment of the gene ygdH: upstream homologous arm-downstream homologous arm.

[0153] A DNA fragment containing the ygdH gene target sequence was obtained by annealing primers gRNA-ygdH-S (SEQ ID NO.128) and gRNA-ygdH-A (SEQ ID NO.129), and the recombinant vector pGRB-ygdH was obtained after recombination with the linearized pGRB vector.

[0154] The upstream homology arm-downstream homology arm of the knockout fragment of the gene ygdH and pGRB-ygdH were electrotransformed into E. coli Ud12 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 pGRB-ygdH and pREDCas9 used for gene editing were eliminated to obtain strain E. coliUd13.

[0155] Agarose gel electrophoresis verification diagram Fig.10 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 of gene ygdH; Lane 4 is the PCR fragment of E.coli W3110; Lane 5 is the PCR fragment of the positive strain.

[0156] 9. Knockout of the global transcriptional regulatory gene ArcA: According to the upstream and downstream sequences of the ArcA gene, upstream homology arm primers UP-ArcA-S (SEQ ID NO.130) and UP-ArcA-A (SEQ ID NO.131) and downstream homology arm primers DN-ArcA-S (SEQ ID NO.132) and DN-ArcA-A (SEQ ID NO.133) were designed, and the upstream and downstream homology arms were amplified by PCR technology using the E. coli W3110 genome as a template.

[0157] By recombinant PCR, the upstream and downstream homologous arms of the ArcA gene obtained by the above PCR were used as templates, and UP-ArcA-S (SEQ ID NO.130) and DN-ArcA-A (SEQ ID NO.133) were used as primers to obtain the knockout fragment of the ArcA gene: upstream homologous arm-downstream homologous arm.

[0158] A DNA fragment containing the ArcA gene target sequence was prepared by annealing primers gRNA-ArcA-S (SEQ ID NO.134) and gRNA-ArcA-A (SEQ ID NO.135), and the recombinant plasmid pGRB-ArcA was obtained after recombination with the linearized pGRB vector.

[0159] The knockout fragment of ArcA gene upstream homology arm-downstream homology arm and pGRB-ArcA were electrotransformed into E. coli Ud13 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 pGRB-ArcA and pREDCas9 used for gene editing were eliminated to obtain strain E. coli Ud14. Agarose gel electrophoresis verification is shown in the figure below. Fig.11 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 of ArcA gene; Lane 4 is the PCR fragment of E. coliW3110; Lane 5 is the PCR fragment of the positive strain. Example 2

[0160] Construction of recombinant strain expressing functional protein Using pET28A plasmid as expression vector, the promoter Ptrc and EUGT11 gene were integrated into 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 expression vector, the promoter Ptrc and SrUGT76G4 gene were integrated into the plasmid using SacⅠ and SalⅠ enzymes to obtain the recombinant expression plasmid pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4. The recombinant expression plasmid pET28A-Ptrc-EUGT11-Ptrc-SrUGT76G4 was used as the expression vector, and the promoter Ptrc and susy (nmu) gene, the promoter Ptrc and susy (nta) gene, and the promoter Ptrc and susy (nth) gene were integrated into 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).

[0161] Among them, the susy (nmu) gene is a sucrose synthase gene susy derived from polymorphic nitrosobacter, and its nucleotide sequence is shown in SEQ ID NO.23; the susy (nta) gene is a sucrose synthase gene susy derived from tobacco, and its nucleotide sequence is shown in SEQ ID NO.24; the susy (ath) gene is a 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.

[0162] 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 transformed into Escherichia coli BL21(DE3) cells by CaCl2 chemical transformation, and 100 μL / L of the bacterial solution was spread on LB medium containing kanamycin resistance for culture after recovery culture. Single colonies after transformation were picked and verified by PCR, and 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. colilep4 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) were obtained respectively. Example 3

[0163] Fermentation of engineered strains producing uridine diphosphate Prepare the following culture medium: LB medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar powder 20 g / L.

[0164] 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 100 μL / L, adjust the pH to 7.0 with NaOH solution.

[0165] Fermentation medium: initial glucose 20 g / L, anhydrous sodium citrate 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 0.001 g / L each, V H 0.0005 g / L; LD-XP3020 defoamer 100 μL / L, adjust the pH to 7.0 with NaOH solution.

[0166] Take the bacterial liquid of the uridine diphosphate genetically engineered strain E. coli Ud14 in Example 1, evenly spread it on LB medium, culture it at 37°C for 9 h, pick a single colony into physiological saline, and inoculate it into 50 mL of seed culture medium. During the culture process, ammonia water is added to maintain the pH value at 7. Culture it at 37°C for 6.5 h to obtain seed liquid.

[0167] The seed liquid was inoculated at a volume ratio of 2% into the fermentation medium for fermentation culture. After 11 hours of fermentation at a temperature of 37°C and a pH of 7, glucose was added using a feedback pulse feeding strategy, that is, the pulse rate of feeding on the feeding pump was set at 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 and the fermentation culture continued for 36 hours.

[0168] After the fermentation, 10 mL of the fermentation broth was taken, the cells were broken using an ultrasonic disruptor, and the mixture was centrifuged at 13500 rpm for 10 min. The precipitate was discarded to obtain a supernatant containing UDP (uridine diphosphate). The UDP yield in the supernatant was measured by HPLC to be 38 g / L.

[0169] The remaining fermentation liquid was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected to obtain the fermented engineered strain E. coli Ud15 for producing uridine diphosphate. Example 4

[0170] Fermentation of recombinant strains expressing functional proteins Prepare the following culture medium: LB medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar powder 20 g / L.

[0171] 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 defoamer 100 μL / L.

[0172] Fermentation 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 defoamer 100 μL / L.

[0173] Fed-batch medium: glucose 600 g / L, MgSO4·7H2O 2 g / L, yeast powder 75 g / L.

[0174] The bacterial liquid of the functional protein-expressing recombinant strain E. coli lep3 in Example 2 was evenly spread on LB medium containing 50 μg / ml kanamycin, and cultured at 37°C for 8 h. A single colony was picked into physiological saline and inoculated into 50 mL of seed medium containing 50 μg / ml kanamycin, and cultured at 37°C for 6.5 h. During the culture process, ammonia water was added to maintain the pH value at 7 to obtain a seed liquid.

[0175] The seed liquid was inoculated with 2% volume ratio into the fermentation medium containing 50μg / ml kanamycin for fermentation and culture. During the culture process, the pH value was controlled to 7 by adding ammonia water. The culture was cultured at 37℃. When the OD value of the strain reached 10, the fermentation temperature was controlled at 25℃. When the dissolved oxygen soared, the medium was added to control the residual sugar (glucose) in the fermentation broth within 1g / L, and the fermentation culture was continued for 26h.

[0176] After the fermentation was completed, the fermentation broth was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the bacteria were collected to obtain the fermented functional protein expression recombinant strain E. coli lep3-1.

[0177] The functional protein expressing recombinant strains E. coli lep4 and E. coli lep5 in Example 2 were fermented and cultured 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

[0178] Whole-cell catalytic synthesis of rebaudioside M 1. Use E.coli Ud15 and E.coli lep3-1 to prepare the reaction system as follows: 2.0 g / L potassium dihydrogen phosphate, 25 g / L potassium hydrogen phosphate, 3.0 g / L ammonium sulfate, 2.0 g / L magnesium chloride, 8 g / L glycerol, 2.0 g / L CTAB, 33 g / L E.coliUd15, 115 g / L E.coli lep3-1, 150 g / L sucrose, and 65 g / L rebaudioside A.

[0179] The reaction system was reacted for 20 hours at a speed of 250 rpm, a temperature of 37°C, and a pH value of 7. After the reaction, the obtained reaction solution was diluted 200 times, oscillated and mixed, and centrifuged at 13000 r / min for 10 min at room temperature to collect the supernatant containing rebaudioside M.

[0180] The supernatant was passed 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. HPLC used a Waters C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase was acetonitrile: water (pH 7.0) = 30:70, the flow rate was 1 mL / min, and the column temperature was 40°C. Using an ultraviolet detector for detection, at a wavelength of 210 nm and an injection volume of 10 μL, the content of rebaudioside M was measured to be 56.55 g / L, and the conversion rate was 87%.

[0181] 2. Use E.coli Ud15 and E.coli lep4-1 to configure the reaction system as follows: 2.5 g / L potassium dihydrogen phosphate, 35 g / L potassium hydrogen phosphate, 3.5 g / L ammonium sulfate, 2.5 g / L magnesium chloride, 12 g / L glycerol, 2.5 g / L CTAB, 40 g / L E.coliUd15, 130 g / L E.coli lep4-1, 200 g / L sucrose, and 80 g / L rebaudioside A.

[0182] The reaction system was reacted for 20 hours at a speed of 250 rpm, a temperature of 37°C, and a pH value of 7. After the reaction, the obtained reaction solution was diluted 200 times, oscillated and mixed, and centrifuged at a speed of 13000 r / min for 10 min at room temperature to collect the supernatant.

[0183] The supernatant was passed 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. HPLC used a Waters C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase was acetonitrile: water (pH 7.0) = 30:70, the flow rate was 1 mL / min, and the column temperature was 40°C. Using an ultraviolet detector for detection, 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%.

[0184] 3. Use E.coli Ud15 and E.coli lep5-1 to configure the reaction system as follows: 2.2 g / L potassium dihydrogen phosphate, 30 g / L potassium hydrogen phosphate, 3.1 g / L ammonium sulfate, 2.1 g / L magnesium chloride, 10 g / L glycerol, 2.2 g / L CTAB, 25 g / L E.coliUd15, 100 g / L E.coli lep5-1, 100 g / L sucrose, and 50 g / L rebaudioside A.

[0185] The reaction system was reacted for 20 hours at a speed of 250 rpm, a temperature of 37°C, and a pH value of 7. After the reaction, the obtained reaction solution was diluted 200 times, oscillated and mixed, and centrifuged at 13000 r / min for 10 min at room temperature to collect the supernatant.

[0186] The supernatant was passed through a 0.22 μm organic membrane, and the content of rebaudioside M in the filtrate was determined by HPLC. HPLC used a Waters C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase was acetonitrile: water (pH 7.0) = 30:70, the flow rate was 1 mL / min, and the column temperature was 40 ° C. The detection was performed by an ultraviolet detector, the wavelength was 210 nm, and the injection volume was 10 μL. The content of rebaudioside M was measured to be 48.5 g / L, and the conversion rate was 97%. Comparative Example 1

[0187] An engineered strain for producing uridine diphosphate was constructed according to the method of Example 1, except that pyrBCAKDFE was not integrated into the pseudogene site yghX of E. coli W3110, and the integration fragment of cynT (upstream homology arm-Ptrc-cynT-downstream homology arm) and the recombinant plasmid pGRB-purR were electroporated into competent cells of E. coli W3110 containing the pREDCas9 plasmid, and after screening positive strains, the two plasmids used for gene editing were eliminated to obtain strain E. coli Ud2′, and the strain E. coli Udg2′ was subsequently subjected to gene editing. Comparative Example 2

[0188] The engineered strain producing UDP was constructed according to the method of Example 1, except that pyrAA / AB was not integrated into the gene locus lacI. E951* The integrated fragment of the pyrB gene (upstream homology arm-Ptrc-pyrB-downstream homology arm) and the recombinant plasmid pGRB-pepA were electroporated into the competent cells of E. coli Ud2 containing the pREDCas9 plasmid. After screening the positive strains, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Ud4′, and the strain E. coli Udg4′ was subsequently subjected to gene editing. Comparative Example 3

[0189] An engineered strain for producing uridine diphosphate was constructed according to the method of Example 1, except that pyrB was not integrated into the gene site pepA, and the integrated fragment of the pyrE gene (upstream homology arm-Ptrc-pyrE-downstream homology arm) and the recombinant plasmid pGRB-argR were electroporated into competent cells of E. coli Ud3 containing the pREDCas9 plasmid, and after screening the positive strains, the two plasmids used for gene editing were eliminated to obtain the strain E. coli Ud5′, and the strain E. coli Udg5′ was subsequently subjected to gene editing. Comparative Example 4

[0190] The engineering strain producing UDP was constructed according to the method of Example 1, except that pyrE was not integrated into the gene locus argR, and prs D128A Integration fragment of gene thrA upstream homology arm - Ptrc-prs D128A -thrA downstream homology arm and pGRB-thrA were electroporated into E. coli Ud4 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. coliUd6′, and subsequent gene editing was performed on strain E. coli Udg6′. Comparative Example 5

[0191] An engineered strain for producing uridine diphosphate was constructed according to the method of Example 1, except that the pyrimidine nucleotidase gene ygdH was not knocked out, the upstream homology arm-downstream homology arm of the knockout fragment of the ArcA gene and pGRB-ArcA were electroporated into E. coli Ud12 competent cells containing the pREDCas9 plasmid, and a single colony was obtained by resuscitation culture. A positive strain was obtained by PCR colony verification, and then the two plasmids used for gene editing were eliminated to obtain the strain E. coli Ud14′. Comparative Example 6

[0192] An engineered strain producing uridine diphosphate was constructed according to the method of Example 1, except that the global transcriptional regulatory gene ArcA was not knocked out. Test Case

[0193] The engineered strains for producing uridine diphosphate constructed in Example 1 and Comparative Examples 1 to 6 were fermented and cultured according to the method of Example 3, and the yield of UDP was detected. The results are shown in Table 1.

[0194] Table 1

[0195] The engineered strains for producing uridine diphosphate constructed in Example 1 and Comparative Examples 1 to 6 were fermented and cultured according to the method of Example 3 to obtain fermented uridine diphosphate-producing strains E. coli Ud15, E. coli Ud15-1, E. coli Ud15-2, E. coli Ud15-3, E. coli Ud15-4, E. coli Ud15-5, and E. coli Ud15-6, respectively. Rebaudioside M was synthesized according to the method of Example 5, and the yield and conversion rate of rebaudioside M (RM) were detected. The results are shown in Tables 2 and 3.

[0196] Table 2

[0197] Table 3

[0198] From the results in Table 1, Table 2 and Table 3 above, it can be seen that: Comparative Example 1 did not integrate the pyrBCAKDFE gene, and Comparative Example 2 did not integrate the pyrAA / AB gene at the lacI gene site. E951* After the pyrB gene was not integrated into the pepA gene site in comparative example 3 and the pyrE gene was not integrated into the argR gene site in comparative example 4, the yield of UDP, the yield of RM and the conversion rate all decreased, indicating that after the above-mentioned specific gene was integrated into the above-mentioned specific gene site, the yield of UDP and the yield and conversion rate of RM could be increased. After the pyrimidine nucleotidase gene ygdH was not knocked out in comparative example 5, the yield of UDP, the yield of RM and the conversion rate all decreased, indicating that not expressing the pyrimidine nucleotidase gene ygdH could increase the yield of UDP and thus increase the yield and conversion rate of RM. After the global transcriptional regulatory gene ArcA was not knocked out in comparative example 6, the yield of UDP, the yield of RM and the conversion rate all decreased, indicating that not expressing the global transcriptional regulatory gene ArcA could increase the yield of UDP and thus increase the yield and conversion rate of RM.

[0199] 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. 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 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.

2. The engineered strain for producing uridine diphosphate according to claim 1, characterized in that: 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 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.

3. A method for constructing an engineered strain, for constructing the engineered strain according to claim 1, characterized in that: The following steps are involved: 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.

4. The construction method according to claim 3, 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.

5. 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 or 2 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 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.

6. The method according to claim 5, characterized in that 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 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).

7. The method according to claim 6, 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.

8. The method according to claim 5, 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, culture it at 36.5-37.5°C for 8-10 hours, pick a single colony and inoculate it into the seed medium containing kanamycin, 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 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 completed, the obtained fermentation liquid is centrifuged, and the supernatant is discarded to obtain the fermented functional protein expression recombinant strain.

9. The method according to claim 5, 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.

10. The method according to claim 9, 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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