Sinorhizobium meliloti strain with transcription factor knocked out as well as construction method and application thereof

Abstract

The invention provides a transcription factor knockout sinorhizobium meliloti strain as well as a construction method and application thereof. According to the application disclosed by the invention, transcription factor genes orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and orf4207 are successfully knocked out in a genome of the sinorhizobium meliloti through a genetic engineering method, and a new strategy is provided for improving the yield of vitamin B12. Compared with wild-type sinorhizobium meliloti, the yield of vitamin B12 of the sinorhizobium meliloti recombinant strain constructed by the invention is increased by 50% or above at most.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to gene modification of a Sinorhizobium meliloti strain, and in particular to a transcription factor knocked-out Sinorhizobium meliloti strain and a construction method and application thereof. Background Art

[0002] Vitamin B12, also known as cobalamin, is a corrinoid compound that is widely used in the pharmaceutical and food industries. Vitamin B12 has a large molecular weight and a complex chemical structure. Its chemical synthesis is time-consuming and labor-intensive, with high costs and a long synthesis cycle. In addition, it places high demands on operators during the synthesis process, making it unsuitable for industrial production. Some microorganisms in nature can synthesize vitamin B12 by themselves to meet their growth needs and are natural cell factories for vitamin B12 synthesis. Currently, microbial fermentation is the cheapest method for producing vitamin B12 and is widely used in industrial production. Rhizobium, as a natural vitamin B12 production strain, has great potential in industrial production [Dong H, Li S, Fang H, Xia M, ZhengP, Zhang D, Sun J. A newly isolated and identified vitamin B12 producingstrain: Sinorhizobium meliloti 320. Bioproc Biosyst Eng 2016;39:1527-37.].

[0003] Regarding the improvement of vitamin B12 production, current research mainly focuses on strain evolution and increasing the expression of genes in the vitamin B12 synthesis pathway to increase vitamin B12 production. However, there are few reports on increasing vitamin B12 production by modifying transcriptional regulatory factors [Kang Z, Zhang J, Zhou J, Qi Q, Du G, Chen J. Recentadvances in microbial production of δ-aminolevulinic acid and vitamin B12. Biotechnol Adv 2012;30: 1533-42].

[0004] There are many genes in Sinorhizobium alfalfa that are closely related to the synthesis of the strain's metabolites, but it is still unknown which genes are related to the production of vitamin B12. Further research is also needed to determine what genetic modification method can be used to obtain engineered bacteria with high vitamin B12 production. Summary of the invention

[0005] The present invention found that after stimulating Sinorhizobium alfalfa with vitamin B12, transcriptome gene expression difference analysis was performed, and some transcription factors were significantly up-regulated or down-regulated, indicating that these transcription factors may be related to the synthesis of vitamin B12 by the strain. Therefore, these transcription factors were knocked out at the genome level, and it was found that knocking out these transcription factors can increase the production of vitamin B12 in the strain.

[0006] The present invention provides a Chinese meliloti rhizobium with knocked out or weakened transcription factors, wherein the transcription factors are knocked out or weakened in the original Chinese meliloti rhizobium. orf 149. orf 993, orf1177, orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and orf One or more of the 4207 genes.

[0007] Preferably, the starting Sinorhizobium meliloti is Sinorhizobium medicae TIB.SM.2013.

[0008] Specifically, transcription factors orf 149. orf 993, orf1177, orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and orf The amino acid sequences encoded by the 4207 gene are: WP_127888815.1, WP_034803756.1, KSV74715.1, KSV65705.1, WP_127889366.1, KSV74068.1, WP_127889532.1, WP_034802440.1, WP_113504689.1 and WP_029742798.1.

[0009] More specifically, transcription factors orf 149. orf 993, orf1177, orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and orfThe nucleotide sequences of the 4207 genes are shown in SEQ ID NOs: 1-10, respectively.

[0010] The present invention provides a method for constructing Sinorhizobium meliloti, which comprises the following steps: S1 was respectively converted into orf 149. orf 993, orf1177, orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and / or orf The upstream homology arms of 4207 gene and orf 149. orf 993, orf1177, orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and / or orf The downstream homology arms of the 4207 gene were connected together to form the target gene fragment; S2 was PCR amplified with targeted orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and / or orf4207 Gene knockout plasmid vector PBBR-CasX-sgRNA; S3 used Gibson assembly to connect the knockout fragment obtained in S1 and the linear plasmid fragment obtained in S2, respectively, to obtain knockout plasmids PBBR-CasX-sgRNA-Δorf149, PBBR-CasX-sgRNA-Δorf993, PBBR-CasX-sgRNA-Δorf1177, PBBR-CasX-sgRNA-Δorf1867, PBBR-CasX-sgRNA-Δorf2327, PBBR-CasX-sgRNA-Δorf2340, PBBR-CasX-sgRNA-Δorf3280, PBBR-CasX-sgRNA-Δorf3296, PBBR-CasX-sgRNA-Δorf4169 and PBBR-CasX-sgRNA-Δorf4207, respectively; S4: respectively transform the knockout plasmids PBBR-CasX-sgRNA-Δorf149, PBBR-CasX-sgRNA-Δorf993, PBBR-CasX-sgRNA-Δorf1177, PBBR-CasX-sgRNA-Δorf1867, PBBR-CasX-sgRNA-Δorf2327, PBBR-CasX-sgRNA-Δorf2340, PBBR-CasX-sgRNA-Δorf3280, PBBR-CasX-sgRNA-Δorf3296, PBBR-CasX-sgRNA-Δorf4169 and / or PBBR-CasX-sgRNA-Δorf4207 into Sinorhizobium meliloti, use apramycin as a selection marker, and screen to obtain positive transformants; S5: positive transformants were cultured at 30°C-32°C until OD 600 0.6-0.8, add inducer, dilute and plate, and perform colony PCR test to obtain orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and orf4207 The upstream homology arm and downstream homology arm of the gene produce a strain of homologous recombination with the genomic DNA of Sinorhizobium meliloti.

[0011] Optionally, the method further comprises the following steps: S6 Select the strain that has correctly undergone homologous recombination and inoculate it into a medium without apramycin. After several transfer cultures, the plasmid is lost. After plating, the plate is cultured in a 30℃-32℃ incubator. The grown colonies are copied on plates containing apramycin resistance and plates without resistance to obtain plasmid-free and plasmid-free strains. orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and orf4207 Sinorhizobium meliloti Δ orf149, Δ orf993, Δ orf1177, Δ orf1867, Δ orf2327, Δ orf2340, Δ orf3280, Δ orf3296, Δ orf4169 and Δ orf4207 .

[0012] The invention provides the application of the Sinorhizobium alfalfa in the production of vitamin B12.

[0013] The present invention further provides a method for preparing vitamin B12, which comprises the following steps: after performing seed fermentation on a seed fermentation medium, inoculating the seeds into a fermentation medium to ferment and produce vitamin B12, wherein the medium contains a carbon source.

[0014] Specifically, the carbon source is one or more of sucrose, corn syrup and / or betaine.

[0015] More specifically, the culture medium formula for the seed fermentation is: 40% sucrose, 20% corn steep liquor, 5% betaine, 1% (NH4)2SO4, 2% (NH4)2HPO4, 0.8% MnSO4·H2O, 0.02% CoCl2·6H2O, 0.3% MgO, 0.01% DMBI, 0.01% ZnSO4·7H2O, 0.01% CaCO3 (g / L), and the pH is controlled at 7.0-7.4 by NaOH; the fermentation culture medium formula is: 80% sucrose, 30% corn steep liquor, 15% betaine, 2% (NH4)2SO4, 1.5% MgSO4, 0.75% K2HPO4, 0.14% CoCl2·6H2O, 0.075% DMBI, 0.08% ZnSO4·7H2O, 1 g / L CaCO3, and the pH is controlled at 7.0-7.4 by NaOH.

[0016] Compared with Sinorhizobium meliloti SM, the Sinorhizobium meliloti SMΔ orf149, SMΔ orf993, SMΔ orf1177, SMΔ orf1867, SMΔ orf2327, SMΔ orf2340, SMΔ orf3280, SMΔ orf3296, SMΔ orf4169 and SMΔ orf4207 The vitamin B12 production is increased by more than 50%. The research results of the present invention show that knocking out the transcription factor in the genome DNA of Sinorhizobium alfalfa is a very effective method for increasing the production of vitamin B12. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1. This is the verification band of the S. meliloti strains SMΔorf149, SMΔorf993, SMΔorf1177, SMΔorf1867, SMΔorf2327, SMΔorf2340, SMΔorf3280, SMΔorf3296, SMΔorf4169 and SMΔorf4207 obtained in step (7) of Example 1. Lane 13 is DNA marker, lanes 1, 3, 5, 7, 9, 11, 14, 16, 18, and 20 are PCR verification bands of wild Sinorhizobium meliloti SM, and lanes 2, 4, 6, 8, 10, 12, 15, 17, 19, and 21 are verification bands of Sinorhizobium meliloti SMΔorf149, SMΔorf993, SMΔorf1177, SMΔorf1867, SMΔorf2327, SMΔorf2340, SMΔorf3280, orf3296, SMΔorf4169, and SMΔorf4207 strains in which orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169, and orf4207 genes were successfully knocked out; Among them, the molecular weights corresponding to the bands from top to bottom in the above DNA marker lane are: 5000 bp, 3000 bp, 2000 bp, 1200 bp, 800 bp, 500 bp, and 200 bp. DETAILED DESCRIPTION

[0018] The preferred embodiments of the present invention are now described in detail with reference to the accompanying drawings, but they do not constitute a limitation to the present invention.

[0019] Example 1: Knockout orf149 The method for constructing the gene-containing Sinorhizobium alfalfa comprises the following steps: (1) Using the genomic DNA of Sinorhizobium alfalfa as a template, PCR amplified orf149 The upstream homology arm and the downstream homology arm of the gene; (2) Overlap extension PCR orf149 The upstream homology arms of the gene and orf149 The downstream homology arms of the gene are connected together to form the target gene fragment; (3) PCR amplification with targeted orf149 Gene knockout plasmid vector PBBR-CasX-sgRNA; (4) Use Gibson assembly to connect the knockout fragment obtained in (2) and the linear plasmid fragment obtained in step (3) to obtain the knockout plasmid PBBR-CasX-sgRNA-Δ orf149 ; (5) Insert the knockout plasmid PBBR-CasX-sgRNA-Δ orf149 Transform into Sinorhizobium meliloti TIB.SM.2013 (see CN104342390A), use apramycin as a selection marker, and screen to obtain positive transformants; (6) Cultivate the positive transformants at 30℃-32℃ until OD 600 The induction agent was added to the plate, and the colony PCR test was performed after dilution and plating. orf149 The upstream homology arm and the downstream homology arm of the gene produce homologous recombination with the genomic DNA of Sinorhizobium meliloti; (7) Pick the strain that has correctly undergone homologous recombination and inoculate it into a medium without apramycin. After several transfer cultures, the plasmid is lost. After smearing the plate, place it in a 30℃-32℃ incubator for culture. The grown colonies are copied on plates with apramycin resistance and plates without resistance to obtain plasmid-free strains. orf1177 Sinorhizobium meliloti SMΔ orf149 ; The genomic DNA sequence of the Chinese meliloti orf149 The gene is shown in SEQ ID NO:1.

[0020] Knockout orf149 The specific implementation method of the method for constructing the gene-containing Sinorhizobium meliloti is as follows: 1. The specific steps of step (1) are: According to the genomic DNA sequence of Sinorhizobium meliloti SM orf149 Genetic sequencing, design orf149 The upstream homology arm primer and the downstream homology arm primer of the gene were used as the template, respectively. orf149 The upstream homology arm primer and the downstream homology arm primer of the gene were used for PCR amplification to obtain orf149 The upstream homology arm of the gene (1025 bp) and orf149 Downstream homology arm of the gene (1050 bp).

[0021] 2. The specific steps of step (2) are: by orf149 The upstream homology arms of the gene and orf149 The downstream homology arm of the gene was used as a template, and the upstream homology arm primer orf149-upF and the downstream homology arm primer orf149-dR were used as primers. orf149 The upstream homology arms of the gene and orf149 The downstream homology arms of the gene were connected together to obtain the target gene fragment (2046 bp).

[0022] 3. The specific steps of step (3) are: Using plasmid PBBR-CasX-sgRNA as a template, primers were designed for PCR amplification to obtain a target orf149 The linearized plasmid vector fragment (6102 bp, 4467 bp) of the 20 nucleotide sequence of the gene.

[0023] 4. The specific steps of step (4) are: The fragment obtained in step (2) and the linear plasmid fragment obtained in step (3) were assembled by Gibson to obtain a ligation product; the product was transformed into Escherichia coli DH5α and screened in a culture medium containing apramycin resistance at 30°C-32°C to obtain transformants, and colonies of the transformants were selected for colony PCR verification. If the PCR verification result of the transformant is initially confirmed and then sent for sequencing, the correct sequence indicates that the knockout vector is successfully constructed, and the above transformant is a positive transformant.

[0024] 5. The specific steps of step (5) are: The knockout vector PBBR-CasX-sgRNA-Δ orf149 The strain was transferred into Sinorhizobium meliloti and screened at 30°C-32°C in a culture medium containing apramycin resistance to obtain transformants, which were then verified by colony PCR. PCR verification of the transformants confirmed the knockout vector PBBR-CasX-Δ orf149 Successful transformation into Sinorhizobium meliloti is a positive transformant (i.e., the knockout vector PBBR-CasX-sgRNA-Δ orf149 of Sinorhizobium meliloti SM).

[0025] 6. The specific steps of step (6) are: The positive transformants obtained in step (5) were cultured at 30°C-32°C on a culture medium containing apramycin resistance. 600 When the pH is about 0.8, add IPTG with a final concentration of 1mM for induction. Dilute the bacteria 24 hours after induction to about 10 5 After fold increase, the transformants were plated and verified by colony PCR to confirm the presence of orf149 The successful knockout of the gene is a positive transformant. Then DNA sequencing is performed on the positive transformants for further verification. orf149 The gene deletion strain (S. meliloti SMΔ orf149 ).

[0026] 7. The specific operation steps of step (7) are: The strain that has correctly undergone homologous recombination was inoculated in a culture medium without apramycin at 30℃-32℃ and cultured several times to lose the plasmid. After plating, the plate was placed in a 30℃-32℃ incubator for culture. The grown colonies were photocopied on the plates with apramycin resistance and without resistance. The colonies that grew on the plates without resistance but did not grow on the plates with apramycin resistance were picked, indicating that the plasmid of this colony had been completely lost during the subculture process, and finally a plasmid-free strain was obtained. orf149 Sinorhizobium meliloti SMΔ orf149 ; Electrophoresis was performed to detect the verification bands of the Sinorhizobium alfalfa SMΔorf149 strain. Figure 1 shown.

[0027] Example 2: Knockout orf Construction of Sinorhizobium meliloti expressing 993 gene A plasmid-free strain was obtained by a method similar to that in Example 1. orf 993 gene knockout Sinorhizobium meliloti SMΔ orf 993; among them, the genomic DNA sequence of Sinorhizobium meliloti orf The 993 gene is shown in SEQ ID NO:2.

[0028] Electrophoresis was performed to detect the verification bands of the alfalfa Sinorhizobium SMΔorf993 strain. Figure 1 shown.

[0029] Example 3: Knockout orf1177 Construction of Sinorhizobium meliloti A plasmid-free strain was obtained by a method similar to that in Example 1. orf1177 Sinorhizobium meliloti SMΔ orf1177 Among them, the genomic DNA sequence of Sinorhizobium meliloti orf1177 The gene is shown in SEQ ID NO:3.

[0030] Electrophoresis was performed to detect the verification bands of the Sinorhizobium alfalfa SMΔorf1177 strain. Figure 1 shown.

[0031] Example 4: Knockout orf1867 Construction of Sinorhizobium meliloti A plasmid-free strain was obtained by a method similar to that in Example 1. orf1867 Sinorhizobium meliloti SMΔ orf1867 Among them, the genomic DNA sequence of Sinorhizobium meliloti orf1867 The gene is shown in SEQ ID NO:4.

[0032] Electrophoresis was performed to detect the verification bands of the alfalfa Sinorhizobium SMΔorf1867 strain. Figure 1 shown.

[0033] Example 5: Knockout orf2327 Construction of Sinorhizobium meliloti A plasmid-free strain was obtained by a method similar to that in Example 1. orf2327 Sinorhizobium meliloti SMΔ orf2327 Among them, the genomic DNA sequence of Sinorhizobium meliloti orf2327 The gene is shown in SEQ ID NO:5.

[0034] Electrophoresis was performed to detect the verification bands of the Sinorhizobium alfalfa SMΔorf2327 strain. Figure 1 shown.

[0035] Example 6: Knockout orf2340 Construction of Sinorhizobium meliloti A plasmid-free strain was obtained by a method similar to that in Example 1. orf2340 Sinorhizobium meliloti SMΔ orf2340 ; wherein the genomic DNA sequence of the Chinese rhizobium alfalfa orf2340 The gene is shown in SEQ ID NO:6.

[0036] Electrophoresis was performed to detect the verification bands of the Sinorhizobium alfalfa SMΔorf2340 strain. Figure 1 shown.

[0037] Example 7: Knockout orf3280 Construction of Sinorhizobium meliloti A plasmid-free strain was obtained by a method similar to that in Example 1. orf3280 Sinorhizobium meliloti SMΔ orf3280 ; wherein the genomic DNA sequence of the Chinese rhizobium alfalfa orf3280 The gene is shown in SEQ ID NO:7.

[0038] Electrophoresis was performed to detect the verification bands of the Sinorhizobium alfalfa SMΔorf3280 strain. Figure 1 shown.

[0039] Example 8: Knockout orf3296 Construction of Sinorhizobium meliloti A plasmid-free strain was obtained by a method similar to that in Example 1. orf3296 Sinorhizobium meliloti SMΔ orf3296 ; wherein the genomic DNA sequence of the Chinese rhizobium alfalfa orf3296The gene is shown in SEQ ID NO: 8. Electrophoresis was performed and the verification band of the Sinorhizobium alfalfa SMΔorf3296 strain was as follows Figure 1 shown.

[0040] Example 9: Knockout orf4169 Construction of Sinorhizobium meliloti A plasmid-free strain was obtained by a method similar to that in Example 1. orf4169 Sinorhizobium meliloti SMΔ orf4169 ; wherein the genomic DNA sequence of the Chinese rhizobium alfalfa orf4169 The gene is shown in SEQ ID NO: 9. Electrophoresis was performed and the verification band of the Sinorhizobium alfalfa SMΔorf4169 strain was as follows Figure 1 shown.

[0041] Example 10: Knockout orf4207 The method for constructing the gene-containing Sinorhizobium alfalfa comprises the following steps: A plasmid-free strain was obtained by a method similar to that in Example 1. orf4207 Sinorhizobium meliloti SMΔ orf4207 ; wherein the genomic DNA sequence of the Chinese rhizobium alfalfa orf4207 The gene is shown in SEQ ID NO: 10. Electrophoresis was performed and the verification band of the Sinorhizobium alfalfa SMΔorf4207 strain was as follows Figure 1 shown.

[0042] Example 11: Production of Vitamin B12 Includes: A seed fermentation, B production fermentation.

[0043] The specific steps of seed fermentation are: first activate Sinorhizobium alfalfa, that is, first spread the frozen bacteria on LB solid culture medium, then inoculate the activated bacteria into the culture medium for seed fermentation, and then culture at 180-250rpm, 30℃-32℃ for 24-36 hours to obtain a seed cultured bacterial solution; the specific steps of production fermentation are: fill 25-30mL of production fermentation culture medium into a 250mL triangular flask, and then ferment the seed cultured bacterial solution with an inoculation amount of 10% (volume percentage), a rotation speed of 180-250rpm, a temperature of 30℃-32℃, and culture for 7 days to obtain a production fermented bacterial solution. The specific steps of seed fermentation and production fermentation are both prior art.

[0044] According to the above-mentioned Sinorhizobium meliloti SMΔ orf 149. SMΔ orf 993, SMΔ orf1177、 SMΔ orf 1867, SMΔ orf 2327, SMΔ orf 2340, SMΔ orf 3280, SMΔ orf 3296, SMΔ orf 4169 and SMΔ orf The application steps of 4207 in the production of vitamin B12 each provide 10 culture methods, and the formulas of the seed culture medium and the fermentation culture medium of the 10 culture methods are listed in Table 1.

[0045] Table 1

[0046] SMΔ orf 149. SMΔ orf 993, SMΔ orf1177、 SMΔ orf 1867, SMΔ orf 2327, SMΔ orf 2340, SMΔ orf 3280, SMΔ orf 3296, SMΔ orf 4169 and SMΔ orf The 4207 strain was fermented according to the above culture method to determine the production of vitamin B12; The inventors used high performance liquid chromatography (HPLC) to determine the yield of vitamin B12 in the fermented bacterial broth. Sample pretreatment: Take 1 mL of fermentation broth, add 8% sodium nitrite solution and 0.1 mL of glacial acetic acid, shake well, and place in a 95-100°C water bath for 30-60 minutes; wait until it cools to room temperature, centrifuge at 10,000 rpm for 1 minute, and filter the supernatant through a 0.22 μm filter membrane filter into a sample bottle. The amount of sodium nitrite solution and glacial acetic acid added can be adjusted accordingly with the amount of fermentation broth. The specific determination conditions are: C18-250A column (Agilent, 4.6mmid 9×250mm, 5μm). The mobile phase is 30% organic phase (methanol) and 70% inorganic phase (water), the absorption wavelength is 361nm, the column temperature is 35°C, the flow rate is 0.8mL / min, and the injection volume is 15 μL. The vitamin B12 production in the fermentation broth was calculated based on the standard curve prepared using the vitamin B12 standard. The value is the average of three parallel experiments (see Table 2-11).

[0047] Table 2

[0048] Table 3

[0049] Table 4

[0050] Table 5

[0051] Table 6

[0052] Table 7

[0053] Table 8

[0054] Table 9

[0055] Table 10

[0056] Table 11

[0057] As can be seen from Table 2-11, under the same seed fermentation and production fermentation conditions, compared with the prior art Chinese meliloti rhizobium SM, the Chinese meliloti rhizobium SM of the present invention is more orf 149. SMΔ orf 993, SMΔ orf1177、 SMΔ orf 1867, SMΔ orf 2327, SMΔ orf 2340, SMΔ orf 3280, SMΔ orf 3296, SMΔ orf 4169 and SMΔ orf The vitamin B12 titer in the fermented bacterial liquid of 4207 was greatly improved (by 9.8-50.8%), indicating that the technical solution of the present invention has great application value in increasing the vitamin B12 yield of Sinorhizobium alfalfa.

Claims

1. A Sinorhizobium medicae with knocked-out or weakened transcription factors, characterized in that: It knocks out or weakens the transcription factor in the original Sinorhizobium meliloti orf 149. orf 993, orf1177、orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and orf One or more of the 4207 genes.

2. The Sinorhizobium meliloti of claim 1, wherein The starting Sinorhizobium meliloti is the Sinorhizobium meliloti strain TIB.SM.2013.

3. The Sinorhizobium meliloti of claim 1, wherein Transcription Factors orf 149. orf 993, orf1177、orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and orf The amino acid sequences encoded by the 4207 gene are: WP_127888815.1, WP_034803756.1, KSV74715.1, KSV65705.1, WP_127889366.1, KSV74068.1, WP_127889532.1, WP_034802440.1, WP_113504689.1 and WP_029742798.

1.

4. The Sinorhizobium meliloti of claim 1, wherein Transcription Factors orf 149. orf 993, orf1177、orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and orf The nucleotide sequences of the 4207 genes are shown in SEQ ID NOs: 1-10, respectively.

5. A method for constructing Sinorhizobium meliloti according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1 was respectively converted into orf 149. orf 993, orf1177、orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and / or orf The upstream homology arms of 4207 gene and orf 149. orf 993, orf1177, orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and / or orf The downstream homology arms of the 4207 gene were connected together to form the target gene fragment; S2 was PCR amplified with targeted orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and / or orf4207 Gene knockout plasmid vector PBBR-CasX-sgRNA; S3 used Gibson assembly to connect the knockout fragment obtained in S1 and the linear plasmid fragment obtained in S2, respectively, to obtain knockout plasmids PBBR-CasX-sgRNA-Δorf149, PBBR-CasX-sgRNA-Δorf993, PBBR-CasX-sgRNA-Δorf1177, PBBR-CasX-sgRNA-Δorf1867, PBBR-CasX-sgRNA-Δorf2327, PBBR-CasX-sgRNA-Δorf2340, PBBR-CasX-sgRNA-Δorf3280, PBBR-CasX-sgRNA-Δorf3296, PBBR-CasX-sgRNA-Δorf4169 and PBBR-CasX-sgRNA-Δorf4207, respectively; S4: respectively transform the knockout plasmids PBBR-CasX-sgRNA-Δorf149, PBBR-CasX-sgRNA-Δorf993, PBBR-CasX-sgRNA-Δorf1177, PBBR-CasX-sgRNA-Δorf1867, PBBR-CasX-sgRNA-Δorf2327, PBBR-CasX-sgRNA-Δorf2340, PBBR-CasX-sgRNA-Δorf3280, PBBR-CasX-sgRNA-Δorf3296, PBBR-CasX-sgRNA-Δorf4169 and / or PBBR-CasX-sgRNA-Δorf4207 into Sinorhizobium meliloti, use apramycin as a selection marker, and screen to obtain positive transformants; S5: positive transformants were cultured at 30°C-32°C until OD 600 0.6-0.8, add inducer, dilute and plate, and perform colony PCR test to obtain orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and orf4207 The upstream homology arm and downstream homology arm of the gene produce a strain of homologous recombination with the genomic DNA of Sinorhizobium meliloti.

6. The construction method according to claim 5, characterized in that: The following steps are also included: S6 Select the strain that has correctly undergone homologous recombination and inoculate it into a medium without apramycin. After several transfer cultures, the plasmid is lost. After plating, the plate is cultured in a 30℃-32℃ incubator. The grown colonies are copied on plates containing apramycin resistance and plates without resistance to obtain plasmid-free and plasmid-free strains. orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and orf4207 Sinorhizobium meliloti Δ orf149, Δ orf993, Δ orf1177, Δ orf1867, Δ orf2327, Δ orf2340, Δ orf3280, Δ orf3296, Δ orf4169 and Δ orf4207 .

7. Use of the Sinorhizobium meliloti according to any one of claims 1 to 4 in the production of vitamin B12.

8. A method for preparing vitamin B12, characterized in that: The method comprises the following steps: after carrying out seed fermentation on a seed fermentation medium, the seeds are inoculated into a fermentation medium for fermentation to produce vitamin B12, wherein the medium contains a carbon source.

9. The method according to claim 8, characterized in that The carbon source is one or more of sucrose, corn syrup and / or betaine.

10. The method according to claim 9, characterized in that The culture medium formula of the seed fermentation is: 20-40 sucrose, 10-20 corn steep liquor, 2.5-5 betaine, 0.5-1 (NH4)2SO4, 1-2 (NH4)2HPO4, 0.4-0.8 MnSO4·H2O, 0.01-0.02 CoCl2·6H2O, 0.15-0.3 MgO, 0.005-0.01 DMBI, 0.005-0.01 ZnSO4·7H2O, 0.75-1.5 CaCO3, pH is controlled at 7.0-7.4 by NaOH; the fermentation culture medium formula is: 40-80 sucrose, 15-30 corn steep liquor, 7.5-15 betaine, 2 (NH4)2SO4, 1.5 MgSO4, 0.75 K2HPO4, 0.14 CoCl2·6H2O, DMBI 0.075, ZnSO4·7H2O 0.08, CaCO3 1 g / L, pH was controlled at 7.0-7.4 by NaOH.

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