Sinorhizobium meliloti strain with knocked-out transcription factor, construction method and application thereof

Abstract

The present invention provides a Sinorhizobium meliloti strain with a knocked-out transcription factor, a construction method and an application thereof. By means of genetic engineering, the present invention successfully knocks out transcription factors orf 149, orf 993, orf1177、 orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and orf 4207 genes in the genome of Sinorhizobium meliloti, providing a new strategy for increasing the production of vitamin B12. Compared with the wild-type Sinorhizobium meliloti, the vitamin B12 production of the recombinant strain of Sinorhizobium meliloti constructed by the present invention is increased by more than 50% at most.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the genetic modification of Sinorhizobium meliloti strains, and more particularly to Sinorhizobium meliloti strains with transcription factors knocked out, and construction methods and applications thereof. Background Art

[0002] Vitamin B12, also known as cobalamin, is a corrinoid compound and has wide applications in the pharmaceutical and food industries. Vitamin B12 has a large molecular weight and a complex chemical structure. Chemical synthesis is time-consuming, laborious, costly, and has a long synthesis cycle. Moreover, it has high requirements for 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, the microbial fermentation method is the cheapest method for producing vitamin B12 and is widely used in industrial production. As a natural vitamin B12-producing strain, rhizobia have great potential in industrial production [Dong H, Li S, Fang H, Xia M, Zheng P, Zhang D, Sun J. A newly isolated and identified vitamin B12 producing strain: 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 gene expression of vitamin B12 synthesis pathways to improve vitamin B12 production, while there are few reports on improving vitamin B12 production by modifying transcriptional regulators [Kang Z, Zhang J, Zhou J, Qi Q, Du G, Chen J. Recent advances in microbial production of δ-aminolevulinic acid and vitamin B12. Biotechnol Adv 2012; 30: 1533-42].

[0004] There are many genes in Sinorhizobium meliloti that are closely related to the synthesis of strain metabolites, and it is still unknown which genes are related to vitamin B12 production. Further research is needed on the way of gene modification to obtain engineering bacteria with high-yield vitamin B12. Summary of the Invention

[0005] It was found in the research of the present invention that after stimulating Sinorhizobium medicae with vitamin B12 and performing differential analysis of transcriptome gene expression, some transcription factors were significantly up-regulated and 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 genomic level, and it was found that the knockout of these transcription factors could increase the yield of vitamin B12 by the strain.

[0006] The present invention provides a Sinorhizobium medicae in which a transcription factor is knocked out or weakened, and the transcription factor is knocked out or weakened in the starting Sinorhizobium medicae. orf 149, orf 993, orf1177, orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and orf one or more of the genes 4207.

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

[0008] Specifically, the 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 genes 4207 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, respectively.

[0009] More specifically, the 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 gene are shown in SEQ ID NO: 1-10 respectively.

[0010] The present invention provides a method for constructing the above-mentioned Sinorhizobium meliloti, which includes the following steps:

[0011] S1 By overlapping extension PCR, respectively orf 149, orf 993, orf1177, orf 1867, orf 2327, orf 2340, orf 3280, orf 3296, orf 4169 and / or orf the upstream homologous arms of the 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 homologous arms of the 4207 gene are ligated together to form a target gene fragment;

[0012] S2 PCR amplify the knockout plasmid vector PBBR-CasX-sgRNA carrying the orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and / or orf4207 gene respectively;

[0013] S3 Use Gibson assembly to ligate 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;

[0014] S4 separately transfers 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, uses apramycin as a screening marker, and screens to obtain positive transformants;

[0015] S5 Cultures the positive transformants at 30°C - 32°C until the OD 600 reaches 0.6 - 0.8, adds an inducer, dilutes and coats the plates, and then performs colony PCR detection to respectively obtain orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and orf4207 strains in which the upstream homologous arm and the downstream homologous arm of the gene undergo homologous recombination with the genomic DNA of Sinorhizobium meliloti.

[0016] Optionally, it further includes the following steps:

[0017] S6 Picks the strains with correct homologous recombination and inoculates them into a medium without apramycin, transfers and cultures them several times to lose the plasmid, coats the plates and cultures them in an incubator at 30°C - 32°C, and then performs replica plating of the grown colonies on plates with apramycin resistance and without resistance to respectively obtain plasmid-free orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and orf4207 gene knockout strain Sinorhizobium meliloti Δ orf149, Δ orf993, Δ orf1177, Δ orf1867, Δ orf2327, Δ orf2340, Δ orf3280, Δ orf3296, Δ orf4169 and Δ orf4207 ..

[0018] The present invention provides the application of the described Sinorhizobium meliloti in the production of vitamin B12.

[0019] The present invention further provides a method for preparing vitamin B12, which includes the following steps: After performing seed fermentation on a seed fermentation medium, inoculate it into a fermentation medium to ferment and produce vitamin B12, and the medium contains a carbon source.

[0020] Specifically, the carbon source is one or more of sucrose, corn steep liquor, and / or betaine.

[0021] More specifically, the medium formula for seed fermentation is: sucrose 40, corn steep liquor 20, betaine 5, (NH4)2SO4 1, (NH4)2HPO4 2, MnSO4·H2O 0.8, CoCl2·6H2O 0.02, MgO 0.3, DMBI 0.01, ZnSO4·7H2O 0.01, CaCO3 1.5 (g / L), and the pH is controlled at 7.0 - 7.4 with NaOH; the medium formula for fermentation is: sucrose 80, corn steep liquor 30, betaine 15, (NH4)2SO4 2, MgSO4 1.5, K2HPO4 0.75, CoCl2·6H2O 0.14, DMBI 0.075, ZnSO4·7H2O 0.08, CaCO3 1 g / L, and the pH is controlled at 7.0 - 7.4 with NaOH.

[0022] 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 has the highest vitamin B12 production increased by more than 50%. The research results of the present invention show that knocking out the transcription factor in the genomic DNA of Sinorhizobium meliloti is a very effective method to improve the vitamin B12 production. Description of the Drawings

[0023] Figure 1. Verification bands of Sinorhizobium meliloti SMΔorf149, SMΔorf993, SMΔorf1177, SMΔorf1867, SMΔorf2327, SMΔorf2340, SMΔorf3280, SMΔorf3296, SMΔorf4169 and SMΔorf4207 strains obtained in step (7) of Example 1. Lane 13 is the DNA marker. Lanes 1, 3, 5, 7, 9, 11, 14, 16, 18, 20 are the PCR verification bands of wild Sinorhizobium meliloti SM. Lanes 2, 4, 6, 8, 10, 12, 15, 17, 19, 21 are the verification bands of Sinorhizobium meliloti SMΔorf149, SMΔorf993, SMΔorf1177, SMΔorf1867, SMΔorf2327, SMΔorf2340, SMΔorf3280, SMΔorf3296, SMΔorf4169 and SMΔorf4207 strains in which orf149, orf993, orf1177, orf1867, orf2327, orf2340, orf3280, orf3296, orf4169 and orf4207 genes have been successfully knocked out;

[0024] 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, 200 bp. Detailed implementation mode

[0025] Now, the preferred embodiments of the present invention will be specifically described according to the attached drawings, but it does not constitute a limitation to the present invention.

[0026] Example 1: Construction of Sinorhizobium meliloti with knocked-out orf149 genes, including the following steps:

[0027] (1) Using the genomic DNA of Sinorhizobium meliloti as a template, PCR amplify the upstream homologous arm and downstream homologous arm of the orf149 gene;

[0028] (2) Connect the upstream homologous arm of the orf149 gene and the downstream homologous arm of the orf149 gene together by overlap extension PCR to form a target gene fragment;

[0029] (3) PCR amplify the knockout plasmid vector PBBR-CasX-sgRNA with a target orf149 gene;

[0030] (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 ;

[0031] (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;

[0032] (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;

[0033] (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, 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 ;

[0034] The genomic DNA sequence of the Chinese meliloti orf149 The gene is shown in SEQ ID NO:1.

[0035] Knockout orf149 The specific implementation method of the method for constructing the gene-containing Sinorhizobium meliloti is as follows:

[0036] 1. The specific steps of step (1) are:

[0037] 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).

[0038] 2. The specific steps of step (2) are:

[0039] byorf149 The upstream homologous arm of the gene and orf149 the downstream homologous arm of the gene were used as templates, and the upstream homologous arm primer orf149-upF and the downstream homologous arm primer orf149-dR were used as primers. Through overlap extension PCR, orf149 the upstream homologous arm of the gene and orf149 the downstream homologous arm of the gene were ligated together to obtain the target gene fragment (2046 bp).

[0040] 3. The specific operation steps of step (3) are as follows:

[0041] Using plasmid PBBR-CasX-sgRNA as a template, primers were designed for PCR amplification to obtain a linearized plasmid vector fragment (6102 bp, 4467 bp) with a 20-nucleotide sequence targeting the orf149 gene.

[0042] 4. The specific operation steps of step (4) are as follows:

[0043] The fragment obtained in step (2) and the linear plasmid fragment obtained in step (3) were subjected to Gibson assembly to obtain a ligation product; it was transformed into Escherichia coli DH5α and screened on a medium containing apramycin resistance at 30°C - 32°C to obtain transformants. Colony PCR verification was performed on the transformants by picking colonies. If the PCR verification result of the transformants was preliminarily confirmed, it was sent for sequencing. A correct sequence indicated that the knockout vector was successfully constructed, and the above transformants were positive transformants.

[0044] 5. The specific operation steps of step (5) are as follows:

[0045] The knockout vector PBBR-CasX-sgRNA-Δ orf149 was transformed into Sinorhizobium meliloti, and screened on a medium containing apramycin resistance at 30°C - 32°C to obtain transformants. Colony PCR verification was performed on the transformants. The PCR verification of the transformants was used to prove that the knockout vector PBBR-CasX-Δ orf149 was successfully transformed into Sinorhizobium meliloti, and it was a positive transformant (i.e., Sinorhizobium meliloti SM into which the knockout vector PBBR-CasX-sgRNA-Δ orf149 was transferred).

[0046] 6. The specific operation steps of step (6) are as follows:

[0047] The positive transformants obtained in step (5) were cultured on a medium containing apramycin resistance at 30°C - 32°C. When the cell density reached OD 600 of about 0.8, IPTG with a final concentration of 1 mM was added for induction. After 24 hours of induction, the cells were diluted, diluted approximately 105 After amplification, plate coating was performed, and colony PCR was carried out on the transformants to prove that the orf149 gene on the genome of Sinorhizobium meliloti SM was successfully knocked out, and the positive transformants were obtained. Subsequently, DNA sequencing was performed on the positive transformants for further verification, and orf149 a gene deletion strain (i.e., Sinorhizobium meliloti SMΔ orf149 ) was obtained.

[0048] 7. The specific operation steps of step (7) are as follows:

[0049] The strain with correct homologous recombination was inoculated into a medium without apramycin at 30°C - 32°C and subcultured several times. The plasmid was lost. After plate coating, it was cultured in an incubator at 30°C - 32°C. The grown colonies were replica plated on plates with and without apramycin resistance. The colonies that grew on the plate without resistance but not on the plate with apramycin resistance were picked. This indicates that all plasmids in this colony have been lost during subculture, and finally, a plasmid-free orf149 gene knockout Sinorhizobium meliloti SMΔ orf149 was obtained;

[0050] And electrophoresis detection was carried out. The verification bands of Sinorhizobium meliloti SMΔorf149 strain are as Figure 1 shown.

[0051] Example 2: Construction of Sinorhizobium meliloti with orf orf993 gene knocked out

[0052] A plasmid-free orf orf993 gene knockout Sinorhizobium meliloti SMΔ orf orf993 was obtained by a method similar to that in Example 1; among them, the orf orf993 gene in the genomic DNA sequence of Sinorhizobium meliloti is as shown in SEQ ID NO: 2.

[0053] And electrophoresis detection was carried out. The verification bands of Sinorhizobium meliloti SMΔorf993 strain are as Figure 1 shown.

[0054] Example 3: Construction of Sinorhizobium meliloti with orf1177 a gene knocked out

[0055] A plasmid-free orf1177 gene knockout strain Sinorhizobium meliloti SMΔ orf1177 was obtained by a method similar to that in Example 1; among them, the orf1177 gene in the genomic DNA sequence of Sinorhizobium meliloti is as shown in SEQ ID NO: 3.

[0056] Electrophoresis detection was carried out, and the verification bands of Sinorhizobium meliloti strain SMΔorf1177 are as Figure 1 shown.

[0057] Example 4: Construction of Sinorhizobium meliloti with orf1867 gene knocked out

[0058] A plasmid-free orf1867 gene knockout strain Sinorhizobium meliloti SMΔ orf1867 was obtained by a method similar to that in Example 1; wherein, the orf1867 gene in the genomic DNA sequence of Sinorhizobium meliloti is as shown in SEQ ID NO: 4.

[0059] Electrophoresis detection was carried out, and the verification bands of Sinorhizobium meliloti strain SMΔorf1867 are as Figure 1 shown.

[0060] Example 5: Construction of Sinorhizobium meliloti with orf2327 gene knocked out

[0061] A plasmid-free orf2327 gene knockout strain Sinorhizobium meliloti SMΔ orf2327 was obtained by a method similar to that in Example 1; wherein, the orf2327 gene in the genomic DNA sequence of Sinorhizobium meliloti is as shown in SEQ ID NO: 5.

[0062] Electrophoresis detection was carried out, and the verification bands of Sinorhizobium meliloti strain SMΔorf2327 are as Figure 1 shown.

[0063] Example 6: Construction of Sinorhizobium meliloti with orf2340 gene knocked out

[0064] A plasmid-free orf2340 gene knockout strain Sinorhizobium meliloti SMΔ orf2340 was obtained by a method similar to that in Example 1; wherein, the orf2340 gene in the genomic DNA sequence of Sinorhizobium meliloti is as shown in SEQ ID NO: 6.

[0065] Electrophoresis detection was carried out, and the verification bands of Sinorhizobium meliloti strain SMΔorf2340 are as Figure 1 shown.

[0066] Example 7: Construction of Sinorhizobium meliloti with orf3280 gene knocked out

[0067] A plasmid-free orf3280Gene knockout strain Sinorhizobium meliloti SMΔ orf3280 ; wherein, the orf3280 gene is shown in SEQ ID NO: 7.

[0068] And electrophoresis detection was carried out. The verification band of the Sinorhizobium meliloti SMΔorf3280 strain is as Figure 1 shown.

[0069] Example 8: Construction of Sinorhizobium meliloti with orf3296 gene knocked out

[0070] A plasmid-free orf3296 gene knockout strain Sinorhizobium meliloti SMΔ orf3296 was obtained by a method similar to that in Example 1; wherein, the orf3296 gene in the genomic DNA sequence of the Sinorhizobium meliloti is shown in SEQ ID NO: 8. And electrophoresis detection was carried out. The verification band of the Sinorhizobium meliloti SMΔorf3296 strain is as Figure 1 shown.

[0071] Example 9: Construction of Sinorhizobium meliloti with orf4169 gene knocked out

[0072] A plasmid-free orf4169 gene knockout strain Sinorhizobium meliloti SMΔ orf4169 was obtained by a method similar to that in Example 1; wherein, the orf4169 gene in the genomic DNA sequence of the Sinorhizobium meliloti is shown in SEQ ID NO: 9. And electrophoresis detection was carried out. The verification band of the Sinorhizobium meliloti SMΔorf4169 strain is as Figure 1 shown.

[0073] Example 10: Method for constructing Sinorhizobium meliloti with orf4207 gene knocked out, comprising the following steps:

[0074] A plasmid-free orf4207 gene knockout strain Sinorhizobium meliloti SMΔ orf4207 was obtained by a method similar to that in Example 1; wherein, the orf4207 gene in the genomic DNA sequence of the Sinorhizobium meliloti is shown in SEQ ID NO: 10. And electrophoresis detection was carried out. The verification band of the Sinorhizobium meliloti SMΔorf4207 strain is as Figure 1 shown.

[0075] Example 11: Production of vitamin B12

[0076] Including: A seed fermentation, B production fermentation.

[0077] The specific steps of seed fermentation are as follows: First, activate Sinorhizobium meliloti, that is, coat the frozen bacteria on the LB solid medium, and then inoculate the activated strain into the seed fermentation medium and culture it at 180 - 250 rpm and 30℃ - 32℃ for 24 - 36 hours to obtain the bacterial liquid for seed culture; The specific steps of production fermentation are as follows: Load 25 - 30 mL of the production fermentation medium into a 250 mL Erlenmeyer flask, and then inoculate the bacterial liquid for seed culture with an inoculation amount of 10% (volume percentage), a rotation speed of 180 - 250 rpm, and a temperature of 30℃ - 32℃, and ferment and culture for 7 d to obtain the bacterial liquid for production fermentation. The above specific steps of seed fermentation and production fermentation are all prior arts.

[0078] According to the above 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 4207, their application steps in vitamin B12 production each provide 10 culture methods, and Table 1 respectively lists the formulations of the seed medium and fermentation medium for the 10 culture methods.

[0079] Table 1

[0080]

[0081] All respectively use 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 4207 strains are fermented according to the above culture methods to determine the yield of vitamin B12;

[0082] The present inventor used high performance liquid chromatography (HPLC) method to determine the yield of vitamin B12 in the fermented bacterial liquid. Sample pretreatment: Take 1 mL of the fermentation broth, add 0.1 mL each of 8% sodium nitrite solution and glacial acetic acid, shake well, and place in a water bath at 95 - 100 °C for 30 - 60 min; after cooling to room temperature, centrifuge at 10000 rpm for 1 min, and filter the supernatant through a 0.22 μm membrane filter into a sample vial. The addition amounts of sodium nitrite solution and glacial acetic acid can be adjusted accordingly according to the amount of the fermentation broth. The specific determination conditions are as follows: C18-250A column (Agilent, 4.6 mm id × 250 mm, 5 μm). The mobile phase is 30% organic phase (methanol) and 70% inorganic phase (water), the absorption wavelength is 361 nm, the column temperature is 35 °C, the flow rate is 0.8 mL / min, and the injection volume is 15 μL. Calculate the yield of vitamin B12 in the fermented bacterial liquid according to the standard curve made from the vitamin B12 standard product, and the value is the average of three parallel experiments (see Table 2-11).

[0083] Table 2

[0084]

[0085] Table 3

[0086]

[0087] Table 4

[0088]

[0089] Table 5

[0090]

[0091] Table 6

[0092]

[0093] Table 7

[0094]

[0095] Table 8

[0096]

[0097] Table 9

[0098]

[0099] Table 10

[0100]

[0101] Table 11

[0102]

[0103] As can be seen from Table 2-11, under the same conditions of seed fermentation and production fermentation, compared with Sinorhizobium meliloti SM of the prior art, the 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 4207 have a significant increase in the titer of vitamin B12 in the fermentation broth of production fermentation (an increase of 9.8% - 50.8%). It shows that: the technical solution of the present invention has great application value in improving the yield of vitamin B12 of Sinorhizobium meliloti.

Claims

1. A transcription factor knocked out Sinorhizobium medicae, characterized in that: It knocked out the transcription factor in the original Sinorhizobium meliloti orf1177 , the transcription factor orf1177 The amino acid sequence encoded by the gene is: KSV74715.

1.

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 orf1177 The nucleotide sequence of the gene is shown in SEQ ID NO:

3.

4. A method for constructing Sinorhizobium meliloti according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1 was respectively converted into orf1177 The upstream homology arms of the gene and orf1177 The downstream homologous arms of the gene are connected together to form the target gene fragment; S2 was PCR amplified with targeted orf1177 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 to obtain knockout plasmids PBBR-CasX-sgRNA-Δorf1177; S4 respectively transferred the knockout plasmid PBBR-CasX-sgRNA-Δorf1177 into Sinorhizobium meliloti, and used apramycin as a selection marker to screen for positive transformants; S5: positive transformants were cultured at 30°C-32°C until OD 600 The induction agent was added to the plate, and the colony PCR test was performed after dilution and plating. orf1177 The upstream homology arm and the downstream homology arm of the gene produce homologous recombination with the genomic DNA of Sinorhizobium meliloti; 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 no resistance to obtain plasmid-free strains. orf1177 Sinorhizobium meliloti Δ orf1177 .

5. Use of the Sinorhizobium meliloti according to any one of claims 1 to 3 in the production of vitamin B12.

6. A method for preparing vitamin B12, characterized in that: The method comprises the following steps: fermenting the Chinese rhizobium alfalfa obtained by the construction method according to claim 4 on a seed fermentation medium, and then inoculating the medium into a fermentation medium for fermentation to produce vitamin B12, wherein the medium contains a carbon source.

7. The method according to claim 6, characterized in that The carbon source is one or more of sucrose, corn syrup and / or betaine.

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