Genetically engineered leucothrix mucor strain for improving vitamin b12 production

Abstract

This invention discloses a genetically engineered *Streptococcus adhesioides* strain that enhances vitamin B12 production, belonging to the field of bacterial engineering technology. The genetically engineered strain contains a recombinant expression plasmid containing the natural *cobPWNO* operon. The *cobPWNO* operon consists of the natural promoter of the *cobP* gene and the coding sequences of the *cobP*, *cobW*, *cobN*, and *cobO* genes in tandem. This invention, by preserving the natural operon structure through overexpression, perfectly maintains the inherent synergistic regulatory relationship between the four key genes. This coordinated overexpression method ensures a balanced enzyme ratio in each step of the metabolic pathway, allowing for smooth metabolic flow. This not only enhances the synthesis of the target product, vitamin B12, but also avoids the accumulation of intermediate products or metabolic disorders caused by excessively strong local metabolic nodes. Furthermore, by utilizing the gene's own natural promoter, it avoids cellular stress responses that may be triggered by strong exogenous promoters, ensuring normal bacterial growth.

Description

Technical Field

[0001] This invention belongs to the field of engineered bacteria technology, specifically relating to a genetically engineered *Strombus amygdalinus* strain that increases vitamin B12 production. Background Technology

[0002] Vitamin B12, also known as cobalamin, is an essential water-soluble vitamin for human and animal physiological activities, playing a crucial role in maintaining nervous system function, erythrocyte production, and DNA synthesis. Since the human body cannot synthesize vitamin B12, it must be obtained through diet or supplements, making it an important product in the pharmaceutical, food additive, and feed industries. With increasing health awareness and the large-scale development of the livestock industry, the global market demand for vitamin B12 continues to grow, placing higher demands on efficient and low-cost production technologies. Currently, large-scale vitamin B12 production mainly relies on microbial fermentation, achieving vitamin B12 accumulation under optimized fermentation conditions through the screening and improvement of high-yield strains. Common production strains include *Pseudomonas denitrificans*, *Propionibacterium fischeri*, and *Streptococcus adheringii*, which has gained attention in recent years. *Streptococcus adheringii*, as a new vitamin B12 production strain, shows great potential for industrial application due to its rapid growth, clear genetic background, and relatively simple fermentation process, making it an important research subject for the breeding of high-yield vitamin B12 strains.

[0003] The biosynthetic pathway of vitamin B12 is extremely complex, involving the co-expression and regulation of nearly 30 genes. In *Strombus mycoides*, *cobP*, *cobW*, *cobN*, and *cobO* are key genes located on the same operon, participating in core steps such as precursor transformation, cobalt ion binding and transport, and cobalt ion insertion and modification. These genes are arranged in tandem on the genome and are regulated by the same upstream promoter. This natural operon structure is crucial for maintaining the stoichiometric balance of gene expression and the coordination of metabolic flux. In recent years, increasing vitamin B12 production through metabolic engineering has become a research hotspot. Common strategies include enhancing precursor supply, optimizing cofactor synthesis, and overexpressing rate-limiting enzyme genes. Regarding gene overexpression, the common approach is to assemble independent strong promoters for one or more genes to increase the transcriptional level of the target gene, thereby increasing the yield of the corresponding enzyme protein. However, this strategy often ignores the inherent co-expression patterns among multiple functionally related genes, potentially leading to metabolic network imbalance, increased metabolic burden on the host cell, and even unpredictable cellular stress responses, which may inhibit bacterial growth and the synthesis of the target product. Therefore, developing a genetic improvement strategy that can effectively enhance the vitamin B12 synthesis pathway while maximally maintaining the balance of endogenous regulation within cells is of great significance for increasing the fermentation yield and industrial production efficiency of vitamin B12.

[0004] Therefore, there is a need for a genetically engineered *Strombus fasciatus* strain that can increase vitamin B12 production, in order to solve the problems existing in the current technology. Summary of the Invention

[0005] The purpose of this invention is to provide a genetically engineered *Strombus amygdalinus* strain that increases vitamin B12 production, thereby addressing the problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a genetically engineered *Strombus adhesiveus* strain that increases vitamin B12 production, wherein the genetically engineered strain contains a recombinant expression plasmid, the recombinant expression plasmid contains the natural operon cobPWNO, the natural operon cobPWNO being composed of the natural promoter of the cobP gene and the coding sequences of the cobP, cobW, cobN and cobO genes in tandem.

[0007] In a preferred embodiment, the vector backbone of the recombinant expression plasmid is the broad-spectrum expression vector pBBR1MCS-5, which contains the origin of replication for autonomous replication in *Strombus amygdalinus* and resistance marker genes for selection.

[0008] As a preferred embodiment, the nucleotide sequence of the cobPWNO natural operon is shown in SEQ ID NO:1.

[0009] In a preferred embodiment, the recombinant plasmid is pBBR-cobPWNO, which is constructed by inserting the natural cobPWNO operon into the multiple cloning site of the pBBR1MCS-5 vector; the natural cobPWNO operon is driven by the natural promoter of the cobP gene, which controls the tandem coding regions of the cobP, cobW, cobN, and cobO genes.

[0010] As a preferred embodiment, the method includes the following steps: (1) Amplify a complete DNA fragment containing the natural promoter of cobP and the genes of cobP, cobW, cobN and cobO from *Strombus amygdalinus*. (2) The DNA fragment obtained in step (1) is cloned into the multiple cloning site of the pBBR1MCS-5 vector to obtain the recombinant plasmid pBBR-cobPWNO. (3) The recombinant plasmid is introduced into the host cell of *Strombus adherentus* to obtain the recombinant *Strombus adherentus*.

[0011] As a preferred embodiment, a method for verifying the vitamin B12 yield of recombinant *Strombus adhesiveus* includes the following steps: (1) Fermentation culture: The recombinant adhesiocytosis was cultured in a fermentation medium under appropriate conditions; (2) Sample preparation: Add glacial acetic acid and sodium nitrite solution to the fermentation broth, hydrolyze in a boiling water bath, dilute and centrifuge, and take the supernatant; (3) Content determination: The vitamin B12 in the sample was quantitatively analyzed by high performance liquid chromatography.

[0012] As a preferred embodiment, recombinant *Streptococcus adhesus* is used in the production of vitamin B12.

[0013] In a preferred embodiment, the fermentation culture temperature is 30-34℃, the culture time is 7-9 days, and the fermentation culture medium contains carbon source, nitrogen source and inorganic salt.

[0014] In one preferred embodiment, the recombinant *Strombus amygdalinus* is fermented, mixed with a freeze-drying protectant, freeze-dried, and then stored at low temperature for a long period of time.

[0015] Compared with the prior art, the *Strombus adhesiveus* genetically engineered strain provided by the present invention for increasing vitamin B12 production has at least the following beneficial effects: Compared to existing strategies that assemble independent strong promoters for each gene, this invention maintains the inherent synergistic regulatory relationship among four key genes by preserving the natural operon structure during overexpression. This coordinated overexpression method keeps the enzyme ratios at each step of the metabolic pathway balanced, allowing for smooth metabolic flow. This enhances the synthesis of the target product, vitamin B12, while avoiding the accumulation of intermediate products or metabolic disorders caused by excessive local metabolic nodes. Furthermore, utilizing the genes' own natural promoters avoids cellular stress responses that might be triggered by exogenous strong promoters, ensuring normal bacterial growth. Experimental data show that, compared to wild-type strains, the engineered bacteria constructed in this invention increase vitamin B12 production by approximately 7% without significantly affecting bacterial growth, demonstrating excellent potential for industrial application. This overexpression strategy based on the natural operon structure provides a more refined and coordinated genetic modification approach for microbial metabolic engineering. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the construction of pBBR-cobPWNO according to the present invention; Figure 2 This is a schematic diagram illustrating the vitamin B12 production increase of the present invention.

[0017] In the diagram: 1. Shell; 101. Shell; 102. Shell; 103. Shell; 104. Shell; 2. Door panel; 201. Door panel; 202. Door panel; 203. Door panel; 3. Waste collection component; 4. Support component; 5. Cylinder; 6. Pressure plate; 7. Protrusion; 8. Discharge door; 9. Wastewater tank; 10. Ventilation hole; 11. Box body; 12. Slide plate; 13. Bottom hole; 14. Spring; 15. Groove; 16. Partition plate; 17. Guide plate; 18. Through hole. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments.

[0019] Please see Figure 1-2This invention provides a genetically engineered *Ensifera adhaerens* strain for increasing vitamin B12 production. The engineered strain contains a recombinant expression plasmid containing the natural operon *cobPWNO*. The *cobPWNO* operon consists of the natural promoter of the *cobP* gene and the coding sequences of the *cobP*, *cobW*, *cobN*, and *cobO* genes in tandem. *Ensifera adhaerens*, a vitamin B12-producing strain with industrial potential, possesses a natural operon on its chromosome containing four key genes: *cobP*, *cobW*, *cobN*, and *cobO*. These four genes play indispensable synergistic roles in the vitamin B12 biosynthesis pathway: *cobP* participates in precursor transformation, *cobW* is responsible for cobalt ion binding and transport, and *cobN* and *cobO* are responsible for cobalt ion insertion and modification. In their natural state, these four genes are regulated by the same upstream promoter and are arranged in tandem. This structure ensures the stoichiometric balance of gene expression and the coordination of metabolic flux. This invention achieves overexpression of the cobPWNO operon, which contains the natural promoter and the complete coding sequence, in a recombinant expression plasmid and introduces it into *Cyclophorus fasciatus* host cells. Because the natural operon structure and promoter regulation are preserved, the expression ratio of the four genes is maintained, and the enhancement of metabolic pathways is carried out in a coordinated manner, avoiding metabolic stress caused by expression imbalance. Compared to existing strategies that assemble independent strong promoters for each gene, this invention maintains the inherent synergistic regulatory relationship among four key genes by preserving the natural operon structure during overexpression. This coordinated overexpression method keeps the enzyme ratios at each step of the metabolic pathway balanced, allowing for smooth metabolic flow. This enhances the synthesis of the target product, vitamin B12, while avoiding the accumulation of intermediate products or metabolic disorders caused by excessive local metabolic nodes. Furthermore, utilizing the genes' own natural promoters avoids cellular stress responses that might be triggered by exogenous strong promoters, ensuring normal bacterial growth. Experimental data show that, compared to wild-type strains, the engineered bacteria constructed in this invention increase vitamin B12 production by approximately 7% without significantly affecting bacterial growth, demonstrating excellent potential for industrial application. This overexpression strategy based on the natural operon structure provides a more refined and coordinated genetic modification approach for microbial metabolic engineering.

[0020] It is worth further elaboration that the vector backbone of the recombinant expression plasmid is the broad-spectrum expression vector pBBR1MCS-5. This backbone includes an origin of replication capable of autonomous replication in *Strombus adherentus* and resistance marker genes for selection. pBBR1MCS-5 is a widely used broad-spectrum expression vector for Gram-negative bacteria, possessing advantages such as stable replication, moderate copy number, and broad host range. The vector backbone contains an origin of replication capable of autonomous replication in *Strombus adherentus*, allowing the recombinant plasmid to exist stably in a free form within the strain without integration into the chromosome. Simultaneously, the resistance marker genes carried on the vector (such as gentamicin resistance genes) provide a convenient means for screening transformants; positive clones successfully introduced with the recombinant plasmid can be effectively screened by adding the appropriate antibiotic. The stable replication characteristics of the pBBR1MCS-5 vector in *Strombus adherentus* ensure that the exogenous cobPWNO operon can be inherited and expressed stably and long-term, avoiding yield reduction problems caused by plasmid loss. The moderate copy number ensures effective gene overexpression without excessively increasing the metabolic burden on the host cell due to an excessively high copy number. The broad-spectrum host characteristics of this vector make it applicable not only to *Streptococcus adherent* but also to other related strains, demonstrating excellent versatility. Compared to chromosome integration, the free plasmid form simplifies and speeds up the construction process, eliminating the need for complex homologous recombination and screening, significantly shortening the construction cycle of engineered bacteria. The presence of resistance markers not only facilitates screening but also ensures plasmid stability during fermentation by maintaining antibiotic pressure, providing a reliable guarantee for industrial production.

[0021] It is worth further elaborating that the nucleotide sequence of the natural cobPWNO operon is shown in SEQ ID NO:1. This sequence contains the natural promoter region of the cobP gene, the complete coding sequences of the four genes cobP, cobW, cobN, and cobO, and the natural spacer sequences between the genes. SEQ ID NO:1 is a specific sequence obtained by sequencing and analyzing the genome of *Strombus amygdalinus*, representing the true structure of the cobPWNO operon in this strain. Cloning and overexpression using this exact sequence ensures that the introduced operon has consistent regulatory characteristics and gene arrangement with the natural operon on the host cell chromosome. Limiting the scope of protection to the specific SEQ ID NO:1 sequence provides a clear technical boundary, making the technical solution of this invention clearly reproducible. Those skilled in the art can accurately obtain the operon fragment through PCR amplification or artificial synthesis based on the provided sequence information, avoiding experimental failures or differences in results due to sequence uncertainty. The natural promoter sequence contained in SEQ ID NO:1 perfectly matches the transcriptional regulatory mechanism of the host cell and can be correctly recognized by the host cell's RNA polymerase, achieving precise transcription initiation and regulation. The natural spacer sequences between genes may contain regulatory elements that affect mRNA stability or translation efficiency, and the preservation of these potential regulatory functions further ensures the coordination of expression of the four genes.

[0022] Further details are worth noting: the recombinant plasmid is pBBR-cobPWNO, constructed by inserting the natural cobPWNO operon into the multiple cloning site of the pBBR1MCS-5 vector. The natural cobPWNO operon is driven by the natural promoter of the cobP gene, which drives the tandem coding regions of the cobP, cobW, cobN, and cobO genes. This plasmid uses pBBR1MCS-5 as its vector backbone, with the cobPWNO natural operon fragment inserted at its multiple cloning site. The insertion direction has been optimized to ensure that the natural promoter in the operon can correctly drive the transcription of the four downstream genes. This plasmid, as an independent genetic element, can be amplified and preserved in common cloning hosts such as *E. coli*, facilitating large-scale preparation and subsequent transformation. When this plasmid is introduced into *Streptococcus adhesioides*, its… The carried cobPWNO operon begins transcription and translation within the host cell, producing four corresponding enzyme proteins. The recombinant plasmid pBBR-cobPWNO is a crucial bridge connecting the gene fragment and the host cell; its design and construction quality directly determine the performance of the subsequent engineered bacteria. By cloning the entire natural operon into the vector at once, multiple independent gene manipulations are avoided, greatly simplifying the construction process and improving experimental efficiency. Plasmid-based genetic tools offer high flexibility and transferability, and can be used not only for *Cyclophorus fasciatus* but also for heterologous expression in other related strains, expanding the application scope of the technology. As a pre-constructed tool, pBBR-cobPWNO can be standardized for preparation and distribution, facilitating the replication and verification of the invention's technical solution by different laboratories, thus promoting the dissemination and application of the technology.

[0023] A method for constructing recombinant *Strombus adhesiveus* includes the following steps: (1) Amplify a complete DNA fragment containing the natural promoter of cobP and the genes of cobP, cobW, cobN and cobO from *Strombus amygdalinus*. (2) The DNA fragment obtained in step (1) is cloned into the multiple cloning site of the pBBR1MCS-5 vector to obtain the recombinant plasmid pBBR-cobPWNO, as shown in the attached figure. Figure 2 As shown; (3) The recombinant plasmid was introduced into the host cells of *Strombus adherentus* to obtain recombinant *Strombus adherentus*; This method first uses the genomic DNA of *Strombus fasciatus* as a template to amplify a large DNA fragment containing the natural cobP promoter and the complete coding regions of the cobP, cobW, cobN, and cobO genes via PCR. The key to this step is that the primer design must cover the entire operon region and ensure the integrity and fidelity of the amplified product. Subsequently, the amplified fragment is ligated in vitro with the pBBR1MCS-5 vector linearized with restriction endonucleases to construct the recombinant plasmid pBBR-cobPWNO. Finally, the recombinant plasmid is introduced into *Strombus fasciatus* host cells using suitable methods such as electroporation. Positive transformants are obtained through antibiotic selection, which constitutes the recombinant *Strombus fasciatus* engineered strain of this invention. This method fully utilizes conventional molecular biology techniques, with a clear and concise operational process, making it easy for those skilled in the art to master and replicate. Compared to the complex process in existing technologies that require independent promoter optimization and assembly for each gene, this invention significantly reduces operational difficulty and workload by cloning the entire natural operon in one step, improving construction efficiency. PCR amplification directly obtains the target fragment from the genome, avoiding the high cost of artificially synthesized genes. Electroporation exhibits high transformation efficiency in *Strombus adherentus*, ensuring successful introduction of recombinant plasmids. The entire method avoids complex gene editing or chromosome integration, provides a clear genetic background, and has a short construction cycle, facilitating the rapid acquisition of large quantities of engineered strains for subsequent fermentation validation. This simple and efficient construction method lays a solid technical foundation for the development and industrial application of high-yield vitamin B12 strains.

[0024] A method for verifying the vitamin B12 yield of recombinant *Strombus adhesiveus* includes the following steps: (1) Fermentation culture: The recombinant adhesiocytosis was cultured in a fermentation medium under appropriate conditions; (2) Sample preparation: Add glacial acetic acid and sodium nitrite solution to the fermentation broth, hydrolyze in a boiling water bath, dilute and centrifuge, and take the supernatant; (3) Content determination: The vitamin B12 in the sample was quantitatively analyzed by high performance liquid chromatography; This method first involves culturing engineered bacteria in an optimized fermentation medium, typically at a specific temperature (e.g., 32℃) and rotation speed (e.g., 270 rpm) for several days to allow sufficient accumulation of vitamin B12. After fermentation, a suitable amount of fermentation broth is pretreated: glacial acetic acid is added for acidification, followed by derivatization with sodium nitrite solution. Heating in a boiling water bath releases the bound vitamin B12 and converts it into a detectable form. The treated sample is diluted, centrifuged to remove precipitate, and the supernatant is injected into a high-performance liquid chromatograph (HPLC) for quantitative analysis. Chromatographic conditions typically use a C18 column with methanol and water as the mobile phase. The absorbance of vitamin B12 is detected at a characteristic wavelength (e.g., 550 nm), and the concentration of vitamin B12 in the sample is calculated by comparing it with a standard curve. This method provides a standardized operating procedure for the accurate determination of vitamin B12 yield. HPLC is characterized by high separation efficiency, good sensitivity, and accurate quantification, effectively eliminating interference from other impurities in the fermentation broth and ensuring the reliability of the detection results. The sample pretreatment step involves conversion with sodium nitrite, uniformly transforming various forms of vitamin B12 within microbial cells into easily detectable derivatives, thus avoiding detection biases caused by different forms. Boiling water bath hydrolysis is simple and mild, fully releasing intracellular products without damaging the target molecule. This method forms a complete chain from fermentation and treatment to detection, providing a scientific basis for evaluating the performance of engineered bacteria and a necessary prerequisite for subsequent fermentation process optimization. Experimental data show that the vitamin B12 yield of the engineered bacteria using this method reached 128.99±5.51 mg / L, significantly higher than the 119.92±2.66 mg / L of the control strain, fully validating the technical effectiveness of this invention.

[0025] It is worth further elaborating on the application of recombinant *Streptococcus adhesus* in vitamin B12 production. The high-yield engineered strain obtained through this process was used as the production strain. Large-scale fermentation was carried out under suitable fermentation media and conditions. By optimizing fermentation process parameters (such as temperature, pH, dissolved oxygen, and carbon-nitrogen source ratio), the vitamin B12 synthesis potential of the engineered strain was fully realized. After fermentation, vitamin B12 was isolated and extracted from the fermentation broth, and after purification, a product meeting quality requirements was obtained. This application covers the complete process flow from strain activation, seed culture, fermentation production to product extraction.

[0026] It is worth further elaborating that the fermentation temperature was 30-34℃, the cultivation time was 7-9 days, and the fermentation medium contained carbon sources, nitrogen sources, and inorganic salts. As a mesophilic bacterium, *Strombus viscera* maintains good growth activity and metabolic capacity within the 30-34℃ range; excessively high or low temperatures will affect cell growth and product synthesis. The 7-9 day cultivation time is an optimized fermentation cycle, ensuring sufficient accumulation of vitamin B12 while avoiding increased energy consumption and the risk of contamination due to excessively long cycles. The composition of the fermentation medium is also a key factor affecting yield. Appropriate ratios of carbon sources (such as glucose and sucrose), nitrogen sources (such as yeast extract and peptone), and inorganic salts (such as phosphates and magnesium salts) provide sufficient nutrition for cell growth and product synthesis. By limiting and optimizing fermentation conditions, the best external environment is provided for the engineered bacteria to perform optimally. Temperature is a key parameter affecting enzyme activity and metabolic rate; the 30-34℃ range covers the optimal growth temperature of *Strombus viscera*, ensuring the coordinated progress of various enzymatic reactions in the metabolic pathway. The 7-9 day culture cycle was determined based on the kinetics of vitamin B12 synthesis. In the early stage, the bacteria rapidly grow and accumulate biomass, while in the middle and later stages, a large amount of product is synthesized. This cycle design maximizes yield. The optimal culture medium provides balanced nutrition for the bacteria: carbon sources provide energy and the carbon skeleton, nitrogen sources supply the protein synthesis, and inorganic salts participate in the formation of enzyme active sites and cofactors. This comprehensive optimization of conditions fully releases the yield potential of the engineered bacteria, further consolidating its dominant position in vitamin B12 production.

[0027] Further details are provided regarding a method for preserving recombinant *Strombus amygdalinus*. After fermentation, the recombinant *Strombus amygdalinus* is mixed with a freeze-drying protectant, freeze-dried, and then stored long-term at low temperatures. This method first involves culturing the recombinant *Strombus amygdalinus* to an appropriate stage in a suitable culture medium. The cells are then collected and mixed with a freeze-drying protectant (such as skim milk powder, trehalose, or glycerol) to ensure uniform coating of the bacterial cells. The mixed bacterial solution is then freeze-dried, allowing water to sublimate under vacuum to obtain dried bacterial powder. Finally, the freeze-dried bacterial powder is sealed and stored long-term at low temperatures (e.g., -20°C or 4°C). When the strain is needed, the freeze-dried bacterial powder is rehydrated and activated in a suitable culture medium to restore its activity for fermentation production. This method allows the constructed high-yield engineered bacteria to be preserved as a stable strain resource for long-term use, facilitating immediate production and avoiding the risk of yield decline due to frequent subculturing. Simultaneously, the freeze-dried bacterial powder is easy to package and transport, facilitating the transfer and distribution of engineered bacteria between different production sites, thus providing convenience for large-scale promotion and application.

[0028] In summary, compared to existing strategies that assemble independent strong promoters for each gene, this invention, by preserving the natural operon structure during overexpression, perfectly maintains the inherent synergistic regulatory relationship among the four key genes. This coordinated overexpression method ensures a balanced ratio of enzyme amounts at each step of the metabolic pathway, allowing for smooth metabolic flow. This not only enhances the synthesis of the target product, vitamin B12, but also avoids the problems of intermediate product accumulation or metabolic disorders caused by excessive local metabolic nodes. Furthermore, by utilizing the genes' own natural promoters, it avoids cellular stress responses that may be triggered by exogenous strong promoters, ensuring normal bacterial growth. Experimental data show that, compared to wild-type strains, the engineered bacteria constructed in this invention increase vitamin B12 production by approximately 7% without significantly affecting bacterial growth, as shown in the attached figure. Figure 2 As shown, this overexpression strategy based on natural operon structures demonstrates excellent potential for industrial applications, providing a more refined and coordinated genetic modification approach for microbial metabolic engineering.

Claims

1. A genetically engineered *Strombus adhesiveus* strain that enhances vitamin B12 production, characterized in that, The genetically engineered bacteria contains a recombinant expression plasmid containing the cobPWNO natural operon, which consists of the natural promoter of the cobP gene and the coding sequences of the cobP, cobW, cobN and cobO genes in tandem.

2. The *Strombus adhesiveus* genetically engineered bacterium for increasing vitamin B12 production according to claim 1, characterized in that, The vector backbone of the recombinant expression plasmid is the broad-spectrum expression vector pBBR1MCS-5, which contains the origin of replication for autonomous replication in *Strombus amygdalinus* and resistance marker genes for selection.

3. The *Strombus adhesiveus* genetically engineered bacterium for increasing vitamin B12 production according to claim 1, characterized in that, The nucleotide sequence of the cobPWNO natural operator is shown in SEQ ID NO:

1.

4. A recombinant plasmid for overexpressing the cobPWNO gene in *Strombus amygdalinus*, characterized in that, The recombinant plasmid is pBBR-cobPWNO, constructed by inserting the natural cobPWNO operon into the multiple cloning site of the pBBR1MCS-5 vector; the natural cobPWNO operon is driven by the natural promoter of the cobP gene, which controls the tandem coding regions of the cobP, cobW, cobN, and cobO genes.

5. A method for constructing the recombinant *Strombus adhesiveus* as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Amplify a complete DNA fragment containing the natural promoter of cobP and the genes of cobP, cobW, cobN and cobO from *Strombus amygdalinus*. (2) The DNA fragment obtained in step (1) is cloned into the multiple cloning site of the pBBR1MCS-5 vector to obtain the recombinant plasmid pBBR-cobPWNO. (3) The recombinant plasmid is introduced into the host cell of *Strombus adherentus* to obtain the recombinant *Strombus adherentus*.

6. A method for verifying the vitamin B12 yield of recombinant *Streptococcus adhesiveus* as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Fermentation culture: The recombinant adhesiocytosis was cultured in a fermentation medium under appropriate conditions; (2) Sample preparation: Add glacial acetic acid and sodium nitrite solution to the fermentation broth, hydrolyze in a boiling water bath, dilute and centrifuge, and take the supernatant; (3) Content determination: The vitamin B12 in the sample was quantitatively analyzed by high performance liquid chromatography.

7. The use of the recombinant Streptomyces adhesiveus according to any one of claims 1-3 in the production of vitamin B12.

8. The application according to claim 7, characterized in that, The fermentation culture is conducted at a temperature of 30-34℃ for 7-9 days, and the fermentation medium contains carbon source, nitrogen source and inorganic salts.

9. The method for preserving recombinant *Strombus adhesiveus* according to any one of claims 1-3, characterized in that, The recombinant *Strombus amygdalinus* was fermented and cultured, then mixed with a freeze-drying protectant, and freeze-dried before being stored at low temperatures for a long period of time.

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