High-yield vitamin B12 strain with improved robustness and application of high-yield vitamin B12 strain
Through adaptive laboratory evolution and fermentation optimization of denitrifying Pseudomonas, a highly robust and high-yielding strain, Mut407-JN, was screened, solving the robustness and yield problems of vitamin B12 production by microbial fermentation and achieving efficient and stable vitamin B12 production.
Patent Information
- Application Number
- CN202512041815.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-03-20
AI Technical Summary
Existing microbial fermentation methods for producing vitamin B12 have poor robustness, resulting in slow growth, unstable yields, and difficulty in exceeding the yield limit of 300 mg/L.
By conducting adaptive laboratory evolution on denitrifying Pseudomonas, and using fermentation supernatant and vitamin B12 structural analogues as selection pressures, a highly robust and high-yielding strain was screened out and named Mut407-JN. Shake flask and fermenter fermentation were then optimized.
It significantly improved the robustness of the strain and the yield of vitamin B12, stabilized the fermentation process, achieved a yield of over 310 mg/L, and reduced production costs.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation and metabolic engineering technology, and in particular to a robust, high-yield vitamin B12 strain and its applications. Background Technology
[0002] Vitamin B12 is an important water-soluble vitamin and enzyme cofactor, with huge demand in the pharmaceutical, food, and feed industries. Currently, vitamin B12 is mainly produced through microbial fermentation, with commonly used strains including *Pseudomonas denitrificans* and *Propionibacterium schwanniferum*.
[0003] In the process of producing vitamin B12 by microbial fermentation, there is a common key bottleneck: the poor robustness of high-yield strains. This is mainly reflected in: (1) heavy metabolic burden: the synthesis pathway of vitamin B12 is extremely complex, consuming a large amount of energy and precursors, resulting in slow growth and fragile physiological state of engineered strains. (2) inhibition of products and byproducts: the high concentration of vitamin B12 and its metabolic byproducts (such as organic acids) accumulated in the fermentation broth will have feedback inhibition and toxic effects on cell growth and metabolism, limiting the final yield. (3) genetic and fermentation instability: during long-term fermentation, high-yield strains are prone to performance degradation, plasmid loss or negative mutation, resulting in short production cycles and large yield fluctuations, increasing the cost of industrial production.
[0004] Traditional rational metabolic engineering strategies (such as overexpression or knockout of specific genes) often fail to systematically address robustness issues due to limited understanding of the global regulatory network of the strain. Furthermore, while existing technologies have been extensively studied for vitamin B12-producing strains, metabolism easily plateaus under high-yield conditions, with the final concentration of vitamin B12 fermentation remaining consistently below 300 mg / L, making it difficult to break through this threshold. Therefore, developing a method to systematically improve the growth capacity and production stability of producing strains under stress conditions is of great significance for the industrial production of vitamin B12. Summary of the Invention
[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of robust, well-growing, and highly efficient microbial strains for synthesizing vitamin B12 in the prior art. Through pressure screening, a denitrifying Pseudomonas strain with improved performance is provided, and the final vitamin B12 yield of this strain is higher than 310 mg / L.
[0006] This invention provides a denitrifying Pseudomonas, named Pseudomonas denitrificans Mut407-JN, with accession number CCTCC NO. M 20252593.
[0007] The present invention provides a microbial inoculant containing the above-mentioned denitrifying Pseudomonas.
[0008] Furthermore, the microbial agent is a liquid agent.
[0009] Furthermore, the microbial agent is a solid microbial agent.
[0010] The present invention provides a culture or a processed product thereof containing the above-mentioned denitrifying Pseudomonas.
[0011] On the other hand, the present invention also provides a method for synthesizing vitamin B12, including a fermentation step using the aforementioned denitrifying Pseudomonas bacteria.
[0012] Furthermore, fermentation was carried out using the aforementioned denitrifying Pseudomonas as a single strain.
[0013] Furthermore, the fermentation is either shake-flask fermentation or fed-batch fermentation.
[0014] Furthermore, the shake-flask fermentation includes the following steps: inoculating the strain into a seed culture medium to prepare a seed liquid, inoculating the seed liquid into a fermentation culture medium, and culturing at 30-32℃ and 200-260r / min.
[0015] Furthermore, the fed-batch fermentation includes the following steps: inoculating the strain into a seed culture medium to prepare a primary seed culture, inoculating the primary seed culture into a fermentation culture medium to prepare a secondary seed culture, and then inoculating the secondary seed culture into a fermenter and culturing it at 30-32℃ and pH 7.0-7.4 with an aeration rate of 1-10 vvm and fed-batch culture medium.
[0016] Furthermore, the seed culture medium contains: glucose 30-60 g / L, corn steep liquor 20-50 g / L, betaine 5 g / L, (NH4)2SO4 1-2.5 g / L, (NH4)2HPO4 1-2 g / L, KH2PO4 2.5-5 g / L, MnSO4·H2O 0.2-0.8 g / L, MgSO4 0.3-1.5 g / L, CoCl2·6H2O 0.02-0.15 g / L, DMBI 0.005-0.01 g / L, ZnSO4·7H2O 0.01-0.02 g / L, and pH 7.0-7.4.
[0017] Furthermore, the fermentation medium contains: glucose 60-80 g / L, corn steep liquor 30-60 g / L, betaine 14-40 g / L, (NH4)2SO4 1-2 g / L, KH2PO4 0.75-8 g / L, MgSO4 1.5-5 g / L, CoCl2·6H2O 0.075-0.15 g / L, DMBI 0.05-0.1 g / L, ZnSO4·7H2O 0.08-0.2 g / L, and pH 7.0-7.4.
[0018] Furthermore, the supplemental culture medium contains: glucose 300~500g / L, betaine 30~39g / L, DMBI 0.15~0.3g / L, and CoCl2·6H2O 0.15~0.4g / L.
[0019] In addition, the present invention also claims protection for the use of the above-mentioned denitrifying Pseudomonas aeruginosa in the preparation of vitamin B12 or products containing vitamin B12.
[0020] Furthermore, the product containing vitamin B12 can be a pharmaceutical, health food, etc.
[0021] Finally, this invention also provides a method for screening vitamin B12-producing strains, including a step of screening using the fermentation supernatant of the previous generation as the selection pressure. The evolutionary method provided by this invention (especially the method using fermentation supernatant as the selection pressure) has strong universality and can be widely applied to different types of vitamin B12-producing strains (such as denitrifying Pseudomonas, Propionibacterium, etc.), and has important promotional value.
[0022] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:
[0023] (1) Systematically improve robustness: The strains screened in this invention have undergone beneficial adjustments at the genomic, transcriptomic and metabolomic levels, systematically improving their adaptability and robustness to fermentation stress environments.
[0024] (2) High yield and stable yield: The evolved strain obtained in this invention not only significantly increases the yield of vitamin B12, but more importantly, it exhibits excellent stability during the scale-up fermentation process, with slow decline in production intensity, effectively extending the high-efficiency production period, making it more suitable for industrial scale-up production.
[0025] (3) The strain grows well: Although the vitamin B12 synthesis pathway is extremely complex and consumes a lot of energy and precursors, the mutant strain of the present invention shows a significant increase in cell number and vigorous growth during fermentation compared to the original strain.
[0026] (4) Reduced production costs: Due to the improved robustness of the strain, the fermentation process is more stable, the risk of contamination is reduced, and the yield per unit time is increased, thus significantly reducing the industrial production cost of vitamin B12.
[0027] Preservation of biological materials
[0028] The denitrific bacteria Mut407-JN was deposited on November 19, 2025, at the China Center for Type Culture Collection (CCTCCNO. M 20252593), located at Wuhan University, China. Attached Figure Description
[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.
[0030] Figure 1 The vitamin B12 yield of the evolved strain screened in Example 1;
[0031] Figure 2 This is the result of the fermentation production of vitamin B12 in the 7L fermenter in Example 2;
[0032] Figure 3 This refers to the fermentation output of four consecutive batches from the 7L fermenter in Example 2. Detailed Implementation
[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0034] The technical solution involved in this invention is as follows:
[0035] In a first aspect, the present invention provides a microbial strain, which is a vitamin B12 producing strain with accession number CCTCC No. M 20252593, obtained through adaptive laboratory evolution with vitamin B12 or its structural analogues as selective pressure; the microbial strain exhibits significantly improved robustness under high concentrations of vitamin B12 stress compared to the original producing strain, specifically in at least one of the following aspects:
[0036] a) The growth rate is faster on plates containing at least 50 mg / L thiamine hydrochloride (the colony size on LB plates containing 50 mg / L thiamine hydrochloride and cultured at 37°C for 48 hours is more than 1.5 times that of the original strain).
[0037] b) In fresh culture medium supplemented with at least 20% (v / v) of the previous generation fermentation supernatant, the maximum biomass is increased by at least 20%;
[0038] c) When fed-batch fermentation is carried out in fermenters of 7L or larger, the fermentation production between batches is stable, and the highest final yield of vitamin B12 is higher than 310 mg / L.
[0039] Preferably, the microbial strain is *Pseudomonas denitrificans*.
[0040] In a second aspect, the present invention provides a method for preparing the microbial strain described in the first aspect, comprising the following steps:
[0041] (1) Provide a vitamin B12 producing bacterium as the original strain;
[0042] (2) Apply selection pressure to the culture medium and continuously passage the original strain to carry out adaptive laboratory evolution; the selection pressure includes: exogenous addition of vitamin B12 or its structural analogues, and / or addition of fermentation supernatant from the previous generation;
[0043] (3) During the evolutionary process, the intensity of the selection pressure is gradually increased;
[0044] (4) From the evolved microbial community, a single colony with a higher growth rate and vitamin B12 production than the original strain under high selection pressure is selected to obtain the highly robust and high-yielding strain.
[0045] Preferably, the structural analogue of vitamin B12 is thiamine hydrochloride.
[0046] Preferably, the continuous subculture is carried out in a shake flask, microplate, or bioreactor, with a subculture number of not less than 50 generations.
[0047] Thirdly, the present invention provides the application of the microbial strains described in the first aspect in the fermentation production of vitamin B12.
[0048] Fourthly, the present invention provides a method for producing vitamin B12 by fermentation, comprising: culturing the microbial strain described in the first aspect in a fermentation medium under suitable fermentation conditions, thereby producing and optionally recovering vitamin B12.
[0049] Preferably, the fermentation is a fed-batch fermentation with a fermentation scale of not less than 7L.
[0050] Fifthly, the present invention provides a culture medium composition for improving the robustness of vitamin B12 producing bacteria, the composition comprising an effective dose of thiamine hydrochloride as a selection pressure, said effective dose being 50-100 mg / L.
[0051] The materials and methods involved in the following embodiments are as follows:
[0052] (1) Culture medium
[0053] The seed culture medium contained: glucose 40 g / L, corn steep liquor 35 g / L, betaine 5 g / L, (NH4)2SO4 1.5 g / L, (NH4)2HPO4 1.5 g / L, KH2PO4 4 g / L, MnSO4·H2O 0.6 g / L, MgSO4 1 g / L, CoCl2·6H2O 0.1 g / L, DMBI 0.005 g / L, ZnSO4·7H2O 0.01 g / L, pH 7.0.
[0054] The fermentation medium contained: glucose 70 g / L, corn steep liquor 40 g / L, betaine 20 g / L, (NH4)2SO4 1 g / L, KH2PO4 0.75 g / L, MgSO4 1.5 g / L, CoCl2·6H2O 0.15 g / L, DMBI 0.05 g / L, ZnSO4·7H2O 0.08 g / L, pH 7.0.
[0055] (2) Shake-flask fermentation:
[0056] The bacterial cells cultured on an agar slant for 48 hours were inoculated into 10 mL of seed culture medium and cultured at 28℃ and 200 rpm for 24 hours. Then, 10% of the seed culture was inoculated into a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium and cultured at 30℃ and 260 rpm for 120 hours.
[0057] (3) Feeding and batch fermentation:
[0058] The bacterial cells cultured on an agar slant for 48 hours were inoculated into 10 mL of primary seed culture medium and cultured at 28°C and 200 rpm for 24 hours. Then, 10% of the primary seed culture was inoculated into a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium and cultured at 30°C and 260 rpm for 24 hours. Next, 10% of the secondary seed culture was inoculated into a fermenter and cultured at 30°C, maintaining the pH between 7.0 and 7.4 throughout the process. Sufficient oxygen supply was maintained in the fermenter at an aeration rate of 1 vvm, with moderate agitation. Feed culture medium was added according to the growth and metabolic progress.
[0059] Example 1: Screening of high-yielding and robust strains
[0060] I. Self-stress Adaptive Laboratory Evolution
[0061] 1. Original strain: Denitrifying Pseudomonas.
[0062] 2. Evolution medium: basic fermentation medium.
[0063] 3. Selective pressure: The initial selective pressure is the addition of 20% (v / v) of the previous generation fermentation supernatant.
[0064] 4. Evolutionary process: Continuous subculturing was carried out in shake flasks. Subculture was performed every 24 hours at a rate of 5% (v / v). Every 10 subcultures, the proportion of fermentation supernatant added was increased by 5% (v / v) until it reached 50% (v / v). Approximately 80 generations were performed in total.
[0065] 5. Sample preservation: Take samples every 20 generations and preserve them in glycerol tubes at -80°C.
[0066] II. Adaptive Laboratory Evolution with Exogenous Addition
[0067] 1. Original strain: Denitrifying Pseudomonas.
[0068] 2. Evolution medium: basic fermentation medium.
[0069] 3. Selected pressure: The initial selected pressure is 50 mg / L of thiamine hydrochloride.
[0070] 4. Evolutionary process: Continuous subculturing was carried out in shake flasks. Subculture was performed every 24 hours at a rate of 5% (v / v). Every 10 subcultures, the concentration of thiamine hydrochloride was increased by 10 mg / L until it reached 100 mg / L. Approximately 60 generations were performed in total.
[0071] 5. Sample preservation: Take samples every 20 generations and preserve them in glycerol tubes at -80°C.
[0072] III. Screening of High-Yielding and Robust Strains
[0073] 1. From the bacterial community at the evolutionary endpoints of steps one and two, samples were taken and streaked onto plates containing 80 mg / L thiamine hydrochloride to isolate single clones.
[0074] 2. Based on the size of the strain, approximately 460 single clones were selected and micro-fermented in 96-well plates using a medium containing 40% fermentation supernatant.
[0075] 3. The vitamin B12 content in the fermentation broth was determined by HPLC. Figure 1 ).
[0076] 4. Sixty-seven candidate strains were screened, with both vitamin B12 production and OD600 increasing by more than 30% compared to the original strain.
[0077] 5. The 67 candidate strains were further screened through shake-flask fermentation, and a strain with the best performance was ultimately identified and named Mut407-JN. Its optimal vitamin B12 yield in shake-flask fermentation was approximately 38.7% higher than that of the original strain.
[0078] Example 2: Performance verification of the evolved strain Mut407-JN
[0079] 1. 7L Fermenter Validation: Fed-batch fermentation was conducted in a 7L fermenter. Results are as follows: Figure 2 As shown, the yield of the original strain (a) slowed down after 130 hours and began to decline after 170 hours; while the Mut407-JN strain (b) maintained high yield throughout the 200-hour fermentation cycle, with the final vitamin B12 yield reaching 314 mg / L, which was 25.6% higher than that of the original strain (250 mg / L), and the production intensity remained stable throughout the process.
[0080] 2. Fermentation batch stability verification: Four consecutive batch fermentations were conducted in a 7L fermenter. The results are as follows: Figure 3 As shown, the yield of multiple fermentation batches is stable.
[0081] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A denitrifying Pseudomonas bacterium, characterized in that, It was named Pseudomonas denitrificans Mut407-JN, with accession number CCTCC NO. M 20252593.
2. A microbial inoculant, characterized in that, It includes the denitrifying Pseudomonas as described in claim 1.
3. The microbial agent according to claim 2, characterized in that, The microbial agent is either a liquid agent or a solid agent.
4. A culture or a processed product thereof comprising the denitrifying Pseudomonas as described in claim 1 or the microbial agent as described in claim 2 or 3.
5. A method for synthesizing vitamin B12, characterized in that, The method includes the step of fermentation using the denitrifying Pseudomonas as described in claim 1 or the microbial agent as described in claim 2 or 3.
6. The method according to claim 5, characterized in that, The fermentation is either shake-flask fermentation or fed-batch fermentation.
7. The method according to claim 6, characterized in that, The shake-flask fermentation includes the following steps: inoculating the strain into a seed culture medium to prepare a seed solution, inoculating the seed solution into a fermentation culture medium, and culturing at 30-32℃ and 200-260r / min; And / or, the fed-batch fermentation includes the following steps: inoculating the strain into a seed culture medium to prepare a primary seed culture, inoculating the primary seed culture into a fermentation culture medium to prepare a secondary seed culture, and then inoculating the secondary seed culture into a fermenter and culturing it at 30-32°C and pH 7.0-7.4 with an aeration rate of 1-10 vvm and fed-batch culture medium.
8. The method according to claim 7, characterized in that, It must contain at least one of the following characteristics: (1) The seed culture medium contains: glucose 30~60 g / L, corn steep liquor 20~50 g / L, betaine 5 g / L, (NH4)2SO4 1~2.5 g / L, (NH4)2HPO4 1~2 g / L, KH2PO4 2.5~5 g / L, MnSO4·H2O 0.2~0.8 g / L, MgSO4 0.3~1.5 g / L, CoCl2·6H2O 0.02~0.15 g / L, DMBI 0.005~0.01 g / L, ZnSO4·7H2O 0.01~0.02 g / L, pH 7.0~7.4; (2) The fermentation medium contains: glucose 60~80 g / L, corn steep liquor 30~60 g / L, betaine 14~40 g / L, (NH4)2SO4 1~2 g / L, KH2PO4 0.75~8 g / L, MgSO4 1.5~5 g / L, CoCl2·6H2O 0.075~0.15 g / L, DMBI 0.05~0.1 g / L, ZnSO4·7H2O 0.08~0.2 g / L, pH 7.0~7.4; (3) The supplemental culture medium contains: glucose 300~500g / L, betaine 30~39g / L, DMBI 0.15~0.3g / L, and CoCl2·6H2O 0.15~0.4g / L.
9. The use of the denitrifying Pseudomonas aeruginosa of claim 1 or the microbial agent of claim 2 or 3 in the preparation of vitamin B12 or products containing vitamin B12.
10. A method for screening vitamin B12-producing strains, characterized in that, This includes the step of using the supernatant from the previous generation of fermentation as the selection pressure for screening.