Preparation method of safe high-yield strain for vitamin B2 production

Abstract

The invention belongs to the technical field of vitamin B2 production, and particularly relates to a preparation method of a safe high-yield strain for vitamin B2 production, which comprises the following steps: S1, strain source and screening: analyzing the current situation and development trend of vitamin B2, and screening the strain; s2, genetic engineering modification: evaluating the production performance and genetic stability of the strain, and carrying out mutagenesis treatment on the screened strain by using a chemical mutagenic agent to obtain a genetically mutated strain; s3, screening and identifying high-yield mutant strains: integrating the fragments containing the spore cereus dry riboflavin operon into a vitamin B2 genome to generate the mutant strains, and performing fine adjustment on the riboflavin operon in the mutant strains by utilizing a multiple genome editing system. And through technical popularization, the manufacturing cost of the product can be reduced, and the social benefit is obvious.

Description

Technical Field

[0001] The present invention relates to the technical field of vitamin B2 production, and in particular to a method for preparing a safe and high-yield bacterial strain for vitamin B2 production. Background Art

[0002] Riboflavin, also known as vitamin B2, was isolated in 1879 by chemist Blyth from milk supernatant. However, this substance was not true riboflavin; he called it lactochrome. In 1930, Kuhn, Eggersdorfer, and others isolated riboflavin. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the most common forms of riboflavin in the body. These two substances act as cofactors for oxidoreductases in cells, transferring electrons during oxidation. While riboflavin does not directly participate in cellular metabolism, its derivatives, FMN and FAD, regulate various physiological processes. Riboflavin deficiency in plants and animals can slow their growth and development. Insufficient riboflavin intake can lead to a variety of diseases, such as cardiovascular disease, angular cheilitis, migraines, angina pectoris, cataracts, and malaria. Therefore, riboflavin is primarily used in medicine as an adjunct therapy. Riboflavin can also be used in the food industry as a pigment and nutritional supplement. The current production methods of vitamin B2 have problems such as low yield, high cost, environmental pollution and possible safety hazards. Summary of the Invention

[0003] The purpose of the present invention is to solve the shortcomings of vitamin B2 production methods such as low yield, high cost, environmental pollution and possible safety hazards, and to propose a method for preparing a safe and high-yield bacterial strain for vitamin B2 production.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for preparing a safe and high-yield bacterial strain for vitamin B2 production comprises the following steps:

[0006] S1. Strain source and screening: Analyze the current status and development trend of vitamin B2 and screen the strains;

[0007] S2. Genetic engineering: Evaluate the production performance and genetic stability of the strains, and use chemical mutagens to mutagenize the selected strains to obtain genetically modified strains;

[0008] S3. Screening and identification of high-yield mutants: A fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome to generate mutants. The riboflavin operon in the mutants was fine-tuned using a multiplex genome editing system to increase vitamin B2 production. By comparing the production performance of different mutants, high-yield vitamin B2 strains were screened.

[0009] S4. Optimization of fermentation process: Optimize the fermentation conditions of high-yield strains and develop downstream separation and purification technologies suitable for high-yield strains;

[0010] S5. Development of downstream separation and purification technologies: Debug and optimize the production line to ensure the stability and efficiency of the production process, establish a comprehensive product quality control system, and strictly test the yield, purity, and safety indicators of vitamin B2;

[0011] S6. Product quality control and safety assessment: Summarize the entire project development process and evaluate the project's success and shortcomings.

[0012] Preferably, in S1, the research status and development trend of vitamin B2 at home and abroad are analyzed, the limitations of current production technology and market demand are understood, technical literature on vitamin B2 strain construction, fermentation process, separation and purification at home and abroad are collected and studied, and the types, dosage forms, prices and application fields of vitamin B2 products on the market are investigated.

[0013] Preferably, in S2, currently commonly used engineered bacteria for producing vitamin B2 are selected, including Ashwagandha yeast and Bacillus subtilis, and their production performance and genetic stability are evaluated. The screened strains are induced to undergo mutation treatment using chemical mutagens to obtain genetically mutated strains. Engineered bacteria with the ability to synthesize purine and riboflavin are screened out using an intelligent computer system, and structural analogs of threonine and proline are used as screening conditions to further obtain ideal engineered strains.

[0014] Preferably, in S3, a fragment containing the riboflavin operon of Bacillus cereus is integrated into the vitamin B2 genome to generate a mutant strain, and the riboflavin operon in the mutant strain is fine-tuned using a multiple genome editing system to increase the production of vitamin B2. By comparing the production performance of different mutant strains, strains with high vitamin B2 production are screened out, and the screened high-yield strains are subjected to multiple rounds of verification to ensure their genetic stability and the reliability of their production performance.

[0015] Preferably, in S4, the fermentation conditions of the high-yield strain are optimized, including the culture medium formula, fermentation temperature, and pH value. Through shake flask experiments, small-scale tests and pilot stages, the fermentation scale is gradually expanded, the fermentation process is evaluated and optimized, and downstream separation and purification technology suitable for the high-yield strain is developed, including cell disruption, extraction, and purification steps. The separation and purification process is optimized to improve the purity and yield of vitamin B2.

[0016] Preferably, in S5, an industrial production line of vitamin B2 is constructed based on the optimized fermentation process and separation and purification technology, the production line is debugged and optimized to ensure the stability and efficiency of the production process, a complete product quality control system is established, and the output, purity and safety indicators of vitamin B2 are strictly tested.

[0017] Preferably, in S6, the development process of the entire project is summarized, the success and shortcomings of the project are evaluated, the project data and information are sorted out, a complete research report and technical documentation are formed, the strain construction and fermentation process of vitamin B2 are further optimized, the output is further increased and the cost is reduced, the application field of vitamin B2 is expanded, more market-competitive products are developed, cooperation and exchanges with relevant domestic and foreign enterprises and research institutions are strengthened, and the sustainable development of the vitamin B2 industry is promoted.

[0018] Preferably, a fragment containing the riboflavin operon of Bacillus cereus is integrated into the vitamin B2 genome, specifically comprising: screening out a complete operon related to riboflavin synthesis from the genome of Bacillus cereus ATCC14579 through bioinformatics comparison, including key genes such as ribGBAH, designing primers to amplify the target fragment, introducing homology arms during PCR amplification, and adding restriction sites, selecting an Escherichia coli-Bacillus subtilis shuttle vector, inserting the amplified riboflavin operon fragment into the vector multiple cloning site, and adding an antibiotic resistance marker, verifying the correctness of the vector by enzyme digestion and sequencing, introducing the recombinant vector into Bacillus subtilis competent cells, setting the electric field strength to 12-15 kV / cm and the pulse time to 5 ms to improve the transformation efficiency, and utilizing the natural homologous recombination system of Bacillus subtilis to site-directedly insert the riboflavin operon into a specific region of the host genome through double exchange.

[0019] Preferably, when the screened strains are induced to undergo mutation treatment using chemical mutagens, a selective culture medium is used to activate the target strain and inhibit the growth of foreign bacteria. The strain is purified by dilution plate method or microscopic single spore separation technology to ensure that the starting strain is a haploid pure strain. The bacteria are transferred to fresh liquid culture medium and cultured with shaking until the logarithmic growth phase to ensure that the cell metabolic state is consistent.

[0020] Preferably, the mutagenesis reaction conditions are: treatment temperature: 25-37°C, treatment time: EMS treatment 30-60 min, NTG treatment 10-30 min.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] Through mutagenesis breeding and gene recombination technology, a high-yield vitamin B2 strain with no antibiotic resistance genes was successfully constructed. This strain showed excellent growth performance and yield during the fermentation process, providing a solid foundation for subsequent industrial preparation. The fermentation process was comprehensively optimized, including the adjustment of key parameters such as culture medium formulation and fermentation conditions. At the same time, downstream separation and purification technology suitable for this high-yield strain was developed, which effectively improved the purity and yield of vitamin B2. An industrial production line for vitamin B2 was built, and large-scale production was successfully achieved. The production line operates stably, and the product quality meets relevant standards and regulatory requirements, meeting market demand.

[0023] In terms of promotion and application, we actively cooperate with pharmaceutical companies, health care product companies and food companies to promote and apply the technical achievements of vitamin B2 strain construction and high-yield and safe industrial production;

[0024] The present invention obtains vitamin B2 strain construction and high-yield and safe industrial preparation technology, which has high yield, safety and reliability. Through technology promotion, product manufacturing costs can be reduced, and social benefits are obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a structural schematic diagram of a method for preparing a safe and high-yield bacterial strain for vitamin B2 production proposed by the present invention. DETAILED DESCRIPTION

[0026] The technical solution of this embodiment will be clearly and completely described below in conjunction with the drawings in this embodiment. Obviously, the described embodiment is only a part of this embodiment, rather than all the embodiments.

[0027] Example 1

[0028] Reference Figure 1 A method for preparing a safe and high-yield strain of vitamin B2 for production comprises the following steps:

[0029] S1. Strain source and screening: Analyze the current status and development trend of vitamin B2 and screen the strains;

[0030] S2. Genetic engineering: Evaluate the production performance and genetic stability of the strains, and use chemical mutagens to mutagenize the selected strains to obtain genetically modified strains;

[0031] S3. Screening and identification of high-yield mutants: A fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome to generate mutants. The riboflavin operon in the mutants was fine-tuned using a multiplex genome editing system to increase vitamin B2 production. By comparing the production performance of different mutants, high-yield vitamin B2 strains were screened.

[0032] S4. Optimization of fermentation process: Optimize the fermentation conditions of high-yield strains and develop downstream separation and purification technologies suitable for high-yield strains;

[0033] S5. Development of downstream separation and purification technologies: Debug and optimize the production line to ensure the stability and efficiency of the production process, establish a comprehensive product quality control system, and strictly test the yield, purity, and safety indicators of vitamin B2;

[0034] S6. Product quality control and safety assessment: Summarize the entire project development process and evaluate the project's success and shortcomings.

[0035] In this embodiment, in S1, the research status and development trend of vitamin B2 at home and abroad are analyzed, the limitations of current production technology and market demand are understood, technical literature on vitamin B2 strain construction, fermentation process, separation and purification at home and abroad are collected and studied, and the types, dosage forms, prices and application fields of vitamin B2 products on the market are investigated.

[0036] In this embodiment, in S2, currently commonly used engineered bacteria for producing vitamin B2, including Ashwagandha yeast and Bacillus subtilis, are selected, their production performance and genetic stability are evaluated, and the screened strains are induced to undergo mutation treatment using chemical mutagens to obtain genetically mutated strains. Engineered bacteria with the ability to synthesize purine and riboflavin are screened out using an intelligent computer system, and structural analogs of threonine and proline are used as screening conditions to further obtain ideal engineered strains.

[0037] In this example, in S3, a fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome to generate a mutant strain. The riboflavin operon in the mutant strain was fine-tuned using a multiple genome editing system to increase the production of vitamin B2. By comparing the production performance of different mutant strains, high-yield vitamin B2 strains were screened, and the screened high-yield strains were subjected to multiple rounds of verification to ensure their genetic stability and the reliability of their production performance.

[0038] In this embodiment, in S4, the fermentation conditions of the high-yield strain are optimized, including the culture medium formula, fermentation temperature, and pH value. Through shake flask experiments, small-scale tests, and pilot stages, the fermentation scale is gradually expanded, the fermentation process is evaluated and optimized, and downstream separation and purification technology suitable for the high-yield strain is developed, including cell disruption, extraction, and purification steps. The separation and purification process is optimized to improve the purity and yield of vitamin B2.

[0039] In this embodiment, in S5, an industrial production line of vitamin B2 is constructed based on the optimized fermentation process and separation and purification technology, the production line is debugged and optimized to ensure the stability and efficiency of the production process, a complete product quality control system is established, and the output, purity, and safety indicators of vitamin B2 are strictly tested.

[0040] In this embodiment, in S6, the development process of the entire project is summarized, the success and shortcomings of the project are evaluated, the project data and information are sorted out, a complete research report and technical documents are formed, the strain construction and fermentation process of vitamin B2 are further optimized, the output is further increased and the cost is reduced, the application field of vitamin B2 is expanded, more market-competitive products are developed, cooperation and exchanges with relevant domestic and foreign enterprises and research institutions are strengthened, and the sustainable development of the vitamin B2 industry is promoted.

[0041] In this example, a fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome. Specifically, the steps included screening the complete operon related to riboflavin synthesis from the Bacillus cereus ATCC14579 genome through bioinformatics comparison, including key genes such as ribGBAH, designing primers to amplify the target fragment, introducing homology arms during PCR amplification, and adding restriction sites. An Escherichia coli-Bacillus subtilis shuttle vector was selected, and the amplified riboflavin operon fragment was inserted into the vector multiple cloning site. An antibiotic resistance marker was added, and the correctness of the vector was verified by enzyme digestion and sequencing. The recombinant vector was introduced into Bacillus subtilis competent cells, and the electric field strength was set to 12 kV / cm and the pulse time was 5 ms to improve the transformation efficiency. The riboflavin operon was site-specifically inserted into a specific region of the host genome by double crossover using the natural homologous recombination system of Bacillus subtilis.

[0042] In this embodiment, when the screened strains are induced to undergo mutation treatment using chemical mutagens, a selective culture medium is used to activate the target strain and inhibit the growth of other bacteria. The strain is purified by dilution plate method or microscopic single spore separation technology to ensure that the starting strain is a haploid pure strain. The bacteria are transferred to fresh liquid culture medium and cultured with shaking until the logarithmic growth phase to ensure that the cell metabolic state is consistent.

[0043] In this example, the mutagenesis reaction conditions are as follows: treatment temperature: 25° C., treatment time: EMS treatment for 30 min, and NTG treatment for 10 min.

[0044] Example 2

[0045] A method for preparing a safe and high-yield bacterial strain for vitamin B2 production comprises the following steps:

[0046] S1. Strain source and screening: Analyze the current status and development trend of vitamin B2 and screen the strains;

[0047] S2. Genetic engineering: Evaluate the production performance and genetic stability of the strains, and use chemical mutagens to mutagenize the selected strains to obtain genetically modified strains;

[0048] S3. Screening and identification of high-yield mutants: A fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome to generate mutants. The riboflavin operon in the mutants was fine-tuned using a multiplex genome editing system to increase vitamin B2 production. By comparing the production performance of different mutants, high-yield vitamin B2 strains were screened.

[0049] S4. Optimization of fermentation process: Optimize the fermentation conditions of high-yield strains and develop downstream separation and purification technologies suitable for high-yield strains;

[0050] S5. Development of downstream separation and purification technologies: Debug and optimize the production line to ensure the stability and efficiency of the production process, establish a comprehensive product quality control system, and strictly test the yield, purity, and safety indicators of vitamin B2;

[0051] S6. Product quality control and safety assessment: Summarize the entire project development process and evaluate the project's success and shortcomings.

[0052] In this embodiment, in S1, the research status and development trend of vitamin B2 at home and abroad are analyzed, the limitations of current production technology and market demand are understood, technical literature on vitamin B2 strain construction, fermentation process, separation and purification at home and abroad are collected and studied, and the types, dosage forms, prices and application fields of vitamin B2 products on the market are investigated.

[0053] In this embodiment, in S2, currently commonly used engineered bacteria for producing vitamin B2, including Ashwagandha yeast and Bacillus subtilis, are selected, their production performance and genetic stability are evaluated, and the screened strains are induced to undergo mutation treatment using chemical mutagens to obtain genetically mutated strains. Engineered bacteria with the ability to synthesize purine and riboflavin are screened out using an intelligent computer system, and structural analogs of threonine and proline are used as screening conditions to further obtain ideal engineered strains.

[0054] In this example, in S3, a fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome to generate a mutant strain. The riboflavin operon in the mutant strain was fine-tuned using a multiple genome editing system to increase the production of vitamin B2. By comparing the production performance of different mutant strains, high-yield vitamin B2 strains were screened, and the screened high-yield strains were subjected to multiple rounds of verification to ensure their genetic stability and the reliability of their production performance.

[0055] In this embodiment, in S4, the fermentation conditions of the high-yield strain are optimized, including the culture medium formula, fermentation temperature, and pH value. Through shake flask experiments, small-scale tests, and pilot stages, the fermentation scale is gradually expanded, the fermentation process is evaluated and optimized, and downstream separation and purification technology suitable for the high-yield strain is developed, including cell disruption, extraction, and purification steps. The separation and purification process is optimized to improve the purity and yield of vitamin B2.

[0056] In this embodiment, in S5, an industrial production line of vitamin B2 is constructed based on the optimized fermentation process and separation and purification technology, the production line is debugged and optimized to ensure the stability and efficiency of the production process, a complete product quality control system is established, and the output, purity, and safety indicators of vitamin B2 are strictly tested.

[0057] In this embodiment, in S6, the development process of the entire project is summarized, the success and shortcomings of the project are evaluated, the project data and information are sorted out, a complete research report and technical documents are formed, the strain construction and fermentation process of vitamin B2 are further optimized, the output is further increased and the cost is reduced, the application field of vitamin B2 is expanded, more market-competitive products are developed, cooperation and exchanges with relevant domestic and foreign enterprises and research institutions are strengthened, and the sustainable development of the vitamin B2 industry is promoted.

[0058] In this example, a fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome. Specifically, the steps included screening the complete operon related to riboflavin synthesis from the Bacillus cereus ATCC14579 genome through bioinformatics comparison, including key genes such as ribGBAH, designing primers to amplify the target fragment, introducing homology arms during PCR amplification, adding restriction sites, selecting an Escherichia coli-Bacillus subtilis shuttle vector, inserting the amplified riboflavin operon fragment into the vector multiple cloning site, adding an antibiotic resistance marker, verifying the correctness of the vector by enzyme digestion and sequencing, introducing the recombinant vector into Bacillus subtilis competent cells, setting the electric field strength to 13 kV / cm and the pulse time to 5 ms to improve the transformation efficiency, and utilizing the natural homologous recombination system of Bacillus subtilis to site-directedly insert the riboflavin operon into a specific region of the host genome by double crossover.

[0059] In this embodiment, when the screened strains are induced to undergo mutation treatment using chemical mutagens, a selective culture medium is used to activate the target strain and inhibit the growth of other bacteria. The strain is purified by dilution plate method or microscopic single spore separation technology to ensure that the starting strain is a haploid pure strain. The bacteria are transferred to fresh liquid culture medium and cultured with shaking until the logarithmic growth phase to ensure that the cell metabolic state is consistent.

[0060] In this embodiment, the mutagenesis reaction conditions are as follows: treatment temperature: 30° C., treatment time: EMS treatment for 40 min, and NTG treatment for 20 min.

[0061] Example 3

[0062] A method for preparing a safe and high-yield bacterial strain for vitamin B2 production comprises the following steps:

[0063] S1. Strain source and screening: Analyze the current status and development trend of vitamin B2 and screen the strains;

[0064] S2. Genetic engineering: Evaluate the production performance and genetic stability of the strains, and use chemical mutagens to mutagenize the selected strains to obtain genetically modified strains;

[0065] S3. Screening and identification of high-yield mutants: A fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome to generate mutants. The riboflavin operon in the mutants was fine-tuned using a multiplex genome editing system to increase vitamin B2 production. By comparing the production performance of different mutants, high-yield vitamin B2 strains were screened.

[0066] S4. Optimization of fermentation process: Optimize the fermentation conditions of high-yield strains and develop downstream separation and purification technologies suitable for high-yield strains;

[0067] S5. Development of downstream separation and purification technologies: Debug and optimize the production line to ensure the stability and efficiency of the production process, establish a comprehensive product quality control system, and strictly test the yield, purity, and safety indicators of vitamin B2;

[0068] S6. Product quality control and safety assessment: Summarize the entire project development process and evaluate the project's success and shortcomings.

[0069] In this embodiment, in S1, the research status and development trend of vitamin B2 at home and abroad are analyzed, the limitations of current production technology and market demand are understood, technical literature on vitamin B2 strain construction, fermentation process, separation and purification at home and abroad are collected and studied, and the types, dosage forms, prices and application fields of vitamin B2 products on the market are investigated.

[0070] In this embodiment, in S2, currently commonly used engineered bacteria for producing vitamin B2, including Ashwagandha yeast and Bacillus subtilis, are selected, their production performance and genetic stability are evaluated, and the screened strains are induced to undergo mutation treatment using chemical mutagens to obtain genetically mutated strains. Engineered bacteria with the ability to synthesize purine and riboflavin are screened out using an intelligent computer system, and structural analogs of threonine and proline are used as screening conditions to further obtain ideal engineered strains.

[0071] In this example, in S3, a fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome to generate a mutant strain. The riboflavin operon in the mutant strain was fine-tuned using a multiple genome editing system to increase the production of vitamin B2. By comparing the production performance of different mutant strains, high-yield vitamin B2 strains were screened, and the screened high-yield strains were subjected to multiple rounds of verification to ensure their genetic stability and the reliability of their production performance.

[0072] In this embodiment, in S4, the fermentation conditions of the high-yield strain are optimized, including the culture medium formula, fermentation temperature, and pH value. Through shake flask experiments, small-scale tests, and pilot stages, the fermentation scale is gradually expanded, the fermentation process is evaluated and optimized, and downstream separation and purification technology suitable for the high-yield strain is developed, including cell disruption, extraction, and purification steps. The separation and purification process is optimized to improve the purity and yield of vitamin B2.

[0073] In this embodiment, in S5, an industrial production line of vitamin B2 is constructed based on the optimized fermentation process and separation and purification technology, the production line is debugged and optimized to ensure the stability and efficiency of the production process, a complete product quality control system is established, and the output, purity, and safety indicators of vitamin B2 are strictly tested.

[0074] In this embodiment, in S6, the development process of the entire project is summarized, the success and shortcomings of the project are evaluated, the project data and information are sorted out, a complete research report and technical documents are formed, the strain construction and fermentation process of vitamin B2 are further optimized, the output is further increased and the cost is reduced, the application field of vitamin B2 is expanded, more market-competitive products are developed, cooperation and exchanges with relevant domestic and foreign enterprises and research institutions are strengthened, and the sustainable development of the vitamin B2 industry is promoted.

[0075] In this example, a fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome. Specifically, the steps included screening the complete operon related to riboflavin synthesis from the Bacillus cereus ATCC14579 genome through bioinformatics comparison, including key genes such as ribGBAH, designing primers to amplify the target fragment, introducing homology arms during PCR amplification, and adding restriction sites. An Escherichia coli-Bacillus subtilis shuttle vector was selected, and the amplified riboflavin operon fragment was inserted into the vector multiple cloning site. An antibiotic resistance marker was added, and the correctness of the vector was verified by enzyme digestion and sequencing. The recombinant vector was introduced into Bacillus subtilis competent cells, and the electric field strength was set to 15 kV / cm and the pulse time was 5 ms to improve the transformation efficiency. The riboflavin operon was site-specifically inserted into a specific region of the host genome by double crossover using the natural homologous recombination system of Bacillus subtilis.

[0076] In this embodiment, when the screened strains are induced to undergo mutation treatment using chemical mutagens, a selective culture medium is used to activate the target strain and inhibit the growth of other bacteria. The strain is purified by dilution plate method or microscopic single spore separation technology to ensure that the starting strain is a haploid pure strain. The bacteria are transferred to fresh liquid culture medium and cultured with shaking until the logarithmic growth phase to ensure that the cell metabolic state is consistent.

[0077] In this example, the mutagenesis reaction conditions are as follows: treatment temperature: 37° C., treatment time: EMS treatment for 60 min, and NTG treatment for 30 min.

[0078] The above is only a preferred specific implementation method of this embodiment, but the protection scope of this embodiment is not limited to this. Any technician familiar with this technical field can make equivalent replacements or changes based on the technical solution and inventive concept of this embodiment within the technical scope disclosed in this embodiment, and they should be covered by the protection scope of this embodiment.

Claims

1. A method for preparing a safe and high-yield strain of vitamin B2 for production, characterized in that: The following steps are involved: S1. Strain source and screening: Analyze the current status and development trend of vitamin B2 and screen the strains; S2. Genetic engineering: Evaluate the production performance and genetic stability of the strains, and use chemical mutagens to mutagenize the selected strains to obtain genetically modified strains; S3. Screening and identification of high-yield mutants: A fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome to generate mutants. The riboflavin operon in the mutants was fine-tuned using a multiplex genome editing system to increase vitamin B2 production. By comparing the production performance of different mutants, high-yield vitamin B2 strains were screened. S4. Optimization of fermentation process: Optimize the fermentation conditions of high-yield strains and develop downstream separation and purification technologies suitable for high-yield strains; S5. Development of downstream separation and purification technologies: Debug and optimize the production line to ensure the stability and efficiency of the production process, establish a comprehensive product quality control system, and strictly test the yield, purity, and safety indicators of vitamin B2; S6. Product quality control and safety assessment: Summarize the entire project development process and evaluate the project's success and shortcomings.

2. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 1, characterized in that: In said S1, the research status and development trend of vitamin B2 at home and abroad are analyzed, the limitations of current production technology and market demand are understood, technical literature on vitamin B2 strain construction, fermentation process, separation and purification at home and abroad are collected and studied, and the types, dosage forms, prices and application fields of vitamin B2 products on the market are investigated.

3. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 2, characterized in that: In S2, currently commonly used engineered bacteria for producing vitamin B2 are selected, including Ashwagandha yeast and Bacillus subtilis, and their production performance and genetic stability are evaluated. The screened strains are induced to undergo mutation treatment using chemical mutagens to obtain genetically mutated strains. Engineered bacteria with the ability to synthesize purine and riboflavin are screened out using an intelligent computer system, and structural analogs of threonine and proline are used as screening conditions to further obtain ideal engineered strains.

4. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 3, characterized in that: In the S3, a fragment containing the riboflavin operon of Bacillus cereus is integrated into the vitamin B2 genome to generate a mutant strain. The riboflavin operon in the mutant strain is fine-tuned using a multiple genome editing system to increase the production of vitamin B2. By comparing the production performance of different mutant strains, high-yield vitamin B2 strains are screened out, and the screened high-yield strains are subjected to multiple rounds of verification to ensure their genetic stability and the reliability of their production performance.

5. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 4, characterized in that: In S4, the fermentation conditions of the high-yield strain are optimized, including the culture medium formula, fermentation temperature, and pH value. Through shake flask experiments, small-scale tests, and pilot stages, the fermentation scale is gradually expanded, the fermentation process is evaluated and optimized, and downstream separation and purification technologies suitable for the high-yield strain are developed, including cell disruption, extraction, and purification steps. The separation and purification process is optimized to improve the purity and yield of vitamin B2.

6. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 5, characterized in that: In S5, based on the optimized fermentation process and separation and purification technology, an industrial production line of vitamin B2 is built, the production line is debugged and optimized to ensure the stability and efficiency of the production process, a complete product quality control system is established, and the output, purity and safety indicators of vitamin B2 are strictly tested.

7. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 6, characterized in that: In S6, the development process of the entire project is summarized, the success and shortcomings of the project are evaluated, the project data and information are sorted out, a complete research report and technical documents are formed, the strain construction and fermentation process of vitamin B2 are further optimized, the output is further increased and the cost is reduced, the application areas of vitamin B2 are expanded, more market-competitive products are developed, cooperation and exchanges with relevant domestic and foreign companies and research institutions are strengthened, and the sustainable development of the vitamin B2 industry is promoted.

8. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 7, characterized in that: A fragment containing the riboflavin operon of Bacillus cereus was integrated into the vitamin B2 genome. Specifically, the method included screening the complete operon related to riboflavin synthesis, including the ribGBAH key gene, from the genome of Bacillus cereus ATCC14579 through bioinformatics comparison, designing primers to amplify the target fragment, introducing homology arms and adding restriction sites during PCR amplification, selecting an Escherichia coli-Bacillus subtilis shuttle vector, inserting the amplified riboflavin operon fragment into the vector's multiple cloning site, adding an antibiotic resistance marker, verifying the correctness of the vector through restriction enzyme digestion and sequencing, introducing the recombinant vector into Bacillus subtilis competent cells, setting the electric field strength to 12-15 kV / cm and the pulse time to 5 ms to improve transformation efficiency, and utilizing the natural homologous recombination system of Bacillus subtilis to site-directedly insert the riboflavin operon into a specific region of the host genome through double crossover.

9. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 8, characterized in that: When using chemical mutagens to mutate the selected strains, use selective culture medium to activate the target strain and inhibit the growth of other bacteria. Purify the strain by dilution plate method or microscopic single spore separation technology to ensure that the starting strain is a haploid pure strain. Transfer the bacteria to fresh liquid culture medium and culture with shaking until the logarithmic growth phase to ensure that the cell metabolic state is consistent.

10. The method for preparing a safe and high-yield strain for vitamin B2 production according to claim 9, characterized in that: Mutagenesis reaction conditions: treatment temperature: 25-37°C, treatment time: EMS treatment 30-60 min, NTG treatment 10-30 min.

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