High-efficiency fermentation production method of 5-aminolevulinic acid based on propionibacterium
Through the efficient fermentation method based on propionibacterium, gene expression and fermentation conditions are optimized, and the problems of environmental pollution and low yield in traditional 5-aminolevulinic acid production are solved, and high yield, low by-products and high conversion rates are achieved. The effect is in line with industrial green environmental protection standards.
Patent Information
- Application Number
- CN202411382663.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2024-12-27
AI Technical Summary
In the traditional 5-aminolevulinic acid production methods, chemical synthesis has problems of environmental pollution and low yields, and microbial fermentation relies on precise gene operation and complex regulation, limiting industrial applications.
Using an efficient fermentation method based on propionibacterium, the glutamate semialdehyde synthase and glutamate deaminase gene were optimized through CRISPR-Cas9 technology, the propionate metabolic gene was knocked out, and the optimized fermentation medium and finely regulated fermentation conditions were constructed. Combined with cation exchange column purification and freeze-drying techniques.
It significantly improves the yield and bioconversion rate of 5-aminolevulinic acid, reduces the generation of by-products, simplifies the purification process, reduces the cost and environmental burden, and complies with green environmental protection standards.
Smart Images
Figure CN119193724A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a fermentation production method, and more specifically to a high-efficiency fermentation production method of 5-aminolevulinic acid based on propionibacterium. Background Art
[0002] 5-Aminolevulinic acid (5-ALA) is an important biosynthetic precursor and is widely used in medicine, agriculture and food industry. It is a key component of many plant growth regulators and photosensitizers, especially in photodynamic therapy and cancer diagnosis. Traditional 5-ALA production methods mainly include chemical synthesis and microbial fermentation.
[0003] Although chemical synthesis can achieve the production of 5-ALA, it usually involves the use of toxic organic solvents and complex purification steps, with low yields and environmental pollution issues. At present, research on the production of 5-ALA through microbial pathways mainly focuses on the use of microorganisms such as Escherichia coli and Saccharomyces cerevisiae. However, these microbial systems often require precise genetic manipulation and complex metabolic engineering regulation, and are highly dependent on fermentation conditions, which limits their application in industrial production. Summary of the invention
[0004] In order to solve the shortcomings of the prior art, a method for efficiently fermenting 5-aminolevulinic acid based on Propionibacterium is provided. The specific scheme is as follows:
[0005] A highly efficient fermentation production method for 5-aminolevulinic acid based on Propionibacterium, the fermentation production method comprising the following steps:
[0006] S1. Optimization of Propionibacterium: including optimization of glutamate semialdehyde synthase gene and glutamate deaminase gene and knockout of propionic acid metabolism gene;
[0007] S2. constructing a fermentation medium: the fermentation medium comprises a carbon source, a nitrogen source, a precursor substance, a phosphorus source and trace elements;
[0008] S3, seed culture: thaw the frozen Propionibacterium strain and inoculate it into the seed culture medium, with an inoculation amount of 5-10%, and shake culture at 37°C and 200 rpm for 12-16 hours;
[0009] S4: Optimize fermentation conditions: transfer the seed culture solution to the fermentation tank, control the fermentation conditions, and the fermentation cycle is 48 to 72 hours;
[0010] S5: Isolation and purification of 5-aminolevulinic acid: After the fermentation is completed, the fermentation liquid is centrifuged to remove the bacteria; the pH value of the supernatant is adjusted to 4.0-4.5, and the precipitate is removed by centrifugation; the cation exchange column is used for separation and purification, and the cation exchange column uses CM-cellulose resin and a low concentration NaCl solution to elute the 5-aminolevulinic acid; the eluate is vacuum concentrated, the pH is adjusted to 5.0-5.5 for crystallization, and finally high-purity 5-aminolevulinic acid is obtained by freeze-drying.
[0011] Furthermore, S1 specifically includes the following steps:
[0012] S101: Optimization of glutamate semialdehyde synthetase gene: CRISPR-Cas9 technology was used to design specific sgRNA for the glutamate semialdehyde synthetase gene in Propionibacterium, and the glutamate semialdehyde synthetase gene was precisely knocked in by co-transfecting sgRNA and Cas9 nuclease into Propionibacterium. The knocked-in glutamate semialdehyde synthetase gene included an expression framework controlled by a strong promoter to ensure high levels of gene expression. The promoter strength was set to synthesize about 200-300 mRNA molecules per second;
[0013] S102: Optimization of glutamate deaminase gene: Homologous recombination technology was used to insert the glutamate deaminase gene into the genome of Propionibacterium;
[0014] S103 Knockout of propionic acid metabolism genes: Using the CRISPR-Cas9 system, sgRNAs targeting key enzyme genes of propionic acid metabolism were designed, and these sgRNAs and Cas9 protein complexes were introduced into Propionibacterium to achieve complete knockout of these genes.
[0015] Furthermore, the carbon source in S2 is glucose or glycerol, and the concentration is 30-40 g / L; the nitrogen source is yeast extract 10-15 g / L, peptone 10-15 g / L, and ammonium sulfate 5-10 g / L; the precursor substance is glutamic acid, and the concentration is 3-5 g / L; the phosphorus source is potassium dihydrogen phosphate 1-2 g / L, and potassium hydrogen phosphate 0.5-1 g / L; and the trace elements are calcium chloride 0.05-0.1 g / L, and magnesium sulfate 0.2-0.5 g / L.
[0016] Furthermore, the pH of the culture medium in S2 is controlled at 6.5-7.
[0017] Furthermore, in S3, when OD 600 When the value reaches 1.0-1.5, the culture is stopped.
[0018] Furthermore, in S4, the fermentation temperature is controlled at 30-37°C, the stirring speed is 300-500rpm; the dissolved oxygen condition is an aerobic state of 20-30% at the initial stage, and is switched to a microaerobic or anaerobic state of less than 5% after the bacteria reach a certain concentration.
[0019] Furthermore, the pH in the fermentation tank in S4 is set to 6.5-7.
[0020] Furthermore, in S5, the centrifugal speed of the fermentation liquid was set to 8000 rpm, and the centrifugal time was 15 min; the centrifugal speed of the supernatant was set to 6000 rpm, and the centrifugal time was 10 min.
[0021] Beneficial effects:
[0022] The present invention provides a method for efficiently fermenting 5-aminolevulinic acid based on Propionibacterium, which has the following beneficial effects:
[0023] (1) The production of 5-ALA was significantly increased by optimizing the expression of glutamate semialdehyde synthase and glutamate deaminase genes. Through gene editing and fine-tuning of fermentation conditions, the production was higher than that of traditional methods, effectively meeting the growing market demand for 5-ALA.
[0024] (2) By knocking out the key enzyme genes related to propionic acid metabolism, the production of propionic acid as a byproduct during the fermentation process was significantly reduced. Reducing byproducts not only simplifies the subsequent product purification process, but also reduces the additional costs and environmental burden that may be incurred by handling byproducts.
[0025] (3) By precisely controlling various parameters in the fermentation process, such as pH, temperature, dissolved oxygen conditions, etc., the conversion efficiency of raw materials to products is improved, and the bioconversion rate reaches more than 90%, which is a significant improvement over traditional methods.
[0026] (4) Due to the reduction of by-products and the improvement of production efficiency, this method reduces waste generation and energy consumption, and meets the green environmental protection standards of current industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a flow chart of the efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium. DETAILED DESCRIPTION
[0028] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.
[0029] Figure 1 is a flow chart of a highly efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium, Figure 1As shown, the efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium includes: S1, optimizing Propionibacterium; S2, constructing a fermentation medium; S3, seed culture; S4: optimizing fermentation conditions; S5: separation and purification of 5-aminolevulinic acid, and the specific embodiments are as follows:
[0030] Example:
[0031] This example describes an efficient fermentation production method for 5-aminolevulinic acid (5-ALA) based on Propionibacterium oxysporum optimized. The following are the specific steps and operating parameters:
[0032] S1: Genetic optimization of Propionibacterium
[0033] (1) Optimization of glutamate semialdehyde synthase gene:
[0034] Using CRISPR-Cas9 technology, specific sgRNA targeting the hemA gene of Propionibacterium was designed.
[0035] sgRNA and Cas9 nuclease were co-transfected into Propionibacterium to achieve precise knock-in of the hemA gene.
[0036] The knocked-in hemA gene includes a strong promoter with a promoter strength of about 200-300 mRNA molecules synthesized per second.
[0037] (2) Optimization of glutamate deaminase gene:
[0038] The gdh gene was inserted into the genome of Propionibacterium by homologous recombination technology.
[0039] The expression of the gdh gene is controlled by a natural promoter with high transcriptional activity, ensuring efficient protein synthesis.
[0040] (3) Knockout of propionate metabolism genes:
[0041] The CRISPR-Cas9 system was used to design sgRNAs targeting the ppk and pct genes.
[0042] The sgRNA and Cas9 protein complex were introduced into Propionibacterium to achieve complete knockout of these genes.
[0043] The success and completeness of the knockout were verified by PCR and sequencing.
[0044] S2: Construction of fermentation medium
[0045] Carbon source: Glucose concentration was 35 g / L.
[0046] Nitrogen source: yeast extract 12g / L, peptone 12g / L, ammonium sulfate 7g / L.
[0047] Precursor substance: Glutamic acid concentration is 4g / L.
[0048] Phosphorus source: potassium dihydrogen phosphate 1.5g / L, dipotassium hydrogen phosphate 0.75g / L.
[0049] Trace elements: calcium chloride 0.075g / L, magnesium sulfate 0.35g / L.
[0050] S3: Seed culture
[0051] Optimized Propionibacterium strains were thawed from glycerol stocks at -80°C.
[0052] The inoculum size was 8% and was inoculated into the seed medium.
[0053] The culture was shaken at 37°C and 200 rpm for 14 h until OD 600 The value reaches 1.2.
[0054] S4: Optimize fermentation conditions
[0055] Transfer the seed culture into a pre-sterilized fermenter.
[0056] The fermentation temperature was set to 34°C and the stirring speed was set to 400 rpm.
[0057] The dissolved oxygen condition was adjusted from the initial aerobic state of 20% to a microaerobic state of less than 5%.
[0058] The fermentation cycle was set to 60 hours, maintaining the pH at 6.8.
[0059] S5: Isolation and purification of 5-aminolevulinic acid
[0060] After fermentation, the fermentation broth was centrifuged at 8000 rpm for 15 minutes to remove the bacteria.
[0061] After adjusting the pH of the supernatant to 4.2, the supernatant was centrifuged again at 6000 rpm for 10 minutes to remove the precipitate.
[0062] 5-ALA was separated and purified using a cation exchange column of CM-cellulose resin and eluted using a 0.1 M NaCl solution.
[0063] The eluate is concentrated in vacuo, the pH value is adjusted to 5.2 for crystallization, and finally high-purity 5-aminolevulinic acid is obtained by freeze-drying.
[0064] Test items Comparative example (not optimized) Embodiment (after optimization) Remark 5-ALA yield (g / L) 1.2 4.5 In the same fermentation cycle Fermentation cycle (hours) 72 60 Fermentation time is shortened after optimization Byproduct propionic acid (g / L) 0.5 0.1 Significantly reduce by-product generation Bioconversion rate (%) 75 90 Increased conversion rate Total biomass (g / L) 8.0 12.0 Indicates that the bacteria grow more vigorously Protein expression (mg / L) 200 500 hemA and gdh gene expression
[0065] From the above table, it can be seen that: the optimized strain can produce more 5-ALA in the same volume of culture medium; the optimized fermentation cycle is shortened, the production efficiency is higher, the production cycle can be carried out faster, saving time and cost; the optimization reduces the generation of by-products such as propionic acid, which helps to reduce the difficulty and cost of handling in subsequent purification steps; the bioconversion rate shows how the optimized gene expression and fermentation conditions improve the conversion efficiency of raw materials to products; the increase in biomass indicates that the optimized strain growth conditions are more ideal, which helps to increase yield; the protein expression amount shows the quantitative results of gene expression, indicating that the optimized promoter and expression system increase the yield of the target protein.
[0066] As a further improvement, the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for efficiently fermenting 5-aminolevulinic acid based on Propionibacterium, characterized in that: The fermentation production method comprises the following steps: S1. Optimization of Propionibacterium: including optimization of glutamate semialdehyde synthase gene and glutamate deaminase gene and knockout of propionic acid metabolism gene; S2. constructing a fermentation medium: the fermentation medium comprises a carbon source, a nitrogen source, a precursor substance, a phosphorus source and trace elements; S3, seed culture: thaw the frozen Propionibacterium strain and inoculate it into the seed culture medium, with an inoculation amount of 5-10%, and shake culture at 37°C and 200 rpm for 12-16 hours; S4: Optimize fermentation conditions: transfer the seed culture solution to the fermentation tank, control the fermentation conditions, and the fermentation cycle is 48 to 72 hours; S5: Isolation and purification of 5-aminolevulinic acid: After the fermentation is completed, the fermentation liquid is centrifuged to remove the bacteria; the pH value of the supernatant is adjusted to 4.0-4.5, and the precipitate is removed by centrifugation; the cation exchange column is used for separation and purification, and the cation exchange column uses CM-cellulose resin and a low concentration NaCl solution to elute the 5-aminolevulinic acid; the eluate is vacuum concentrated, the pH is adjusted to 5.0-5.5 for crystallization, and finally high-purity 5-aminolevulinic acid is obtained by freeze-drying.
2. The efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium according to claim 1, characterized in that: S1 specifically includes the following steps: S101: Optimization of glutamate semialdehyde synthetase gene: CRISPR-Cas9 technology was used to design specific sgRNA for the glutamate semialdehyde synthetase gene in Propionibacterium, and the glutamate semialdehyde synthetase gene was precisely knocked in by co-transfecting sgRNA and Cas9 nuclease into Propionibacterium. The knocked-in glutamate semialdehyde synthetase gene included an expression framework controlled by a strong promoter to ensure high levels of gene expression. The promoter strength was set to synthesize about 200-300 mRNA molecules per second; S102: Optimization of glutamate deaminase gene: Homologous recombination technology was used to insert the glutamate deaminase gene into the genome of Propionibacterium; S103 Knockout of propionic acid metabolism genes: Using the CRISPR-Cas9 system, sgRNAs targeting key enzyme genes of propionic acid metabolism were designed, and these sgRNAs and Cas9 protein complexes were introduced into Propionibacterium to achieve complete knockout of these genes.
3. The efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium according to claim 1, characterized in that: The carbon source in S2 is glucose or glycerol with a concentration of 30-40 g / L; the nitrogen source is yeast extract 10-15 g / L, peptone 10-15 g / L, and ammonium sulfate 5-10 g / L; the precursor is glutamic acid with a concentration of 3-5 g / L; the phosphorus source is potassium dihydrogen phosphate 1-2 g / L, and dipotassium hydrogen phosphate 0.5-1 g / L; the trace elements are calcium chloride 0.05-0.1 g / L, and magnesium sulfate 0.2-0.5 g / L.
4. The efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium according to claim 1, characterized in that: The pH of the culture medium in S2 was controlled at 6.5-7.
5. The efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium according to claim 1, characterized in that: S3 was cheated by OD 600 When the value reaches 1.0-1.5, the culture is stopped.
6. The efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium according to claim 1, characterized in that: In S4, the fermentation temperature is controlled at 30-37°C, and the stirring speed is 300-500rpm; the dissolved oxygen condition is an aerobic state of 20-30% at the initial stage, and is switched to a microaerobic or anaerobic state of less than 5% after the bacteria reach a certain concentration.
7. The efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium according to claim 1, characterized in that: The pH in the fermenter in S4 was set to 6.5-7.
8. The efficient fermentation production method of 5-aminolevulinic acid based on Propionibacterium according to claim 1, characterized in that: In S5, the centrifugal speed of the fermentation broth was set to 8000 rpm, and the centrifugal time was 15 min; the centrifugal speed of the supernatant was set to 6000 rpm, and the centrifugal time was 10 min.