A method for mutagenesis and screening of 5-ala high-yield strain

Abstract

The application discloses a 5-ALA high-yield strain mutagenesis screening method, a) after ARTP mutagenesis, 5-ALA strains are respectively coated on culture mediums with 5-ALA concentrations of 20 g / L-50 g / L to obtain first-generation production strains; b) the production strains are subjected to ARTP mutagenesis again, and then are coated on culture mediums with pH values of 5.5-6.5 to obtain 5-ALA production strains. Through two-step ARTP mutagenesis and two kinds of culture environment pressure induction, the production strains with a titer 25% higher than that of the starting strains are obtained, the production cost is reduced, and the production efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically, it relates to a mutagenesis screening technique for high-yield 5-ALA strains. Background Technology

[0002] 5-Aminolevulinic acid (ALA), also known as 5-amino-4-ketovalerate, is a naturally occurring functional non-protein amino acid. It is a common precursor in the biosynthesis of all porphyrin compounds (heme, chlorophyll, vitamin B12, and cytochromes) and is widely found in bacteria, fungi, animals, and plants, playing a crucial role in cellular energy metabolism. ALA is biodegradable and non-toxic, leaving no residue, and is widely used in medicine, pesticides, and chemicals.

[0003] In agriculture, 5-ALA promotes plant growth and increases crop yield and quality. In medicine, it is used for the prevention, diagnosis, and treatment of diabetes and cancer. In cosmetics, it moisturizes and helps improve skin hydration and elasticity. In animal feed, it accelerates muscle protein synthesis, improves anemia, and enhances the immunity of poultry and livestock. Currently, due to limitations in production technology and cost, medicine and agriculture are the main application areas for 5-ALA; other fields have not yet been effectively developed.

[0004] In recent years, with the rise of biotechnology, microbial fermentation has attracted attention, and some companies have achieved small-scale industrial production. However, due to high production costs, microbial fermentation still cannot meet the needs of the application market, resulting in 5-ALA applications mainly concentrated in the high-end pharmaceutical field. This invention aims to improve the fermentation production level of 5-ALA, reduce production costs, and broaden the application fields of 5-ALA by developing high-yield strains of 5-ALA suitable for industrial production, thereby further developing the 5-ALA market.

[0005] The current level of 5-ALA fermentation production is generally low, resulting in a relatively high price for 5-aminolevulinic acid, and the current production capacity of 5-ALA cannot meet domestic demand. Therefore, improving the fermentation production level of 5-ALA has broad prospects. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for mutagenesis screening of high-yield 5-ALA strains.

[0007] A method for mutagenesis screening of 5-ALA producing strains. a) After ARTP mutagenesis, the 5-ALA strains were plated on medium with 5-ALA concentrations of 20 g / L to 50 g / L, and surviving clones were screened to obtain the first generation of production strains. b) The production strain was then subjected to ARTP mutagenesis and then plated on a medium with pH 5.5-6.5. Surviving clones were screened to obtain the 5-ALA production strain.

[0008] The ARTP mutagenesis was performed as follows: Corynebacterium glutamicum was streaked onto LB agar plates and incubated upside down at 30°C for 2 days. Single colonies were picked and cultured in LB liquid shaker tubes at 30°C and 250 rpm for 14-16 hours. The culture medium was washed away with 1 mM sterile NaCl aqueous solution, and the bacterial cells were diluted to 10⁻⁶. 6 -10 8 cfu / ml; 10 μL of diluted bacterial solution was evenly spread onto the surface of a thin iron sheet. ARTP mutagenesis was performed using an incident power of 120 W, a helium flow rate of 10 L / min, a mutagenesis time of 60 s, and a plasma jet nozzle distance of 2 mm from the sample.

[0009] 5-ALA strain was fermented in shake flasks using M9 medium containing: glucose 25 g / L, disodium hydrogen phosphate dodecahydrate 17.1 g / L, glycine 6 g / L, potassium dihydrogen phosphate 3 g / L, yeast extract 2 g / L, ammonium chloride 1 g / L, magnesium sulfate heptahydrate 0.24 g / L, and calcium chloride 11 mg / L. Single clones of 5-ALA strain were picked from LB agar plates containing 15 mg / L chloramphenicol and placed in liquid shake tubes. The mixture was incubated at 30°C and 250 rpm for 12-16 h. 3%-4% of the culture was inoculated into M9 shake flasks, which were then incubated in a shaker at 30°C and 250 rpm for 20 h. After 20 h, 0.5 mM IPTG was added for induction culture, and the culture was continued for 48 h before sampling and analysis.

[0010] 5-ALA strain fed-batch fermentation in a 5L tank: Fermentation broth: peptone 10g / L, yeast extract 15g / L, sodium chloride 2.5g / L, glycine 7.5g / L, glucose 50g / L; Fermentation process: Initial fermentation temperature 30℃, aeration rate 30L / min, initial rotation speed 400rpm, pH 7.0, dissolved oxygen value linked to rotation speed not less than 30% during cell growth; when cell wet weight reaches 100g / L, add 0.5mM / L IPTG for induction, pH 6.5, add 100g / L glycine and 100g / L succinic acid, cycle 600s, opening speed 1s, after 2h induction, add 0.2g / L calcium pantothenate, 0.2g / L pyridoxal hydrochloride, 10mg / L ascorbic acid, 0.01g / L FeSO4, 0.01g / L MnSO4, and 0.2g / L MgSO4; the dissolved oxygen value linked to rotation speed is set at 10%, and the fermentation time is within 48h.

[0011] The beneficial effects of this invention are: By inducing mutagenesis in two steps and under two different culture environment pressures, a production strain with a potency 25% higher than the starting strain was obtained, which reduced production costs and improved production efficiency. Attached Figure Description

[0012] Figure 1 This is a comparison chart of fermentation potency in 5L tanks. Detailed Implementation

[0013] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0014] Unless otherwise specified, the biological materials, reagents, or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0015] Equipment: Atmospheric pressure room temperature plasma mutagenesis breeding instrument (Wuxi Yuanqing Tianmu), constant temperature shaker (Yiheng), constant temperature incubator (Yiheng), water bath, 5L fermenter (Bailun), centrifuge (Tianmei).

[0016] ARTP mutagenesis screening method: Corynebacterium glutamicum was streaked onto LB agar plates and incubated upside down at 30°C for about 2 days. Single colonies were picked and transferred to LB liquid culture tubes and incubated at 30°C and 250 rpm for 14-16 hours. The culture medium was washed off with 1 mM sterile NaCl aqueous solution, and the bacterial cells were diluted to 10⁻⁶. 6 -10 8 cfu / ml.

[0017] 10 μL of diluted bacterial solution was evenly spread onto the surface of a thin iron sheet, and ARTP mutagenesis was performed under the following conditions: incident power 120 W, helium flow rate 10 L / min, mutagenesis time 60 s, and plasma jet nozzle distance 2 mm from the sample.

[0018] The mutagenized samples were incubated in LB liquid culture containing 0.5% glycine for 2-3 hours, then spread on LB-MOPS solid plates containing high concentrations of 5-ALA (20g / L-80g / L) or acidic (pH 5.0-pH 6.0) and incubated at 30°C for 2-3 days.

[0019] 96-well plate fermentation validation: Select fast-growing (large colony) single clones into 96-well cell culture plates. The culture medium is LB-MOPS with 0.2mM IPTG. Incubate at 30℃ and 190rpm for 72h. Take samples every 24h to detect 5-ALA. Select single clones with high content for shake-flask fermentation validation.

[0020] Shake-flask fermentation of 5-ALA strain: M9 shake flask culture medium: glucose 25 g / L, disodium hydrogen phosphate dodecahydrate 17.1 g / L, glycine 6 g / L, potassium dihydrogen phosphate 3 g / L, yeast extract 2 g / L, ammonium chloride 1 g / L, magnesium sulfate heptahydrate 0.24 g / L, calcium chloride 11 mg / L. Single clones of 5-ALA strain were picked from LB (15 mg / L chloramphenicol) plates and placed in liquid shake tubes. The mixture was incubated at 30°C and 250 rpm for 12-16 h. M9 shake flasks were then inoculated with 3%-4% of the culture and incubated at 30°C and 250 rpm. After 20 h, 0.5 mM IPTG was added for induction culture, and the culture was continued for 48 h before sampling and analysis.

[0021] 5-ALA strain fed-batch fermentation in a 5L tank: Fermentation broth: peptone 10g / L, yeast extract 15g / L, sodium chloride 2.5g / L, glycine 7.5g / L, glucose (sterilized separately) 50g / L.

[0022] Fermentation process: Initial fermentation temperature 30℃, aeration rate 30L / min, initial turbine rotation speed 400rpm, pH 7.0, dissolved oxygen level linked to turbine rotation speed not lower than 30% during cell growth. When the cell wet weight reaches approximately 100g / L (generally after 10 hours of fermentation), IPTG (0.5mM / L) is added for induction, pH 6.5, followed by the addition of glycine (100g / L) and succinic acid (100g / L) (cycle 600s, opening speed 1s). After 2 hours of induction, calcium pantothenate 0.2g / L, pyridoxal hydrochloride 0.2g / L, ascorbic acid (VC) 10mg / L, FeSO4 0.01g / L, MnSO4 0.01g / L, and MgSO4 0.2g / L are added. The dissolved oxygen level linked to turbine rotation speed is set at approximately 10%, and the fermentation time is approximately within 48 hours.

[0023] 5-ALA detection method: A. Preparation of color developer (store at 4 degrees Celsius for no more than 12 hours) Take 9 ml of glacial acetic acid, add 1 ml of perchloric acid, mix well, and then add 0.2 g of p-dimethylaminobenzaldehyde to dissolve.

[0024] B. Sodium acetate solution Take 50mL of pure water, add 5.7mL of glacial acetic acid and 8.2g of sodium acetate, and dilute with pure water to 100mL.

[0025] C. Sample diluent (used to dilute the fermentation broth) Acetic acid solution with pH 3.0.

[0026] Testing steps: (1) Dilute the fermentation broth with solution C to a suitable concentration (approximately 1-10 mg / L); (2) Take 250 μL of the diluted solution, add 125 μL of solution B, add 62.5 μL of acetylacetone, and boil in boiling water for 15 minutes.

[0027] (3) Cool the sample from step (2) to room temperature, add 400 μL of A, mix well, and react for 20 minutes.

[0028] (4) The absorbance of the sample at a wavelength of 554 nm was measured by a spectrophotometer (OD554).

[0029] (5) 5-ALA standard curve Weigh 0.1 g of 5-aminolevulinic acid salt standard, dissolve it in 100 mL of C solution, and dilute it to prepare standard solutions of 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L. Detect the absorbance of the standard at a wavelength of 554 nm (OD554) according to the method in steps (2)-(3), and generate the corresponding linear calculation formula.

[0030] (6) Calculate the concentration of 5-aminolevulinic acid salt in the sample based on the OD554 value and the corresponding formula, and convert it to the concentration of 5-aminolevulinic acid.

[0031] Example 1: Adaptive Screening with High Concentration 5-ALA The 5-ALA strain was mutagenized by ARTP and then plated on LB-MOPS plates with 5-ALA concentrations of 0 g / L, 20 g / L, 50 g / L, and 80 g / L, respectively. After 5 days, the lethality rate was calculated based on the number of clones growing on the plates, and 96 clones were selected from each concentration gradient plate for 96-well plate fermentation verification.

[0032] The initial screening results of 96-well plates showed that clones screened using LB solid medium with 20 g / L 5-ALA had an average titer 21% higher than those screened without 5-ALA. However, when the 5-ALA concentration was increased to 50 g / L, the average titer of the screened clones did not increase, and when the 5-ALA concentration was increased to 80 g / L, no clones grew.

[0033] Five clones with the highest potency were selected from 96-well plates corresponding to concentrations of 0 g / L, 20 g / L, and 50 g / L, respectively, for shake-flask fermentation.

[0034] Based on the secondary screening results, clone 20-2 exhibited the highest titer in shake flasks. This clone was then subjected to fed-batch fermentation in a 5L tank, achieving a maximum titer of 30 g / L, 7% higher than the starting strain. This strain was named R1. Figure 1 (As shown) Example 2: Low pH Adaptability Screening The R1 strain was mutagenized by ARTP and plated on LB-MOPS plates at pH 5.0, pH 5.5, pH 6.0 and pH 6.5, respectively. After 3 days, the lethality rate was calculated based on the number of clones growing on the plates, and 96 clones were selected from each pH gradient plate for 96-well plate fermentation verification.

[0035] Five clones with the highest potency were selected from 96-well plates corresponding to pH 6.5, pH 6.0, and pH 5.5, respectively, for shake-flask fermentation.

[0036] Based on the shake-flask screening results, strain 6.0-1 was selected for fed-batch fermentation in a 5L tank. After 33 hours of fermentation, the highest efficiency of this strain reached 35 g / L, which was 25% higher than the starting strain. This strain was named R2. Figure 1 (As shown) The embodiments described above can be further combined or replaced, and these embodiments are merely descriptions of preferred embodiments of the present invention, not limitations on the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the inventive concept are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalents.

Claims

1. A method for mutagenesis screening of 5-ALA producing strains, characterized in that, a) After ARTP mutagenesis, the 5-ALA strains were plated on medium with 5-ALA concentrations of 20 g / L to 50 g / L, and surviving clones were screened to obtain the first generation of production strains. b) The production strain was then subjected to ARTP mutagenesis and then plated on a medium with pH 5.5-6.

5. Surviving clones were screened to obtain the 5-ALA production strain.

2. The mutagenesis screening method for 5-ALA producing strains according to claim 1, characterized in that, The ARTP mutagenesis was performed as follows: Corynebacterium glutamicum was streaked onto LB agar plates and incubated upside down at 30°C for 2 days. Single colonies were picked and cultured in LB liquid shaker tubes at 30°C and 250 rpm for 14-16 hours. The culture medium was washed away with 1 mM sterile NaCl aqueous solution, and the bacterial cells were diluted to 10⁻⁶. 6 -10 8 cfu / ml; 10 μL of diluted bacterial solution was evenly spread onto the surface of a thin iron sheet. ARTP mutagenesis was performed using an incident power of 120 W, a helium flow rate of 10 L / min, a mutagenesis time of 60 s, and a plasma jet nozzle distance of 2 mm from the sample.

3. The mutagenesis screening method for 5-ALA producing strains according to claim 1, characterized in that, 5-ALA strain was fermented in shake flasks using M9 medium containing: glucose 25 g / L, disodium hydrogen phosphate dodecahydrate 17.1 g / L, glycine 6 g / L, potassium dihydrogen phosphate 3 g / L, yeast extract 2 g / L, ammonium chloride 1 g / L, magnesium sulfate heptahydrate 0.24 g / L, and calcium chloride 11 mg / L. Single clones of 5-ALA strain were picked from LB agar plates containing 15 mg / L chloramphenicol and placed in liquid shake tubes. The mixture was incubated at 30°C and 250 rpm for 12-16 h. 3%-4% of the culture was inoculated into M9 shake flasks, which were then incubated in a shaker at 30°C and 250 rpm for 20 h. After 20 h, 0.5 mM IPTG was added for induction culture, and the culture was continued for 48 h before sampling and analysis.

4. The mutagenesis screening method for 5-ALA producing strains according to claim 1, characterized in that, 5-ALA strain fed-batch fermentation in a 5L tank: Fermentation broth: peptone 10g / L, yeast extract 15g / L, sodium chloride 2.5g / L, glycine 7.5g / L, glucose 50g / L; Fermentation process: Initial fermentation temperature 30℃, aeration rate 30L / min, initial rotation speed 400rpm, pH 7.0, dissolved oxygen value linked to rotation speed not less than 30% during cell growth; when cell wet weight reaches 100g / L, add 0.5mM / L IPTG for induction, pH 6.5, feed 100g / L glycine and 100g / L succinic acid, cycle 600s, opening speed 1s, after 2h induction, add 0.2g / L calcium pantothenate, 0.2g / L pyridoxal hydrochloride, 10mg / L ascorbic acid, 0.01g / L FeSO4, 0.01g / L MnSO4, and 0.2g / L MgSO4; set dissolved oxygen value linked to rotation speed to 10%, fermentation time within 48h.

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