Acid-resistant corynebacterium glutamicum and application thereof

Abstract

The invention discloses acid-resistant corynebacterium glutamicum and application thereof, and relates to the technical field of microorganisms. The acid-resistant corynebacterium glutamicum is named as corynebacterium glutamicum A-8, and is preserved in the Guangdong Microbial Culture Collection Center on June 3, 2025, the preservation number is GDMCC NO: 66452, and the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. The Corynebacterium glutamicum A-8 provided by the invention can normally grow in an acidic environment with the pH value of 4.0 and produce glutamic acid, and the problem of bacterial strain growth inhibition caused by pH value reduction in traditional glutamic acid fermentation is effectively solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of acid-tolerant Corynebacterium glutamicum and its application. BACKGROUND

[0002] Glutamic acid, as an important amino acid, is widely used in food additives, pharmaceutical manufacturing and production of chemical products. It is not only a key component of protein synthesis, but also plays an irreplaceable role in maintaining human health and promoting growth. Currently, the preparation of glutamic acid on an industrial scale mainly relies on microbial fermentation, especially the use of Corynebacterium glutamicum for large-scale cultivation. Through the regulation of metabolic pathways and in-depth research and modification of genetic engineering, the yield of glutamic acid has been successfully improved, while maintaining good biological stability and low cost efficiency. Corynebacterium glutamicum

[0003] However, during the fermentation of glutamic acid, the production of glutamic acid leads to a decrease in the pH of the culture medium, which not only limits the growth rate of the strain, but also may cause changes in the metabolic pathway, resulting in a decrease in glutamic acid production. To solve this problem, in traditional production, alkali (such as sodium hydroxide) is often added to adjust the pH of the culture medium to alleviate acid stress, but frequent pH adjustment operations not only increase the production cost, but also may interfere with the metabolic balance of the strain; and in the subsequent glutamic acid extraction process, a large amount of acid (such as hydrochloric acid) needs to be added to reduce the pH to precipitate the product, which not only consumes a large amount of acid, has low extraction efficiency, but also produces a large amount of acid-containing wastewater, polluting the environment and possibly affecting the quality of the product due to acid and alkali residues. Therefore, breeding a strain of Corynebacterium glutamicum with stronger acid tolerance, which can maintain normal growth and high production performance at low pH, has become a key requirement to improve the production efficiency of glutamic acid and reduce environmental burden. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art and provide a strain of acid-tolerant Corynebacterium glutamicum and its application, to solve the technical problem of relatively low acid tolerance of existing glutamic acid-producing strains.

[0005] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows: A strain of acid-tolerant Corynebacterium glutamicum, the acid-tolerant Corynebacterium glutamicum is named Corynebacterium glutamicum (Corynebacterium glutamicum) A-8, and was preserved in Guangdong Microbial Culture Collection Center on June 3, 2025, with the preservation number GDMCC NO: 66452 and the preservation address being 5th floor, No. 59 Building, 100, Junyi Road, Guangzhou. Corynebacterium glutamicum Further, the acid-tolerant Corynebacterium glutamicum is Corynebacterium glutamicum (Corynebacterium glutamicum) A-8.

[0006] Corynebacterium glutamicum ​​Z0 is an original strain obtained by heavy ion irradiation mutagenesis combined with directional screening.

[0007] Further, the corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum Z0 was preserved in the Guangdong Microbial Culture Collection Center on August 29, 2025, with a preservation number of GDMCC NO: 66897 and a preservation address of 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100 Courtyard.

[0008] The application also provides a corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum application of the corynebacterium glutamicum (Corynebacterium glutamicum)

[0009] Further, the corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum A-8 as a fermentation strain to produce glutamic acid, the fermentation base medium comprises: glucose 55-65 g / L, corn syrup 4-6 g / L, urea 8-12 g / L, potassium chloride 2-4 g / L, magnesium sulfate 0.8-1.0 g / L, dipotassium hydrogen phosphate 0.8-1.2 g / L, sucrose 3.5-3.8 g / L, ferrous sulfate 4-6 mg / L, manganese sulfate 4-6 mg / L, VB1 2.2-2.8 mg / L, and deionized water is added to 1L.

[0010] Further, the corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum A-8 as a fermentation strain to produce glutamic acid, the fermentation temperature is 30-37℃, and the fermentation time is 36-48h.

[0011] Compared with the prior art, the application has the following beneficial effects: (1) The corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum A-8 can grow normally and produce glutamic acid in an acidic environment with a pH of 4.0, while the original strain corynebacterium glutamicum Z0 is inhibited under the same pH conditions and has low glutamic acid production, effectively solving the problem of strain growth inhibition caused by pH reduction in traditional glutamic acid fermentation.

[0012] (2) The corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum A-8 under acidic fermentation conditions with a pH of 4.0, the glutamic acid yield reaches 13.5 g / L, breaking the limitation that the original strain cannot produce acid in an acidic environment, and significantly improving the glutamic acid production capacity under acidic conditions.

[0013] (3) The acid resistance of the strain can reduce the frequency and amount of alkali pH adjustment during fermentation, thereby effectively avoiding the disturbance of frequent pH adjustment to the metabolic balance of the strain, and reducing the operation cost and labor burden of the production process.

[0014] (4) The acid-resistant strain provided by the application can directly ferment to produce acid under acid conditions when producing glutamic acid, so that the use amount of acid in the subsequent extraction process (acid needs to be added to precipitate the product in the traditional process) can be reduced, thereby reducing the generation of acid-containing wastewater and alleviating the environmental burden.

[0015] (5) The acid-resistant strain provided by the application is verified by 10 generations of continuous passage, and the glutamic acid yield has no significant difference, indicating that the acid-resistant and high-yield characteristics are stably inherited, thereby providing a reliable strain guarantee for industrial large-scale application. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the growth curve of the Corynebacterium glutamicum Z0 strain; Figure 2 is a lethal rate curve diagram of the original strain Corynebacterium glutamicum Z0 after heavy ion mutagenesis; Figure 3 is a result diagram of the absorbance (OD 600 ) of the primary screening strain after heavy ion mutagenesis; Figure 4 is a glutamic acid yield content diagram of the secondary screening strain after heavy ion mutagenesis; Figure 5 is a columnar analysis diagram of the genetic stability of the glutamic acid yield of the Corynebacterium glutamicum Z1 strain; Figure 6 is a strain domestication diagram; Figure 7 is a columnar analysis diagram of the genetic stability of the glutamic acid yield of the Corynebacterium glutamicum A-8 strain; Figure 8 is an acid resistance comparison diagram. DETAILED DESCRIPTION

[0017] The application will be further described below in combination with the accompanying drawings and examples, and the modes of the application include but are not limited to the following examples.

[0018] The example provides an acid-resistant Corynebacterium glutamicum strain, and the culture medium used in the construction process of the strain is as follows: The primary seed culture medium: glucose 25 g / L, urea 5 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 1.2 g / L, corn syrup 33 g / L, ferrous sulfate 2 mg / L, and manganese sulfate 2 mg / L, and deionized water is added to 1 L; The seed solid culture medium: 20 g / L agar is added on the basis of the components of the primary seed culture medium; Fermentation basal medium: glucose 60 g / L, corn steep liquor 5 g / L, urea 10 g / L, potassium chloride 3 g / L, magnesium sulfate 0.9 g / L, dipotassium hydrogen phosphate 1 g / L, sucrose 3.6 g / L, ferrous sulfate 5 mg / L, manganese sulfate 5 mg / L, vitamin B1 2.5 mg / L, diluted to 1 L with deionized water.

[0019] In the above culture medium, glucose and urea are sterilized separately at 115°C for 30 min, and the remaining components are sterilized at 121°C for 20 min. They are then mixed before use.

[0020] This embodiment utilizes the Lanzhou Heavy Ion Research Facility (HIRFL) at the Lanzhou Heavy Ion Accelerator National Laboratory of the Institute of Modern Physics, Chinese Academy of Sciences, to screen acid-resistant strains through irradiation mutagenesis. The specific method is as follows: 1. Liquid seed culture The original strain Corynebacterium glutamicum ( Corynebacterium glutamicum Z0 (previously screened from soil in the laboratory and deposited at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC NO: 66897) was taken out of a -80℃ freezer and allowed to thaw slowly at room temperature. It was then inoculated into 100 mL of primary seed culture medium at a 1% inoculum rate and cultured in a constant temperature shaker at 30℃ and 180 rpm for 18 h. Its OD was then measured. 600 , to obtain OD 600 The bacterial solution with a concentration of 0.8 is denoted as the starting bacterial solution.

[0021] 2. Determine the growth curve Corynebacterium glutamicum Z0 culture, which had been passaged 2-3 times, was inoculated at a rate of 1% into 100 mL of primary seed culture medium and incubated at 30℃ and 180 r / min in a shaker. The absorbance of the primary seed culture medium at 600 nm was measured at 0 h after inoculation. Subsequently, samples were taken every 2 h to measure the OD of the original strain Corynebacterium glutamicum Z0 at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, and 32 h. 600 Values, record data and plot growth curves.

[0022] Corynebacterium glutamicum is a Gram-positive, strictly aerobic bacterium with an optimal growth temperature of 30-37℃. The growth curve of Corynebacterium glutamicum Z0 is shown below. Figure 1 As shown, the growth lag phase of the strain is from 0 to 8 hours, and from 8 to 18 hours, the strain grows rapidly in a logarithmic manner, which is the logarithmic growth phase. At 18 hours, the OD... 6006.08, which is the logarithmic growth phase of the strain, then the growth continues but the growth rate decreases, and reaches the peak between 24-30 h, which is the stationary phase of the strain, and then the strain enters the decline phase in the following culture, and the OD 600 of the strain slowly decreases, and the decline of the strain reduces the number of bacterial cells in the culture medium.

[0023] 3. Heavy ion irradiation mutagenesis 2 mL of the starting bacterial liquid in the logarithmic growth phase was taken into a sterile irradiation dish and sealed with a sealing film, and then irradiated with an 80 MeV / u ion beam at the Shallow Therapy and Biological Irradiation Terminal (TR4) of the Heavy Ion Research Facility in Lanzhou (HIRFL) of the Chinese Academy of Sciences, and the irradiation doses were 0 Gy, 25 Gy, 50 Gy, 75 Gy, 100 Gy, 125 Gy, 150 Gy, 175 Gy, 200 Gy and 225 Gy, respectively. 12 C 6+

[0024] 4. Calculation of lethal rate The bacterial liquid irradiated at the irradiation doses of 0 Gy, 25 Gy, 50 Gy, 75 Gy, 100 Gy, 125 Gy, 150 Gy, 175 Gy, 200 Gy and 225 Gy was gradiently diluted to 10 -6 times, and then 100 μL of the diluted bacterial suspension was spread on the seed solid culture medium, and cultured in a 30°C constant temperature incubator for 24 h, and each group had 3 parallel experiments, and then the number of viable bacteria on the plate at different irradiation doses was recorded, the number of colonies after irradiation was divided by the number of colonies of the blank control, and the lethal rate was calculated, and the lethal rate curve was drawn with the irradiation dose as the abscissa and the lethal rate as the ordinate.

[0025] The lethal rate of the strain Corynebacterium glutamicum Z0 under different irradiation doses is shown in Table 1. Figure 2 As can be seen from Table 1, Figure 2 in the range of 0-100 Gy, the lethal rate of the strain is relatively low, and the strain has a good tolerance to the irradiation dose, and the strain can grow normally under the irradiation dose of 100 Gy. 12 C 6+ ​The lethal rate curve increased first and then decreased with the increase of irradiation dose, forming a curve similar to "saddle" when the heavy ion irradiation dose was within 0 Gy-225 Gy. The lethal rate reached the highest value of 61.23% when the irradiation dose was 150 Gy. With the increase of irradiation dose, the lethal rate of the bacteria showed a significant downward trend. This unique "saddle" curve was mainly due to the dynamic balance of DNA damage and repair mechanism and the complexity of physiological response of the bacteria. At a medium dose, the damage accumulation exceeded the repair threshold, a large number of key genes were inactivated, and the lethal rate climbed to the peak value. While high dose mutagenesis caused extreme damage, a small number of strains survived by homologous recombination, recessive repair or stress-activated stress resistance mechanism, so that the lethal rate rose slightly.

[0026] 5. Primary screening of heavy ion mutagenic strains According to the morphological characteristics and growth of the colonies, strains with acid tolerance and high glutamate production capacity were screened. The specific method is as follows: the bacterial liquid of different irradiation doses was diluted to 10 -6 times to obtain bacterial suspensions of different irradiation doses; 50 μL of bacterial suspensions of different irradiation doses were respectively coated on seed solid medium with pH of 5, and incubated at 30°C for 36 h; after the colonies grew, the colonies with large diameters were selected and transferred to well plate medium, and incubated at 30°C and 180 rpm for 18 h; the growth rate of the bacteria was determined by measuring the OD 600 value (OD value at wavelength 600 nm) to select strains with acid tolerance and fast growth rate for further screening; The results of primary screening of heavy ion mutagenic strains are shown in Figure 3 Table 1. Among the 1340 single colonies, 40 strains with larger diameters than the original strain were screened, and the strains were named according to the irradiation dose; 12 strains (Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z12, Z14, Z16) with higher OD 600 values were selected as further screening mutagenic strains.

[0027] 6. Secondary screening of heavy ion mutagenic strains The 12 strains with higher OD 600 values obtained by primary screening were activated, and then inoculated into fermentation base medium with pH of 5 at a volume ratio of 10%, and incubated at 30°C and 180 rpm for 48 h to obtain the culture solution, and then the glutamate content was measured. The specific method is as follows: (1) The bacterial suspension was centrifuged at 12000 rpm for 5 min, and the supernatant was taken for measurement; (2) The glutamate content was measured by using biological sensor analyzer SBA-40D.

[0028] The results of glutamate content determination of the 12 strains are shown inFigure 4 As shown in Figure 4 As can be seen, the screened strains can grow in acidic environment, and the ability to produce glutamic acid at pH 5 is improved to different degrees compared with the original strain (Corynebacterium glutamicum Z0 is inhibited at pH 5). Among them, when the irradiation dose reaches 75 Gy, a positive mutant strain with the highest glutamic acid production is obtained, which is named Corynebacterium glutamicum Z1, and the glutamic acid production reaches 10.3 g / L.

[0029] 7. Genetic stability analysis of heavy ion mutagenesis strain The genetic stability of the mutagenized Corynebacterium glutamicum Z1 was analyzed. The specific method is as follows: Corynebacterium glutamicum Z1 was continuously subcultured for 10 generations. The mutagenized strain was recorded as generation 0. Generation 0 liquid was inoculated in a pH 5 seed solid culture medium at an inoculation amount of 1%, and was cultured at 30°C and 180 rpm for 18 h to obtain generation 1 seed liquid. Generation 1 seed liquid was inoculated in a pH 5 seed solid culture medium at an inoculation amount of 1%, and was cultured at 30°C and 180 rpm for 18 h to obtain generation 2 seed liquid. In this way, 12 generations were continuously cultured, and the glutamic acid production was determined every 2 generations. The results are shown in Figure 5 .

[0030] As can be seen from Figure 5 , after 10 generations of Corynebacterium glutamicum Z1, the glutamic acid production of each generation has no significant difference, which shows that the mutagenized Corynebacterium glutamicum has stable acid-producing performance.

[0031] 8. Strain domestication The method of gradually reducing the pH value of the culture medium (such as Figure 6 ) was used to domesticate Corynebacterium glutamicum Z1. The initial pH of domestication was set to 5, and the Corynebacterium glutamicum Z1 culture liquid was inoculated in a pH 5 seed culture medium at an inoculation amount of 1%, and was incubated at 30°C and 180 rpm for 24 h, then was transferred to fresh pH 4.5 seed culture medium, and the operation was continuously performed for one month, and then the pH was gradually reduced to 4.5 and 4 according to the growth of the strain. Each stage was streaked on low-pH plate medium and cultured for 20-24 h, and the strains with good growth were selected, preserved and used for the next generation. At the same time, the preserved strains at each stage were inoculated into a shake flask fermentation medium, with pH=7 as a control, and were cultured at low pH for 48 h. The strains with low glutamic acid production were eliminated, and the high-yield strains were preserved. Finally, a low-pH-resistant high-yield glutamic acid-producing Corynebacterium glutamicum was obtained, which was named Corynebacterium glutamicum A-8.

[0032] The genetic stability analysis of Corynebacterium glutamicum A-8 is as follows: Figure 7After 10 generations, there was no significant difference in glutamate production between generations, indicating that the mutagenized Corynebacterium glutamicum has stable heritable acid-producing ability.

[0033] The strain that underwent mutagenesis screening and acid-resistant domestication was named Corynebacterium glutamicum A-8 and deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 3, 2025, with accession number GDMCCNO: 66452. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0034] 9. Comparison of acid resistance of strains Growth comparisons were performed in liquid shake flasks: (1) The plate-activated Corynebacterium glutamicum Z0 and A-8 were inoculated into 30 mL of primary seed medium with pH 4.0 at a volume ratio of 10%, and cultured at 30℃ and 180 rpm for 18 h. After the culture was completed, the OD was measured. 600 The measurement results are as follows Figure 8 As shown; (2) The activated Corynebacterium glutamicum Z0 and A-8 were inoculated at a volume ratio of 10% into a primary seed culture medium containing 30 mL of pH 7.0 and cultured at 30℃ and 180 rpm for 18 h. After the culture was completed, the OD was measured. 600 The measurement results are as follows Figure 8 As shown; (3) The activated Corynebacterium glutamicum Z0 and A-8 were inoculated into the fermentation basal medium at pH 4 at a volume ratio of 10%, and cultured at 30℃ and 180 rpm for 48 h to obtain the culture solution. The glutamic acid content was then measured, as shown in Table 1.

[0035] Table 1 Comparison of glutamate production capacity at pH=4 Strain designations Corynebacterium glutamicum Z0 Corynebacterium glutamicum A-8 Glutamic acid yield (g / L) 1.3 13.5 from Figure 8 It can be seen that after 18 h of cultivation, both the original strain *Corynebacterium glutamicum* Z0 and the mutagenized *Corynebacterium glutamicum* A-8 could grow in an environment with a pH of 7. However, when the pH of the culture medium was adjusted to 4, the original strain Z0 could not grow, while the screened acid-resistant strain *Corynebacterium glutamicum* A-8 still grew well. Table 1 shows a comparison of glutamate production capacity after 48 h of fermentation at pH 4. The original strain *Corynebacterium glutamicum* Z0 showed inhibited growth and low glutamate production at pH 4, while the screened acid-resistant strain *Corynebacterium glutamicum* A-8 had a glutamate production capacity of 13.5 g / L.

[0036] In summary, the present invention utilizes 12 C 6+The heavy ion beam and acid resistance domestication are used for breeding of Corynebacterium glutamicum, and finally, a positive mutant Corynebacterium glutamicum (A-8) with acid resistance, high glutamic acid yield and stable performance is obtained. Corynebacterium glutamicum The positive mutant not only provides a new idea for breeding and application of the acid-resistant high-yield glutamic acid strain, but also lays a theoretical and application foundation for the acid-resistant strain for fermenting glutamic acid.

[0037] The above embodiment is only one of the preferred embodiments of the present application, and should not be used to limit the protection scope of the present application, but any modification or polishing without substantial meaning made within the main design idea and spirit of the present application, and the technical problems solved are still consistent with the present application, should be included in the protection scope of the present application.

Claims

1. A strain of Corynebacterium glutamicum which is acid-tolerant, characterized in that, The acid-resistant Corynebacterium glutamicum is named Corynebacterium glutamicum ( Corynebacterium glutamicum A-8 was deposited on June 3, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66452, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The acid-tolerant Corynebacterium glutamicum strain of claim 1, characterized in that, The acid-resistant Corynebacterium glutamicum is obtained by heavy ion irradiation mutagenesis combined with directional screening with Corynebacterium glutamicum (Z0) as the original strain. Corynebacterium glutamicum )Z0 as the original strain, through heavy ion irradiation mutagenesis combined with directional screening.

3. The acid-tolerant Corynebacterium glutamicum strain of claim 2, characterized in that, The corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum ) Z0 was preserved in Guangdong Microbial Culture Collection Center on August 29, 2025, with a preservation number of GDMCC NO: 66897 and a preservation address of 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100 Courtyard.

4. Corynebacterium glutamicum according to claim 1 or 2 or 3, Corynebacterium glutamicum ) Use of A-8 as a fermentation strain in the production of glutamic acid.

5. Use according to claim 4, characterized in that, Use Corynebacterium glutamate ( Corynebacterium glutamicum When A-8 is used as a fermentation strain to produce glutamic acid, the basic fermentation medium includes: glucose 55~65 g / L, corn steep liquor 4~6 g / L, urea 8~12 g / L, potassium chloride 2~4 g / L, magnesium sulfate 0.8~1.0 g / L, dipotassium hydrogen phosphate 0.8~1.2 g / L, sucrose 3.5~3.8 g / L, ferrous sulfate 4~6 mg / L, manganese sulfate 4~6 mg / L, and vitamin B1 2.2~2.8 mg / L, and then diluted to 1L with deionized water.

6. Use according to claim 5, characterized in that, Coryneform bacteria (Corynebacterium glutamicum) (ATCC 13826) was used as the fermentation strain to produce glutamic acid. Corynebacterium glutamicum The fermentation temperature was 30-37°C and the fermentation time was 36-48h when Coryneform bacteria (Corynebacterium glutamicum) (ATCC 13826) was used as the fermentation strain to produce glutamic acid.

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