Acid-tolerant corynebacterium glutamicum and application thereof

Abstract

The application discloses an acid-resistant Corynebacterium glutamicum and application thereof, and relates to the technical field of microorganisms. The acid-resistant Corynebacterium glutamicum is named Corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum ) A-8, and is preserved in the Guangdong Microbial Culture Collection Center on June 3, 2025, with a preservation number of GDMCC NO: 66452 and a preservation address of 5th Floor, Building 59, 100, Middle Liangma Street, Guangzhou. The Corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum ) A-8 provided in the application can grow normally and produce glutamic acid in an acidic environment with a pH of 4.0, and effectively solves the problem of growth inhibition of a strain caused by pH reduction in traditional glutamic acid fermentation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an acid-resistant Corynebacterium glutamate and its applications. Background Technology

[0002] Glutamic acid, an important amino acid, is widely used in food additives, pharmaceutical manufacturing, and the production of chemical products. It is not only a key component in protein synthesis but also plays an irreplaceable role in maintaining human health and promoting growth. Currently, the industrial-scale production of glutamic acid mainly relies on microbial fermentation, especially using Corynebacterium glutamicum (…). Corynebacterium glutamicum Large-scale cultivation was carried out. Through in-depth research and modification of the metabolic pathways of these strains and genetic engineering, the yield of glutamic acid was successfully increased, while maintaining good biological stability and low cost.

[0003] However, during glutamic acid fermentation, the production of glutamic acid leads to a decrease in the pH of the culture medium. This not only limits the growth rate of the strain but may also cause changes in metabolic pathways, resulting in a decrease in glutamic acid yield. To address this issue, traditional production often requires adding alkaline solutions (such as sodium hydroxide) to adjust the pH of the culture medium to alleviate acid stress. However, frequent pH adjustments not only increase production costs but may also disrupt the metabolic balance of the strain. Furthermore, in the subsequent glutamic acid extraction process, large amounts of acid (such as hydrochloric acid) are added to lower the pH to precipitate the product. This process not only consumes large amounts of acid and has low extraction efficiency but also generates a large amount of acidic wastewater, causing environmental pollution. Additionally, acid and alkali residues may affect product quality. Therefore, selecting Corynebacterium glutamicum strains with stronger acid tolerance, enabling them to maintain normal growth and high yield performance in low pH environments, has become a key requirement for improving glutamic acid production efficiency and reducing environmental burden. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an acid-resistant Corynebacterium glutamicum and its application, so as to solve the technical problem that the acid resistance of existing glutamic acid producing strains is relatively low.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A strain of acid-resistant Corynebacterium glutamicum, 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.

[0006] Furthermore, this acid-resistant Corynebacterium glutamicum is Corynebacterium glutamicum ( Corynebacterium glutamicumZ0 is the original strain, obtained through heavy ion irradiation mutagenesis combined with directional screening.

[0007] Furthermore, the Corynebacterium glutamicum ( Corynebacterium glutamicum Z0 was deposited on August 29, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66897, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The present invention also provides a Corynebacterium glutamicum as described above ( Corynebacterium glutamicum The application of A-8 as a fermentation strain in the production of glutamic acid.

[0009] Furthermore, using Corynebacterium glutamicum ( 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.

[0010] Furthermore, using Corynebacterium glutamicum ( Corynebacterium glutamicum When A-8 is used 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 present invention has the following beneficial effects: (1) The Corynebacterium glutamicum provided by this invention ( 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 in growth and produces low glutamic acid under the same pH conditions, effectively solving the problem of strain growth inhibition caused by pH drop in traditional glutamic acid fermentation.

[0012] (2) The Corynebacterium glutamicum provided by this invention ( Corynebacterium glutamicum Under acidic fermentation conditions with a pH of 4.0, A-8 produced 13.5 g / L of glutamic acid, breaking through the limitation that the original strain could not 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 provided by the present invention can reduce the frequency and amount of alkali solution used to adjust pH during fermentation, thereby effectively avoiding the interference of frequent pH adjustment on the metabolic balance of the strain, reducing the operating cost and labor burden of the production process.

[0014] (4) The acid-resistant strain provided by the present invention can directly ferment and produce acid under acidic conditions when producing glutamic acid, which can reduce the amount of acid used in the subsequent extraction process (the traditional process requires adding acid to precipitate the product), thereby reducing the generation of acidic wastewater and reducing the environmental burden.

[0015] (5) The acid-resistant strain provided by the present invention has been verified to have no significant difference in glutamic acid production after 10 generations of continuous subculturing, indicating that its acid-resistant and high-yield characteristics can be stably inherited, providing a reliable strain guarantee for large-scale industrial application. Attached Figure Description

[0016] Figure 1 The growth curve of Corynebacterium glutamicum strain Z0; Figure 2 The lethality curve of the original strain Corynebacterium glutamicum Z0 after heavy ion mutagenesis; Figure 3 The absorbance (OD) of the initial screening strains after heavy ion mutagenesis 600 Resulting figure; Figure 4 Figure showing the glutamic acid content of strains re-screened after heavy ion mutagenesis; Figure 5 A bar chart showing the genetic stability of glutamate production by Corynebacterium glutamicum Z1. Figure 6 Diagram showing the domestication of the strain; Figure 7 A bar chart showing the genetic stability of glutamate production by Corynebacterium glutamicum A-8. Figure 8 This is a comparison chart of acid resistance. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0018] This embodiment provides an acid-resistant Corynebacterium glutamicum. The culture medium used in the construction of this strain is as follows: 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 steep liquor 33 g / L, ferrous sulfate 2 mg / L, manganese sulfate 2 mg / L, diluted to 1L with deionized water; Seed solid culture medium: Based on the components of primary seed culture medium, 20 g / L agar is added; 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... 600The OD value reaches 6.08, indicating the logarithmic growth phase of the strain. It then continues to grow, but the growth rate decreases, peaking between 24-30 hours, marking the stationary phase of the strain's growth. During subsequent cultivation, the strain enters its death phase, and its OD value... 600 As the bacterial population gradually decreases, the death of the bacteria will reduce the number of bacterial cells in the culture medium.

[0023] 3. Heavy ion irradiation-induced mutagenesis Two mL of the starting bacterial culture in the logarithmic growth phase was placed into a sterile irradiation dish and sealed with sealing film. The treatment was administered via a superficial therapy and bio-irradiation terminal (TR4) at the Lanzhou Heavy Ion Research Facility (31111.02.HIRFL), Institute of Modern Physics, Chinese Academy of Sciences, at a concentration of 80 MeV / u. 12 C 6+ Ion beam irradiation was performed at doses of 0 Gy, 25 Gy, 50 Gy, 75 Gy, 100 Gy, 125 Gy, 150 Gy, 175 Gy, 200 Gy, and 225 Gy.

[0024] 4. Calculate the mortality rate The bacterial cultures irradiated with doses of 0 G·u, 25 G·u, 50 G·u, 75 G·u, 100 G·u, 125 G·u, 150 G·u, 175 G·u, 200 G·u, and 225 G·u were serially diluted to a concentration of 10⁻⁶ g·u. -6 After diluting the bacterial suspension by 100 μL, spread it onto seed solid culture medium and incubate at 30℃ for 24 h. Each group had 3 parallel experiments. Then, the number of viable bacteria on the plate was recorded at different irradiation doses. The lethality rate was calculated by dividing the number of colonies after irradiation by the number of colonies in the blank control. A lethality rate curve was plotted with the irradiation dose as the x-axis and the lethality rate as the y-axis.

[0025] The lethality of strain Corynebacterium glutamicum Z0 under different irradiation doses is as follows: Figure 2 As shown. By Figure 2 It can be seen that, 12 C 6+When the heavy ion irradiation dose is between 0 Gy and 225 Gy, the lethality curve initially increases and then decreases with increasing irradiation dose, forming a "saddle"-shaped curve. The lethality rate reaches its highest value of 61.23% at an irradiation dose of 150 Gy. With further increases in irradiation dose, the bacterial lethality rate shows a significant decreasing trend. This unique "saddle"-shaped curve is mainly due to the dynamic balance between DNA damage and repair mechanisms and the complexity of bacterial physiological responses. At moderate doses, accumulated damage exceeds the repair threshold, leading to the inactivation of numerous key genes and a peak lethality rate. While high-dose mutagenesis causes extreme damage, a very small number of strains survive unexpectedly through homologous recombination, recessive repair, or stress-activated resistance mechanisms, resulting in a slight recovery in the lethality rate.

[0026] 5. Initial screening of heavy ion-mutated strains Based on the morphological characteristics and growth of the colonies, strains with acid-resistant and high glutamic acid-producing capabilities were screened. The specific method involved diluting bacterial solutions with different irradiation doses to 10⁻⁶. -6 Different irradiation doses of bacterial suspension were obtained. 50 μL of each irradiation dose bacterial suspension was spread onto seed culture medium at pH 5 and incubated at 30℃ for 36 h. After colony growth, colonies with larger diameters were selected and transferred to well plates, where they were incubated at 30℃ and 180 rpm for 18 h. The bacterial cell concentration (OD) was measured. 600 (OD value at 600 nm) to determine the bacterial growth rate, and select acid-resistant and fast-growing strains for further screening; The initial screening results of heavy ion mutagenesis are as follows: Figure 3 As shown, in this embodiment, 40 strains with a diameter larger than the original strain were screened from 1340 single colonies, and named according to the irradiation dose of the strains, thus identifying the OD strains. 600 The 12 highest-quality mutant strains (Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z12, Z14, Z16) were used for further screening.

[0027] 6. Secondary screening of heavy ion-mutated strains The 12 OD strains obtained from the initial screening 600 After activation, the strains with higher concentrations were inoculated into a fermentation basal medium at pH 5 at a volume ratio of 10%, and cultured at 30℃ and 180 rpm for 48 h to obtain the culture solution. Then, the glutamic acid content was measured. The specific method is as follows: (1) Centrifuge the bacterial suspension at 12,000 rpm for 5 min and take the supernatant for testing; (2) The content of glutamic acid was determined using a biosensor analyzer SBA-40D.

[0028] The results of the determination of glutamic acid content of 12 strains are as follows: Figure 4 As shown, by Figure 4 It can be seen that the screened strains can grow in an acidic environment, and their ability to produce glutamate at pH 5 is improved to varying degrees compared with the original strain (Corynebacterium glutamicum Z0, whose growth is inhibited at pH 5). Among them, a positive mutant strain with the highest glutamate production capacity was obtained when the irradiation dose reached 75 Gy, which was named Corynebacterium glutamicum Z1, and its glutamate production reached 10.3 g / L.

[0029] 7. Analysis of the genetic stability of heavy ion-mutated strains Genetic stability analysis was performed on the mutagenized Corynebacterium glutamicum Z1. The specific method was as follows: Corynebacterium glutamicum Z1 was continuously passaged for 10 generations, with the mutagenized strain designated as generation 0. Generation 0 was inoculated at 1% onto seed culture medium at pH 5 and cultured at 30℃ and 180 rpm for 18 h to obtain generation 1 seed culture. Generation 1 seed culture was then inoculated at 1% onto seed culture medium at pH 5 and cultured at 30℃ and 180 rpm for 18 h to obtain generation 2 seed culture. This process was repeated for 12 generations, with changes in glutamate production measured every two generations. The results are shown below. Figure 5 As shown.

[0030] Depend on Figure 5 It can be seen that after 10 generations of passage, the glutamate production of Corynebacterium glutamicum Z1 did not differ significantly, which indicates that the mutagenized Corynebacterium glutamicum has stable heritable acid-producing ability.

[0031] 8. Microbial strain domestication The method of gradually decreasing the pH of the culture medium (e.g.) Figure 6 The *Corynebacterium glutamicum* Z1 strain was domesticated. The initial pH was set at 5. *Corynebacterium glutamicum* Z1 culture was inoculated at a 1% inoculum into seed culture medium at pH 5 and incubated at 30℃ and 180 rpm for 24 h. Afterward, it was transferred to fresh seed culture medium at pH 4.5. This process was repeated for one month, and then the pH was gradually lowered to 4.5 and 4 based on the growth of the strains. Each stage was streaked on low-pH plates for 20-24 h, and strains with good growth were selected for preservation and subsequent subculture. Simultaneously, the preserved strains from each stage were inoculated into shake-flask fermentation medium, with pH 7 as a control, and cultured at low pH for 48 h. Strains with low acid production capacity were eliminated, and high-producing strains were retained for further preservation. Finally, a low-pH-tolerant, high-glutamate-producing *Corynebacterium glutamicum* strain was obtained and named *Corynebacterium glutamicum* A-8.

[0032] Genetic stability analysis of Corynebacterium glutamicum A-8, 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 number Corynebacterium glutamicum Z0 Corynebacterium glutamicum A-8 Glutamic acid production (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+Heavy ion beam and acid-resistant acclimatization were used to select and breed Corynebacterium glutamicum, ultimately obtaining an acid-resistant, high-glutamic-producing, and stable positive mutant strain of Corynebacterium glutamicum. Corynebacterium glutamicum A-8 not only provides new ideas for the selection and application of acid-resistant high-glutamic acid-producing strains, but also lays the theoretical and applied foundation for acid-resistant strains for fermentation production of glutamic acid.

[0037] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. An acid-resistant Corynebacterium glutamate strain, characterized in that, The acid-resistant Corynebacterium glutamicum is named Corynebacterium glutamicum ( Corynebacterium glutamicum The strain 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. The strain A-8 was able to grow normally and produce glutamic acid in a fermentation medium with a pH of 4.

0.

2. The Corynebacterium glutamicum as described in claim 1 ( Corynebacterium glutamicum The application of A-8 as a fermentation strain in the production of glutamic acid.

3. The application according to claim 2, characterized in that, Use Corynebacterium glutamate ( Corynebacterium glutamic 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.

4. The application according to claim 3, characterized in that, Use Corynebacterium glutamate ( Corynebacterium glutamic When A-8 is used as a fermentation strain to produce glutamic acid, the fermentation temperature is 30-37℃ and the fermentation time is 36-48h.

Images