Thermostable Corynebacterium glutamicum strains, culture media, domestication and screening methods and applications

Abstract

This invention discloses a thermotolerant Corynebacterium glutamicum strain, culture medium, domestication and screening method, and its application. The thermotolerant Corynebacterium glutamicum strain is named MHZ-0115, with the preservation number CGMCC No. 25175; it can be used for fermentation production of glutamic acid. The domestication and screening method includes the following steps: (I) activation and expansion culture of the original strain to obtain a seed culture; (II) taking the seed culture obtained in step (I) and subjecting it to two-stage temperature domestication to obtain thermotolerant Corynebacterium glutamicum. The strain of this invention can ferment at 45℃, and the cells can grow and produce glutamic acid. This can significantly reduce energy consumption and fermentation costs, making it suitable for industrial production and achieving the comprehensive goals of "increased production, reduced consumption, and reduced emissions."

Description

Technical fields:

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a thermotolerant strain of Corynebacterium glutamicum, a culture medium, a domestication and screening method, and its application. Background technology:

[0002] Glutamic acid is one of the structural amino acids of proteins and an important free amino acid, widely used in food, medicine, daily chemical and feed industries.

[0003] The main method for producing glutamic acid currently is fermentation, which utilizes microorganisms to synthesize glutamic acid. This method primarily includes the glycolysis pathway (EMP), the pentose phosphate pathway (PPP), the tricarboxylic acid cycle (TCA), the glyoxylate cycle, and CO2 fixation. The strain used in the fermentation production of glutamic acid is Corynebacterium glutamicum.

[0004] The optimal growth temperature for Corynebacterium glutamicum is 30-33℃. When the fermentation broth temperature exceeds 33℃, cooling water must be used to lower the temperature; otherwise, the activity of Corynebacterium glutamicum will be inhibited, affecting the yield of glutamic acid. In glutamic acid fermentation plants, it is difficult to control the temperature between 30-33℃ in the early stages of fermentation during hot summer weather. High temperatures in the early stages cause premature aging of the strain, resulting in insufficient vigor and low or stagnant acid production in the later stages, leading to decreased yield and increased costs. Furthermore, production under high-temperature conditions requires extensive cooling measures, resulting in high energy consumption. Therefore, it is necessary to conduct high-temperature acclimatization screening of existing Corynebacterium glutamicum to enable it to tolerate high temperatures, allowing the bacteria to grow normally and produce glutamic acid even under high-temperature conditions. Summary of the Invention:

[0005] The first objective of this invention is to provide a thermoresistant strain of Corynebacterium glutamicum.

[0006] A second object of the present invention is to provide the use of a thermoresistant Corynebacterium glutamicum.

[0007] The third objective of this invention is to provide a method for the domestication and screening of thermotolerant Corynebacterium glutamicum strains.

[0008] A fourth object of the present invention is to provide an acclimatization culture medium for an acclimatization screening method for thermotolerant Corynebacterium glutamicum strains.

[0009] The first objective of this invention is achieved by the following technical solution: a thermoresistant strain of Corynebacterium glutamicum, wherein the strain is Corynebacterium glutamicum, named MHZ-0115, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: Beijing, China, accession number: CGMCC No. 25175, deposit date: June 23, 2022.

[0010] Furthermore, the strain was used to ferment and produce glutamic acid at 41℃-45℃, with an acid production of more than 43g / L.

[0011] Furthermore, the strain was used to ferment glutamic acid at 33℃-41℃, with an acid production of more than 45g / L and a fermentation cycle of 20h-24h.

[0012] The second objective of this invention is achieved by the following technical solution: the thermoresistant Corynebacterium glutamicum strain is used for fermentation production of glutamic acid.

[0013] The third objective of this invention is achieved by the following technical solution: a method for the domestication and screening of the thermotolerant Corynebacterium glutamicum strain, comprising the following steps:

[0014] (a) The original strain was activated by inoculating it on LB agar plates. The bacterial growth was picked from the freshly activated plates and inoculated into seed culture medium to expand the culture and obtain seed liquid.

[0015] (II) Take the seed liquid obtained in step (I) and acclimate it to obtain high-temperature acclimatized Corynebacterium glutamicum: The temperature acclimatization is divided into two stages. The temperature range of the first stage of temperature acclimatization is 35℃-41℃, the initial acclimatization temperature is 35℃, and the temperature is gradually increased by 2℃ each time. The temperature range of the second stage of temperature acclimatization is 42℃-45℃, the initial acclimatization temperature is 42℃, and the temperature is gradually increased by 1℃ each time.

[0016] Furthermore, the specific method for activating and expanding the original strain to obtain seed culture in step (I) includes the following steps:

[0017] (1) The original strain was inoculated onto LB agar plates at 33℃ and activated for 24 hours.

[0018] (2) From the activated strains obtained in step (1), select the single colony with dominant growth, take a loopful and inoculate it onto the seed culture medium in a shake flask, and place it in a reciprocating shaker for culture at 200 rpm and 33°C for 24 h to obtain the seed liquid.

[0019] Furthermore, the specific operation method for the first stage of temperature acclimatization in step (II) is as follows:

[0020] (1) Take a loopful of the seed culture obtained in step (1), dilute it, spread it on LB plate medium, place it in a biochemical incubator, and incubate at 35°C for 24 hours; then, select the dominant single colony, take a loopful of it and inoculate it in a shake flask acclimatization medium, place it on a reciprocating shaker, and incubate at 200 rpm and 35°C.

[0021] (2) After the bacteria in step (1) have grown to the logarithmic phase, take a loopful, dilute it, spread it on LB agar plates, place it in a biochemical incubator, and incubate at 37°C for 24 hours; then, select the dominant single colony, take a loopful, inoculate it into the shake flask acclimatization medium, place it on a reciprocating shaker, and incubate at 200 rpm and 37°C.

[0022] (3) After the bacteria in step (2) have grown to the logarithmic phase, take a loopful of diluted solution and spread it on LB agar plate medium. Place it in a biochemical incubator and incubate at 39°C for 24 hours. Then select the dominant single colony, take a loopful of inoculated into the shake flask acclimatization medium, place it on a reciprocating shaker at 200 rpm and incubate at 39°C.

[0023] (4) After the bacteria in step (3) have grown to the logarithmic phase, take a loopful, dilute it, spread it on LB agar plates, and place it in a biochemical incubator at 41°C for 72 hours. Then, select the dominant single colony, take a loopful, inoculate it into the shake flask acclimatization medium, place it on a reciprocating shaker at 200 rpm and 41°C for incubation. After the seed cells grow to the logarithmic phase, the first stage of temperature acclimatization is completed.

[0024] Furthermore, the specific operation method for the second stage of temperature acclimatization in step (ii) is as follows:

[0025] (1) Take a loopful of the seed liquid that has completed the first stage of temperature acclimatization, dilute it, spread it on LB plate medium, place it in a biochemical incubator, and incubate at 42℃ for 72h; then select the single colony with dominant growth, take a loopful of it and inoculate it in a shake flask acclimatization medium, place it on a reciprocating shaker, and incubate at 200rpm and 42℃.

[0026] (2) After the bacteria in step (1) have grown to the logarithmic phase, take a loopful of diluted solution and spread it on LB agar plate medium, place it in a biochemical incubator, and incubate at 43°C for 72 hours; then select the dominant single colony, take a loopful of inoculated into the shake flask acclimatization medium, place it on a reciprocating shaker, and incubate at 200 rpm and 43°C.

[0027] (3) After the bacteria in step (2) have grown to the logarithmic phase, take a loopful, dilute it, spread it on LB agar plates, place it in a biochemical incubator, and incubate at 44°C for 72 hours. Then, select the dominant single colony, take a loopful, inoculate it into the shake flask acclimatization medium, place it on a reciprocating shaker at 200 rpm, and incubate at 44°C.

[0028] (4) After the bacteria in step (3) have grown to the logarithmic phase, take a loopful of diluted solution and spread it on LB agar plates. Place the plates in a biochemical incubator and incubate at 45°C for 72 hours. Then select the dominant single colony and inoculate it with a loopful of solution into a shake flask acclimatization medium. Place the flask on a reciprocating shaker at 200 rpm and incubate at 45°C. After the seed cells have grown to the logarithmic phase, the second stage of temperature acclimatization is complete.

[0029] Furthermore, the LB plate culture medium consists of: 10 g / L fish peptone, 5 g / L yeast extract, 10 g / L NaCl, 18 g / L agar, and 5 g / L glucose.

[0030] Furthermore, the seed culture medium consists of: 20-25 g / L glucose, 15-20 g / L soybean meal extract, 10-15 g / L yeast powder, 5-10 g / L urea, 0.5-1.0 g / L magnesium sulfate heptahydrate, and 0.1-0.2 g / L biotin.

[0031] Furthermore, the acclimatization culture medium consists of: 60-70 g / L glucose, 5-10 g / L yeast extract, 2-4 g / L lysine, 1-2 g / L potassium dihydrogen phosphate, 0.5-10 g / L magnesium sulfate heptahydrate, 0.1-0.2 g / L biotin, and 2-5 g / L trehalose.

[0032] The fourth objective of this invention is achieved by the following technical solution: an acclimatization culture medium for the acclimatization and screening method of the thermotolerant Corynebacterium glutamicum strain, comprising: 60-70 g / L glucose, 5-10 g / L yeast extract, 2-4 g / L lysine, 1-2 g / L potassium dihydrogen phosphate, 0.5-10 g / L magnesium sulfate heptahydrate, 0.1-0.2 g / L biotin, and 2-5 g / L trehalose.

[0033] Advantages of this invention:

[0034] (1) The present invention adopts a two-stage temperature gradient acclimatization to gradually enhance the tolerance of Corynebacterium glutamicum to high temperature, and solves the problems of intolerant to high temperature, slow growth rate and low activity of strains.

[0035] (2) The present invention uses a specific acclimatization culture medium for temperature acclimatization. Trehalose is added to the culture medium to maintain the activity of the strain, which can accelerate the metabolism and nutrient utilization of Corynebacterium glutamicum. While facilitating the high-temperature acclimatization of Corynebacterium glutamicum, the target strain can be obtained more quickly.

[0036] (3) The strain of the present invention can ferment at a high temperature of 45°C, and the cells can grow and produce glutamic acid. This can greatly reduce energy consumption and fermentation costs, making it suitable for industrial production and achieving the comprehensive goal of "increasing production, reducing consumption, and reducing emissions". Attached image description:

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The fermentation OD and acid production curves of the original strain and the domesticated strain at 33℃ and 45℃ are shown.

[0039] The thermotolerant Corynebacterium glutamicum strain of this invention was deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 25175, on June 23, 2022. Detailed implementation method:

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The original strain was *Corynebacterium glutamicum* MHZ-0112-8, with accession number CGMCC No. 11941.

[0042] Example 1: This example specifically illustrates the screening method for the strains of the present invention.

[0043] (I) Activation and scale-up culture of the original strain

[0044] (1) At 33℃, the original strain was inoculated onto LB plate medium and then placed in a biochemical incubator for activation culture for 24 hours.

[0045] (2) From the activated strains obtained in step (1), select the single colony with dominant growth, take a loopful and inoculate it onto the seed culture medium in a shake flask, and place it in a reciprocating shaker for culture at 200 rpm and 33°C for 24 h to obtain the seed liquid.

[0046] In this embodiment, the LB plate culture medium consists of: 10 g / L fish peptone, 5 g / L yeast extract, 10 g / L NaCl, 18 g / L agar, and 5 g / L glucose.

[0047] In this embodiment, the seed culture medium consists of: 20 g / L glucose, 15 g / L soybean meal extract, 10 g / L yeast powder, 5 g / L urea, 0.5 g / L magnesium sulfate heptahydrate, and 0.1 g / L biotin.

[0048] (II) Temperature Acclimation

[0049] Temperature acclimatization is divided into two stages. The temperature range for the first stage of temperature acclimatization is 35℃-41℃, with an initial acclimatization temperature of 35℃, and the temperature is gradually increased by 2℃ each time. The temperature range for the second stage of temperature acclimatization is 42℃-45℃, with an initial acclimatization temperature of 42℃, and the temperature is gradually increased by 1℃ each time.

[0050] The specific steps are as follows:

[0051] 1. First stage of temperature gradient acclimatization

[0052] (1) Take a loopful of the seed culture obtained in step (1), dilute it, spread it on LB plate medium, place it in a biochemical incubator, and incubate at 35°C for 24 hours; then, select the dominant single colony, take a loopful of it and inoculate it in a shake flask acclimatization medium, place it on a reciprocating shaker, and incubate at 200 rpm and 35°C.

[0053] (2) After the bacteria in step (1) have grown to the logarithmic phase, take a loopful, dilute it, spread it on LB agar plates, place it in a biochemical incubator, and incubate at 37°C for 24 hours; then, select the dominant single colony, take a loopful, inoculate it into the shake flask acclimatization medium, place it on a reciprocating shaker, and incubate at 200 rpm and 37°C.

[0054] (3) After the bacteria in step (2) have grown to the logarithmic phase, take a loopful of diluted solution and spread it on LB agar plate medium. Place it in a biochemical incubator and incubate at 39°C for 24 hours. Then select the dominant single colony, take a loopful of inoculated into the shake flask acclimatization medium, place it on a reciprocating shaker at 200 rpm and incubate at 39°C.

[0055] (4) After the bacteria in step (3) have grown to the logarithmic phase, take a loopful, dilute it, spread it on LB agar plates, and place it in a biochemical incubator at 41°C for 72 hours. Then, select the dominant single colony, take a loopful, inoculate it into the shake flask acclimatization medium, place it on a reciprocating shaker at 200 rpm and 41°C for incubation. After the seed cells grow to the logarithmic phase, the first stage of temperature acclimatization is completed.

[0056] 2. Second stage temperature gradient acclimatization

[0057] (1) Take a loopful of the seed liquid that has completed the first stage of temperature acclimatization, dilute it, spread it on LB plate medium, place it in a biochemical incubator, and incubate at 42℃ for 72h; then select the single colony with dominant growth, take a loopful of it and inoculate it in a shake flask acclimatization medium, place it on a reciprocating shaker, and incubate at 200rpm and 42℃.

[0058] (2) After the bacteria in step (1) have grown to the logarithmic phase, take a loopful of diluted solution and spread it on LB agar plate medium, place it in a biochemical incubator, and incubate at 43°C for 72 hours; then select the dominant single colony, take a loopful of inoculated into the shake flask acclimatization medium, place it on a reciprocating shaker, and incubate at 200 rpm and 43°C.

[0059] (3) After the bacteria in step (2) have grown to the logarithmic phase, take a loopful, dilute it, spread it on LB agar plates, place it in a biochemical incubator, and incubate at 44°C for 72 hours. Then, select the dominant single colony, take a loopful, inoculate it into the shake flask acclimatization medium, place it on a reciprocating shaker at 200 rpm, and incubate at 44°C.

[0060] (4) After the bacteria in step (3) have grown to the logarithmic phase, take a loopful of diluted solution and spread it on LB agar plates. Place the plates in a biochemical incubator and incubate at 45°C for 72 hours. Then select the dominant single colony and inoculate it with a loopful of solution into a shake flask acclimatization medium. Place the flask on a reciprocating shaker at 200 rpm and incubate at 45°C. After the seed cells have grown to the logarithmic phase, the second stage of temperature acclimatization is complete.

[0061] In this embodiment, in step (ii), the LB plate culture medium consists of: 10 g / L fish peptone, 5 g / L yeast extract, 10 g / L NaCl, 18 g / L agar, and 5 g / L glucose.

[0062] In this embodiment, the composition of the acclimatization culture medium in step (ii) is as follows: glucose 60g / L, yeast powder 7g / L, lysine 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate heptahydrate 0.8g / L, biotin 0.1g / L, and trehalose 3g / L.

[0063] The obtained thermostable Corynebacterium glutamicum strain was preserved as MHZ-0115 at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 25175, on June 23, 2022.

[0064] Example 2: This example specifically illustrates the method of producing glutamic acid by fermentation using Corynebacterium glutamicum.

[0065] (1) Seed culture

[0066] The strain was activated by inoculating it onto a slant culture medium; bacterial growth was picked from the freshly activated slant and inoculated into a seed culture medium, and cultured at 33℃ and 220 rpm with shaking until the mid-to-late logarithmic growth stage, with a culture time of 24 h, to obtain the seed culture.

[0067] The seed culture medium consisted of: 20 g / L glucose, 15 g / L soybean meal extract, 10 g / L yeast powder, 5 g / L urea, 0.5 g / L magnesium sulfate heptahydrate, and 0.1 g / L biotin.

[0068] The slant culture medium consisted of: 10 g / L fish peptone, 5 g / L yeast extract, 10 g / L NaCl, 18 g / L agar, and 5 g / L glucose.

[0069] (2) Fermentation culture

[0070] The seed culture was inoculated into the fermentation medium at a 20% inoculation rate and cultured on a reciprocating shaker at 200 rpm and 33°C. Fermentation was stopped when the glucose in the fermentation medium was depleted.

[0071] The fermentation medium consisted of: 70 g / L glucose, 10 g / L corn steep liquor, 1 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, and 0.1 g / L biotin.

[0072] Example 3: This example specifically illustrates the comparison of shake-flask fermentation performance between the domesticated strain (strain with accession number CGMCC No. 25175) and the original strain (strain with accession number CGMCC No. 11941).

[0073] Domesticated strains that grew well at 45℃ were selected and fermented at 33℃, 37℃, 41℃, 43℃ and 45℃ using the fermentation method of Example 2.

[0074] The original strain was fermented using the fermentation method described in Example 2 at 33°C, 37°C, 41°C, 43°C, and 45°C, respectively.

[0075] The fermentation performance (glutamate cell growth and acid production capacity) of the domesticated strain and the original strain were compared at different temperatures. Fermentation time, fermentation OD, and glutamate content were recorded. The fermentation parameters of the domesticated strain and the original strain are compared in Table 1.

[0076] Table 1 Comparison of fermentation parameters between the original strain and the domesticated strain at different temperatures.

[0077]

[0078]

[0079] The data in Table 1 show that, under the same initial sugar content and a 24-hour fermentation cycle: the sugar consumption capacity of the original strain gradually decreased with increasing temperature, reaching extremely low levels above 43℃; the acid production capacity of the original strain gradually slowed down with increasing temperature, reaching extremely low levels above 43℃; and the OD growth of the original strain gradually slowed down with increasing temperature, reaching extremely slow levels above 43℃. The acclimated strain showed significant improvements in sugar consumption capacity, acid production capacity, and OD growth. Under the same temperature conditions, all the above indicators were improved in the acclimated strain. Furthermore, it could still grow and produce acid at 45℃, reaching over 85% of the normal level, which is 5-6 times that of the original strain. At the normal culture temperature of 33℃, the fermentation time of the acclimated strain was 4 hours shorter than that of the original strain, indicating that the acclimated strain had stronger activity and a stronger sugar consumption capacity.

[0080] Example 4: This example specifically illustrates the comparison of the fed-batch fermentation effects of the domesticated strain (strain with accession number CGMCC No. 25175) and the original strain (strain with accession number CGMCC No. 11941) based on Example 2.

[0081] Domesticated bacterial strains exhibiting excellent growth at 45℃ were selected and fermented using the method described in Example 2. Based on the shake-flask fermentation of Example 2, glucose-fed fermentation was carried out. A 660 g / L glucose solution was prepared in an Erlenmeyer flask, sterilized, and used as the feed solution. The glucose concentration was maintained at 0.5-2.0 g / L during fermentation. Fermentation was conducted at 33℃ and 45℃, respectively.

[0082] The original bacterial strain was used, and the fermentation method of Example 2 was employed, with glucose-fed fermentation carried out based on the shake-flask fermentation of Example 2. A 660 g / L glucose solution was prepared in an Erlenmeyer flask, sterilized, and used as the feed solution. The glucose concentration was maintained at 0.5-2.0 g / L during fermentation. Fermentation was carried out at 33°C and 45°C, respectively.

[0083] The fermentation parameters are compared in Table 2, and the process OD and acid production growth curves are shown in the figure. Figure 1 As shown.

[0084] Table 2 Comparison of fed-feed fermentation parameters between the original strain and the domesticated strain

[0085]

[0086] The data in Table 2 and Figure 1 This indicates that when fermentation conditions are 33℃, the domesticated strain produces 150 g / L of acid with a peak OD value of [missing value]. 562 The concentration was 100, and the culture time was 20 hours; the original strain produced 144 g / L of acid, with a peak OD value of 100. 562Compared to the previous method, which used a culture time of 24 hours and an acid production rate of 98%, the acclimatized strain produced 6 g / L more acid and shortened the fermentation cycle by 4 hours. At a fermentation temperature of 45℃, the acclimatized strain produced 130 g / L of acid with a peak OD value of [missing value]. 562 The initial culture time was 24 hours, and 260 g of 660 g / L glucose solution was added. The original strain produced 20 g / L of acid, with a peak OD value of 84. 562 Compared to strains with 16, 24-hour culture time, and incomplete depletion of base sugars, the acclimatized strain showed a significant increase in acid production and sugar consumption capacity, with OD... 562 The growth reached 84% of the normal level, and the performance improvement was 5-6 times that of the original strain. This indicates that the strain domesticated by this invention showed significantly increased growth and sugar consumption at 33℃. When the temperature was increased to 45℃, OD growth, sugar consumption, and acid production all increased significantly. The domesticated strain was significantly less affected, thus increasing sugar input and acid production, improving yield while reducing production costs.

[0087] As can be seen from the above, the strain of this invention can not only grow at high temperatures, but also has a significantly increased growth rate and a shortened fermentation cycle. The challenge lies in the fact that organisms possess a series of heat shock proteins that can be induced to synthesize new proteins under stimulation of temperatures higher than normal growth temperatures. Under continuous high-temperature stimulation, cells are forced to highly express large amounts of heat shock proteins to adapt to the harsh environment, and only strains with high expression levels can survive at high temperatures. Trehalose has the characteristic of protecting biological activity, preventing protein dehydration and deformation under high-temperature conditions, and restoring its original spatial conformation and biological activity. Adding trehalose to the acclimatization culture medium can improve…

[0088] The survival rate of strains under high-temperature conditions, and the activities of citrate synthase, aconitase, isocitrate dehydrogenase, and glutamate dehydrogenase in Corynebacterium glutamicum, were investigated. With increasing temperature gradients, the strains gradually adapted to the high-temperature environment, maintaining very high activity. Therefore, this study aimed to simultaneously screen for glutamate-producing bacteria capable of cell growth and glutamate production under high-temperature conditions.

[0089] The above description 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 within the protection scope of the present invention.

Claims

1. A thermoresistant strain of Corynebacterium glutamicum, characterized in that, The strain is Corynebacterium glutamicum ( Corynebacterium glutamicum The sample, named MHZ-0115, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located in Beijing, China, with accession number CGMCC No. 25175, on June 23, 2022.

2. The application of the thermoresistant Corynebacterium glutamicum strain as described in claim 1 in the fermentation production of glutamic acid.

Images