CN114908079A - Breeding method of corynebacterium glutamicum capable of producing isoleucine at high yield - Google Patents
Breeding method of corynebacterium glutamicum capable of producing isoleucine at high yield Download PDFInfo
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Abstract
The invention provides a breeding method of corynebacterium glutamicum for high isoleucine yield, which comprises the steps of taking corynebacterium glutamicum as an object, culturing the corynebacterium glutamicum in a shaker at 30-37 ℃ and 200r/min for a certain time, carrying out normal-temperature normal-pressure plasma mutagenesis treatment for 50-180 s, inoculating the corynebacterium glutamicum to a sulfaguanidine culture medium with the concentration of 0.05-0.4mg/mL, culturing for 1-2 days at 30-37 ℃, carrying out high-throughput screening on 96-pore plates by utilizing the specific reaction of amino acid and ninhydrin and corresponding strain fermentation supernatant fluid, and optionally breeding high-yield strainsCorynebacterium glutamicum B1. The strain is fermented in a 100mL shake flask 3The isoleucine yield is improved by 62.03% compared with the original strain under the condition of 0-48 h, and the hereditary character is stable. The method establishes a high-throughput screening strategy for sulfaguanidine marker primary screening and ninhydrin multi-plate detection, can be widely applied to high-throughput breeding of corynebacterium glutamicum with high isoleucine yield, improves screening efficiency, and has great application value.
Description
Technical Field
The invention relates to a high-throughput screening method for high-yield amino acid, in particular to a high-throughput screening method for corynebacterium glutamicum of isoleucine, belonging to the fields of biological medicine and health industry.
Background
L-isoleucine is one of eight essential amino acids in human body, and is one of three branched chain amino acids, and has a particularly important position in human life metabolism due to its special structure and function. Therefore, the method is widely applied to industries such as food, animal feed and medicine. The production method of L-isoleucine mainly includes protein hydrolysis method, chemical synthesis method and biological fermentation method. The biological fermentation method is a preferred method for industrial production due to low raw material cost, easy control, energy conservation and environmental protection. At present, the yield of isoleucine is not high in China, the production technology has a large gap compared with that of foreign countries, and the breeding of high-yield strains becomes an important link for improving the yield of isoleucine.
An ordinary temperature and pressure plasma (ARTP) mutation breeding technology is a microorganism genome rapid mutation technology independently developed by Qinghua university. The technology has the advantages of low cost, simple and convenient operation, high safety, mild plasma generating condition, abundant active particles, wide mutation spectrum, high mutation rate and the like, and is widely used for biological breeding of microorganisms such as bacteria, fungi, microalgae and the like.
Disclosure of Invention
Aiming at the problems, the invention uses an ARTP mutagen to process Corynebacterium glutamicum (Corynebacterium glutamicum), then performs prescreening by adding sulfaguanidine resistance markers with proper concentration, inoculates the prescreened strain to a porous plate for fermentation culture, and then takes supernatant fluid to perform ninhydrin color reaction detection to realize high-throughput screening, which is not reported at home and abroad.
The technical purpose of the invention is to provide a corynebacterium glutamicum strain with high isoleucine yield, namely corynebacterium glutamicum B1, with the preservation number: CCTCC NO: m2019118, classification name Corynebacterium glutamicum B1, preservation date: no. 3/1 in 2019, storage location: hubei, Wuhan university, depository: china center for type culture Collection. The invention establishes a set of high-throughput screening process, and the high-yield strain ARTP mutagenesis screening mainly comprises the following steps: after culturing an original strain, optimizing an ARTP mutagenesis condition, then primarily screening through a sulfaguanidine resistance marker, selecting a colony with full abundance, inoculating the colony into a porous plate for culturing, extracting a fermentation supernatant to react with a ninhydrin reagent under a certain condition, and selecting OD 570 Fermentation supernatant corresponding to strain with larger lower light absorption value [7] Performing thin-layer chromatography for targeted control detection, performing shake flask re-screening culture, extracting supernatant, and detecting isoleucine content with amino acid analyzer. The breeding process of Corynebacterium glutamicum with high isoleucine yield includes the following steps and the flow chart is shown in FIG. 1.
(1) Culturing corynebacterium glutamicum serving as an object in a shaking table at the temperature of 30-37 ℃ and at the speed of 100-200r/min for a period of time, and then carrying out plasma mutagenesis treatment at normal temperature and normal pressure for 30-180 s;
(2) inoculating to sulfaguanidine culture medium after mutagenesis treatment, and culturing at 30-37 deg.C for 1-2 days;
(3) and (3) carrying out specific color reaction on the strain obtained in the step (2) by using ninhydrin to identify a dominant strain, culturing the dominant strain in a seed culture medium, then culturing in a fermentation culture medium, carrying out amino acid analysis detection and rescreening, and screening to obtain the strain Corynebacterium glutamicum B1 with the highest yield.
The radiation power of the normal temperature and pressure plasma mutagenesis is 100-120W, the flow rate of the helium gas is 3-10L/min, and the mutagenesis time is 120-150 s.
The sulfaguanidine culture medium contains 0.05-0.4mg/mL sulfaguanidine.
In the step (3), the strain and ninhydrin are subjected to specific color reaction by using a multi-well plate, wherein the multi-well plate comprises a 48-96 well plate. The dominant strain of the multi-hole plate is firstly cultured in a seed culture medium for 1 to 2 days at the temperature of between 30 and 37 ℃ and at the speed of 100 and 200 r/min; inoculating 5-10% of the strain into a fermentation culture medium, culturing at 30-37 deg.C and 200r/min for 1-2 days, and performing amino acid analysis, detection and rescreening to obtain high-yield strain Corynebacterium glutamicum B1 with stable performance. The seed liquid culture medium g/L comprises: 5-10g/L of glucose, 1-5g/L of peptone, 1-5g/L of sodium chloride, 1-3g/L of beef extract powder and 0.03-1g/L of disodium hydrogen phosphate;
the fermentation medium g/L comprises: 5-10g/L of glucose, 2-7g/L of peptone, 1-5g/L of sodium chloride, 1-3g/L of beef extract powder, 0.3-1g/L of disodium hydrogen phosphate, 1-5g/L of yeast extract powder and 1-6g/L of ammonium sulfate. Aiming at the process steps, the technical scheme system of the invention examines the influence of the ARTP mutagenesis lethality rate of the method
Mutagenesis was performed for various periods of time (0, 60, 90, 120, 150, 180, 210, 240s), the mutagenized cells were put into 1mL of physiological saline and shaken for 1min, 100uL of the cells were spread on a plate medium and cultured at 37 ℃ for 2 days, and the lethality was calculated.
Research shows that active particles in the plasma act on microorganisms, so that the structure and permeability of the cell wall/membrane of the microorganisms can be changed, gene damage is caused, the gene sequence and the metabolic network of the microorganisms are obviously changed, and finally the microorganisms are mutated. The influence of mutagenesis time and action intensity on the lethality of the microorganism is large. Either too low or too high lethality is detrimental to screening. The results are shown in FIG. 2.
As can be seen from FIG. 2, ARTP has a large killing power on Corynebacterium glutamicum, and is in a linear growth mode between 60 and 120s, the lethality reaches about 88% when the treatment time is 120s, the lethality reaches 98.44% when the treatment time is 180s, and basically all thalli can not survive when the treatment time reaches 240 s. To ensure a certain mutation rate, it is important to select the corresponding mutagenesis time. Experiments show that the corresponding radiation time with the lethality rate of more than 95 percent is suitable for selection.
Furthermore, the invention also systematically examines the influence of the sulfaguanidine concentration on the inhibition rate of the strain
Sulfaguanidine is a structural analogue of aspartic acid, and feedback inhibition of aspartic acid on phosphoenolpyruvate carboxylase can be solved by breeding sulfaguanidine resistance mutant strains. Meanwhile, sulfaguanidine with different concentrations of 0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4mg/mL and the like has different inhibition degrees on cells, so that the sulfaguanidine concentration needs to be optimized to achieve the optimal screening effect. Different sulfaguanidine concentrations were selected for the study. The inhibition rate of various sulfaguanidine concentrations on Corynebacterium glutamicum is shown in FIG. 3.
From fig. 3, it can be seen that sulfaguanidine has an obvious inhibitory effect on corynebacterium glutamicum. The inhibition effect is small at low concentration (0.1mg/mL), and when the concentration of sulfaguanidine reaches 0.3mg/mL, the inhibition effect is enhanced, and the inhibition rate reaches about 97%. When the sulfaguanidine concentration reaches 0.4mg/mL, the thalli can not grow. In order to achieve the screening effect while enabling the thalli to grow, the sulfaguanidine concentration addition amount of about 0.3mg/mL is selected as the optimal addition amount of the screening culture medium and is used for screening the sulfaguanidine resistance marker.
In addition, the technical scheme of the invention performs high-throughput screening on dominant strains
Because of the large number of mutants generated by mutagenesis, a high-throughput rapid screening method needs to be established. Firstly, inoculating a mutagenized strain on a plate into a 24-hole 1mL culture medium by using a bamboo stick, culturing in a microplate constant-temperature shaking table at 37 ℃ and 300r/min, centrifuging a bacterial solution cultured for 24 h, 36h and 48h, taking 20 mu L of a supernatant to a 96-hole deep-hole plate, adding 240 mu L of 0.5% ninhydrin solution to each hole plate, carrying out water bath at 70 ℃ for 8min, cooling to room temperature, measuring the absorbance of the solution by using an enzyme-labeling instrument, and screening out a mutant strain with higher amino acid content by using the absorption light intensity without the ninhydrin solution as a blank control. And continuously passaging the mutagenic strain on a plate culture medium for 10 times, then carrying out fermentation culture, detecting the acid production capacity of the strain, and researching the genetic stability of the strain.
FIG. 4 shows the relative absorbance intensity of 42 mutagenized strains when cultured for 48h at 1.0 absorbance intensity of the original strain. As can be seen from the figure, the relative light absorption intensity of most of the mutant strains is higher than that of the original strain, the relative light absorption values of three mutant strains B1, D6 and E5 are more than 20 percent higher than that of the original strain, the mutant strains B1, D6 and E5 and the original strain W are subjected to shake flask fermentation culture, and the isoleucine yield at 48h is detected by an amino acid analyzer. The results of the rescreening are shown in Table 1.
TABLE 1 results of mutagenesis and rescreening of Corynebacterium glutamicum
As can be seen from Table 1, the shake flask rescreening yields of the mutagenized strains B1, D6 and E5 still show better isoleucine production performance than the original strains, and the yields are respectively increased by 62.03%, 30.89% and 14.19%. It can be seen that the screening method of this experiment is effective.
Finally, the strain obtained by the technical scheme of the invention is subjected to genetic stability investigation
In order to test whether the dominant mutant strain Corynebacterium glutamicum B1 can maintain stable genetic characteristics, the mutant strain B1 is continuously subcultured for 10 times and simultaneously fermented, and the absorption intensity of fermentation supernatant at 570nm is detected, so that the absorption intensity of the strain from 1 st generation to 10 th generation is basically stable, and the mutant strain has good genetic stability.
The technical proposal of the invention establishes a high-efficiency screening method of high-yield isoleucine strains, which is a difficult point of ARTP mutation breeding. In the synthesis path of isoleucine, aspartic acid has a feedback inhibition effect on phosphoenolpyruvate carboxylase, and if the feedback inhibition is removed, metabolic flow can be more smooth, so that the yield of isoleucine is increased. Sulfaguanidine is a structural analogue of aspartic acid, and if sulfaguanidine is added into a culture medium for screening strains, a sulfaguanidine resistant mutant strain can be expected to be obtained, so that the feedback inhibition of aspartic acid on phosphoenolpyruvate carboxylase can be genetically relieved, and the aspartic acid can be synthesized in a large amount. The content of free amino acid in fermentation liquor can be preliminarily compared by utilizing the color reaction of ninhydrin and amino acid, and then the high-throughput screening of the porous plate can be realized by combining with a multifunctional microplate reader.
By combining ARTP random mutagenesis and key enzyme in the pathway of directionally inhibiting microorganism acid production metabolism by sulfaguanidine, the mutation probability of a mutagenic strain to the isoleucine accumulation direction is increased, the probability of screening excellent strains is increased, the screening workload is reduced, an efficient screening method is provided for breeding acid production strains, and important references are provided for screening other high-yield isoleucine strains.
Drawings
FIG. 1 is a process flow diagram of a high throughput screening method for Corynebacterium glutamicum of isoleucine.
FIG. 2 is a graph showing the lethality of Corynebacterium glutamicum ARTP.
FIG. 3 is a graph of the inhibition rate of sulfaguanidine on Corynebacterium glutamicum at different concentrations.
FIG. 4 is the relative absorbance intensity of the mutant strain.
FIG. 5 shows the absorbance intensity of the mutagenized strain after ten serial passages.
Detailed Description
Example 1
Starting from an original strain Corynebacterium glutamicum, performing mutagenesis treatment on the strain through ARTP at the radiation power of 110W and the helium flow of 8L/min for 130s, inoculating the strain into a sulfaguanidine culture medium with the concentration of 0.2mg/mL at the temperature of 34 ℃ for 1 day, selecting a strain with full abundance, then inoculating the strain into a liquid culture medium of a 96-pore plate for culture, performing high-flux screening on the strain fermentation supernatant by utilizing the specific reaction of amino acid and ninhydrin, selecting the strain with the maximum light absorption intensity, performing seed culture on 8g/L glucose, 3g/L peptone, 2g/L sodium chloride, 2g/L beef extract powder and 0.04g/L disodium hydrogen phosphate in a seed culture medium at the temperature of 35 ℃ and 180r/min for 36 hours, then inoculating the strain into a fermentation culture medium at the glucose concentration of 8% (v/v), 5g/L of peptone, 4g/L of sodium chloride, 2g/L of beef extract powder, 0.8g/L of disodium hydrogen phosphate, 4g/L of yeast extract powder and 5g/L of ammonium sulfate, culturing for 36h at 35 ℃ at 180r/min, and then carrying out amino acid analysis detection and rescreening to obtain a high-yield strain Corynebacterium glutamicum B1 with stable performance, wherein the isoleucine yield is improved by 55.6% compared with the original strain.
Example 2
Starting from an original strain Corynebacterium glutamicum, performing mutagenesis treatment for 150s by ARTP at the radiation power of 100W and the helium flow of 7L/min, inoculating the strain into a sulfaguanidine culture medium with the concentration of 0.3mg/mL at 33 ℃ for 1 day, selecting a strain with full abundance, then inoculating the strain into a liquid culture medium of a 96-well plate for culture, performing high-flux screening by utilizing the specific reaction of amino acid and ninhydrin and the fermentation supernatant of the corresponding strain, selecting the strain with the maximum light absorption intensity, performing seed culture on 8g/L glucose, 3g/L peptone, 2g/L sodium chloride, 2g/L beef extract powder and 0.04g/L disodium hydrogen phosphate in the seed culture medium at 34 ℃ and 150r/min for 1-2 days, and then inoculating the strain with the quantity of 7% (v/v) into 8g/L glucose, 5g/L peptone, 8g/L glucose and the like, 4g/L of sodium chloride, 2g/L of beef extract powder, 0.5g/L of disodium hydrogen phosphate, 4g/L of yeast extract powder and 5g/L of ammonium sulfate in a fermentation medium at 35 ℃ at 150r/min for 40h, and after amino acid analysis, detection and rescreening, the high-yield strain Corynebacterium glutamicum B1 with stable performance can be obtained, and the isoleucine production is improved by 60.8 percent compared with the original strain.
Example 3
Starting from an original strain Corynebacterium glutamicum, performing mutagenesis treatment for 120s by ARTP under the conditions that the radiation power is 120W and the helium flow is 3L/min, inoculating the strain into a sulfaguanidine culture medium with the concentration of 0.08mg/mL at the temperature of 30 ℃ for 1 day, selecting a strain with full abundance, then inoculating the strain into a liquid culture medium of a 96-pore plate for culture, performing high-flux screening by utilizing the specific reaction of amino acid and ninhydrin and the fermentation supernatant of the corresponding strain, selecting the strain with the maximum light absorption intensity, performing seed culture on 6g/L glucose, 1g/L peptone, 5g/L sodium chloride, 3g/L beef extract powder and 0.5g/L disodium hydrogen phosphate under the seed culture medium of 35 ℃ at the temperature of 120r/min for 36 hours, then inoculating the strain into a fermentation culture medium of 5g/L glucose with the inoculation amount of 10% (v/v), 2g/L of peptone, 2g/L of sodium chloride, 1g/L of beef extract powder, 0.2g/L of disodium hydrogen phosphate, 2g/L of yeast extract powder and 3g/L of ammonium sulfate, culturing at 30 ℃ for 39h at 150r/min, and then carrying out amino acid analysis, detection and rescreening on the results to obtain a high-yield strain Corynebacterium glutamicum B1 with stable performance, wherein the isoleucine yield is improved by 61.7 percent compared with the original strain.
Example 4
Starting from an original strain Corynebacterium glutamicum, performing mutagenesis treatment on the original strain Corynebacterium glutamicum by ARTP at the radiation power of 100W and the helium flow of 10L/min for 140s, inoculating the strain into a sulfaguanidine culture medium with the concentration of 0.4mg/mL at the temperature of 37 ℃ for culturing for 2 days, selecting a strain with full abundance, then inoculating the strain into a 96-pore plate liquid culture medium for culturing, performing high-flux screening by utilizing the specific reaction of amino acid and ninhydrin and the fermentation supernatant of the corresponding strain, selecting the strain with the maximum light absorption intensity, performing seed culture on 10g/L glucose, 5g/L peptone, 1g/L sodium chloride, 1g/L beef extract powder, 0.07g/L disodium hydrogen phosphate in the seed culture medium at the temperature of 34 ℃ and 200r/min for 1-2 days, and then inoculating the strain with the inoculation amount of 5% (v/v) into the glucose with the concentration of 10g/L peptone, 4g/L of peptone, 5g/L of sodium chloride, 3g/L of beef extract powder, 0.6g/L of disodium hydrogen phosphate, 3g/L of yeast extract powder and 6g/L of ammonium sulfate in a fermentation medium, culturing for 45 hours at 37 ℃ at 170r/min, and after amino acid analysis, detection and rescreening, obtaining a high-yield strain Corynebacterium glutamicum B1 with stable performance, wherein isoleucine is increased by 63.5% compared with the original strain.
Claims (7)
1. A breeding method of corynebacterium glutamicum with high isoleucine yield is characterized by comprising the following steps:
(1) culturing corynebacterium glutamicum serving as an object in a shaking table at the temperature of 30-37 ℃ and at the speed of 100-200r/min for a period of time, and then carrying out plasma mutagenesis treatment at normal temperature and normal pressure for 30-180 s;
(2) inoculating to sulfaguanidine culture medium after mutagenesis treatment, and culturing at 30-37 deg.C for 1-2 days;
(3) identifying the dominant strain after the specific color reaction of the strain obtained in the step (2) by using ninhydrin, culturing the dominant strain in a seed culture medium, culturing in a fermentation culture medium, performing amino acid analysis and detection, re-screening, and screening to obtain the strain with the highest yieldStrains, i.e. Corynebacterium glutamicum producing isoleucine at a high yieldCorynebacterium glutamicumB1。
2. The breeding method of Corynebacterium glutamicum for high isoleucine production according to claim 1, wherein the radiation power of the normal temperature and pressure plasma mutagenesis is 100-120W, the flow rate of the helium gas is 3-10L/min, and the mutagenesis time is 120-150 s.
3. The method for breeding Corynebacterium glutamicum capable of highly producing isoleucine as claimed in claim 1, wherein sulfaguanidine culture medium contains sulfaguanidine 0.05-0.4 mg/mL.
4. The method for breeding Corynebacterium glutamicum producing isoleucine at a high yield in accordance with claim 1, wherein in step (3), the strain and ninhydrin are subjected to a specific chromogenic reaction using a multi-well plate, which comprises 48-96 well plates.
5. The method for breeding Corynebacterium glutamicum for high isoleucine production according to claim 4, wherein the dominant strain in the multi-well plate is cultured in the seed culture medium at 30-37 ℃ and 200r/min for 1-2 days; inoculating 5-10% of the inoculum size to a fermentation medium, culturing at 30-37 deg.C and 200r/min for 1-2 days, and performing amino acid analysis and detection for re-screening to obtain high-yield strain with stable performanceCorynebacterium glutamicum B1。
6. The method for breeding Corynebacterium glutamicum capable of producing isoleucine at high yield of claim 5, wherein the seed culture medium g/L comprises: 5-10g/L of glucose, 1-5g/L of peptone, 1-5g/L of sodium chloride, 1-3g/L of beef extract powder and 0.03-1g/L of disodium hydrogen phosphate;
the fermentation medium g/L comprises: 5-10g/L of glucose, 2-7g/L of peptone, 1-5g/L of sodium chloride, 1-3g/L of beef extract powder, 0.3-1g/L of disodium hydrogen phosphate, 1-5g/L of yeast extract powder and 1-6g/L of ammonium sulfate.
7. A Corynebacterium glutamicum strain, Corynebacterium glutamicum B1, having a high isoleucine-producing yield, which was selected by the method according to any one of claims 1 to 6, deposited under accession number: CCTCC NO: m2019118, Classification nomenclatureCorynebacterium glutamicumB1, date of deposit: no. 3/16 in 2019, north of Hu, Wuhan.
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| CN112795607B (en) * | 2020-12-31 | 2023-06-23 | 河南巨龙生物工程股份有限公司 | Method for improving adenosine fermentation yield |
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| CN116121135B (en) * | 2022-12-29 | 2024-04-09 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant strain and application thereof in L-isoleucine fermentation |
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| CN109971676B (en) | 2022-04-08 |
| CN109971676A (en) | 2019-07-05 |
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