Escherichia coli tolerant to high-concentration L-histidine and application thereof

Abstract

The invention relates to an Escherichia coli ALE-HIS-CL strain tolerant to high-concentration L-histidine and an application of the Escherichia coli ALE-HIS-CL strain. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31770. The ALE-HIS-CL strain can be used for efficiently producing L-histidine, the test of a 5 L fermentation tank shows that the culture period is 44-52 hours, the accumulation amount of the L-histidine reaches 60-68 g / L, the conversion rate is 0.24 g L-histidine / g glucose, and the L-histidine yield and the conversion rate are respectively improved by 54.5% and 19.3% compared with those of an original strain. The strain has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an Escherichia coli tolerant to high-concentration L-histidine and application thereof. Background Art

[0002] L-histidine is a basic amino acid. Due to its special physiological properties, it can be used in medicine, feed, nutritional supplements, cosmetics and other fields. At present, the production methods of L-histidine include protein hydrolysis, chemical synthesis and microbial fermentation. Compared with the first two synthesis methods, microbial fermentation has the advantages of simple process, less environmental pollution, high purity and suitability for large-scale production. Therefore, the production of L-histidine by microbial fermentation has high economic benefits.

[0003] The key to producing L-histidine by microbial fermentation is the selection of strains. Currently, there are two aspects for the selection of L-histidine strains: irrational mutagenesis screening and rational metabolic engineering. Rational metabolic engineering includes relieving feedback inhibition, enhancing the supply of precursors, strengthening the expression of key genes and cofactor balance, etc.; irrational breeding methods include traditional mutagenesis, which is to mutagenize wild-type strains by physical or chemical methods. After multiple rounds of mutagenesis and screening, excellent strains with the ability to synthesize L-histidine are finally obtained. Among them, Kino et al. obtained an L-histidine production strain with excellent performance by mutagenesis screening of strains resistant to structural analogs. After fed-batch fermentation, the concentration of L-histidine produced can reach 46.5 g / L. With the rapid development of gene editing technology, people have gradually enhanced the production of L-histidine by using rational breeding strategies. Wu et al. constructed an efficient L-histidine production strain by removing the feedback regulation of histidine synthesis, enhancing the histidine synthesis pathway, and promoting the extracellular secretion of histidine. After 48 h of fermentation in a 5 L fermenter, the L-histidine concentration reached 66.5 g / L, and the conversion rate was 0.22 g L-histidine / g glucose. Genetic modification of strains at the molecular level has achieved a rational metabolic engineering breeding method. Although the L-histidine yield has been improved compared to irrational metabolic engineering breeding methods, the yield and conversion rate still do not meet the requirements of industrial production.

[0004] It is reported that Wu et al. obtained a strain that efficiently produces L-histidine through metabolic modification. In the late fermentation cycle in the fermenter, the activity of the strain decreased, affecting the fermentation production of the strain. Therefore, the stress resistance analysis of the strain found that high concentrations of L-histidine would inhibit the growth of Escherichia coli, but there are few studies on the tolerance mechanism of Escherichia coli to high concentrations of L-histidine. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a strain that can tolerate high concentrations of L-histidine and efficiently produce L-histidine and its application, and the strain has high industrial application value.

[0006] The technical solution of the present invention is summarized as follows: The present invention provides an Escherichia coli (also known as Escherichia coli, E. coli ) ALE-HIS-CL strain, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 31770, deposit date: August 28, 2024, classification name: Escherichia coli Escherichia coli .

[0007] Said E. coli The ALE-HIS-CL strain is E. coli WHY2-3 was used as the starting strain, and the adaptive laboratory evolution method was used to continuously domesticate Escherichia coli under the condition of high concentration of L-histidine as the tolerance factor. Combined with high-throughput culture methods and screening methods, tolerant mutants were screened, and finally an evolved strain E. coli ALE-HIS-CL that can tolerate L-histidine concentration of 40 g / L was obtained.

[0008] Said E. coli The ALE-HIS-CL strain was used to produce L-histidine by fermentation.

[0009] The present invention also provides a method of using the above E. coli Method for producing L-histidine by ALE-HIS-CL strain.

[0010] Beneficial effects: The key point of the present invention is to obtain a strain that can tolerate 40 g / L L-histidine and can efficiently produce L-histidine through adaptive laboratory evolution. The high-tolerance strain of the present invention can efficiently produce L-histidine. After testing in a 5 L fermenter, the culture cycle is 44-52 h, the accumulation of L-histidine reaches 60-68 g / L, and the conversion rate is 0.24 g L-histidine / g glucose, which is 54.5% and 19.3% higher than the L-histidine yield and conversion rate of the starting strain WHY2-3, respectively. The strain has good industrial application prospects.

[0011] The present invention relates to the deposit information of biological materials: Deposit date: August 28, 2024; Depository: China General Microbiology Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No. 31770; Classification name: Escherichia coli Escherichia coli . BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Graph showing the number of generations of the dual-tolerance pressure alternating adaptive evolution in Example 1.

[0013] Figure 2 : Growth curve of the strain in Example 1 in a medium containing 40 g / L L-histidine.

[0014] Figure 3 : L-histidine production and OD of strains in shake flask fermentation in Example 2 600 Test results.

[0015] Figure 4A : Fermentation process curve of WHY2-3 strain in 5 L fermenter in Example 2.

[0016] Figure 4B : Fermentation process curve of ALE-HIS-CL strain in 5 L fermenter in Example 2. DETAILED DESCRIPTION

[0017] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0018] The present invention provides an evolved strain E. coli ALE-HIS-CL, with the deposit number CGMCC No. 31770, can tolerate high concentrations of L-histidine, grow and metabolize normally in a culture medium with an L-histidine concentration of 40 g / L, and exhibit growth characteristics that are significantly better than those of the starting strain.

[0019] The present invention is based on engineering bacteria E. coli WHY2-3 was used as the starting strain. The engineered bacteria and its construction process have been published in the invention patent CN202010151245.X.

[0020] The present invention adopts the method of adaptive laboratory evolution, continuously domesticates Escherichia coli under the condition of high concentration of L-histidine as tolerance factor, combines high-throughput culture method and screening means, screens tolerant mutants, and finally obtains an evolved strain with high tolerance and high yield. E. coli ALE-HIS-CL. The overall technical route is as follows: (1) Test the growth curve of the starting strain in a medium containing high concentration of L-histidine Currently, there are few studies on the effect of high concentration L-histidine on the growth and metabolism of Escherichia coli. In order to understand the effect of high concentration L-histidine on the growth and metabolism of Escherichia coli, it is necessary to test the growth curve of Escherichia coli in the presence of different concentrations of L-histidine, to determine the L-histidine concentration threshold for initial tolerance and domestication, and to conduct domestication experiments based on this concentration threshold.

[0021] (2) Establishment of a high-throughput screening method for mutant strains Adaptive laboratory evolution experiments will obtain a large number of mutant libraries, and it takes a lot of time to screen out strains with outstanding performance. In order to screen out high-performance strains more quickly, it is necessary to construct a high-throughput screening method, including a high-throughput culture method and a detection method. L-histidine can react with diazobenzenesulfonic acid in an alkaline environment to form an azo compound, and its solution will show an orange-red color reaction and have a maximum absorption value at 476 nm on a spectrophotometer. Therefore, after the mutant strains isolated from the plate have been cultured in a 96-well plate with high throughput, the histidine concentration in the well plate culture medium is subjected to high-throughput screening according to the above-mentioned color reaction principle.

[0022] (3) Adaptive evolution and screening of strains under high concentration of L-histidine The starting strain was subcultured in a medium containing an appropriate concentration of L-histidine. During the process, the concentration of L-histidine in the medium was adjusted in time according to the growth status of the strain, and finally a strain with good growth performance under 40 g / L histidine was obtained. E. coli ALE-HIS-CL.

[0023] The present invention E. coli The ALE-HIS-CL strain can be used for fermentation production of L-histidine.

[0024] The present invention also provides a method for preparing L-histidine, comprising: culturing the E. coli The invention further comprises the steps of producing an ALE-HIS-CL strain, and collecting L-histidine from the strain and / or the culture medium.

[0025] According to the present invention, the L-histidine includes not only free form of L-histidine but also salts or hydrates of L-histidine.

[0026] According to the present invention, the cultivation of the strain can be carried out by conventional methods in the art. The culture medium used to produce L-histidine can be a synthetic or natural culture medium, such as a typical culture medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions and other organic and inorganic components required.

[0027] As a specific embodiment, the culture medium composition is: glucose 20-30 g / L, yeast extract 2-5 g / L, peptone 2-4 g / L, KH 2 PO 4 1-3 g / L, MgSO 4 7H 2 O 1-2 g / L, FeSO 4 7H 2 O 5-20 mg / L, MnSO 4 7H 2 O 5-20 mg / L, VB1, VB3, VB5, VB12, VH 1-3 mg / L each, the rest is water, pH 7.0-7.2.

[0028] According to the present invention, the strain can be cultured under aerobic conditions for 16 to 72 hours, or 24 to 60 hours, or 40 to 50 hours; the culture temperature can be controlled within 30 to 45°C, or 30 to 37°C; and the pH can be adjusted between 5.0 and 8.0, or between 6.0 and 7.5, or between 6.8 and 7.2. The pH can be adjusted by using inorganic or organic acidic or alkaline substances, as well as ammonia gas.

[0029] As another specific embodiment, the method further comprises: maintaining the culture temperature at a constant 37° C., and / or maintaining the pH at about 7.0.

[0030] As another specific implementation, the method further includes: maintaining the dissolved oxygen at about 35%.

[0031] As another specific embodiment, the method further comprises: during the culture process, when glucose is exhausted, a glucose solution of a certain concentration needs to be added to maintain the residual glucose concentration in the fermentation medium at 0.1-5 g / L.

[0032] After cultivation, solids such as cells and cell debris can be removed from the liquid culture medium by conventional techniques (e.g., centrifugation, membrane filtration), and L-arginine can then be recovered from the fermentation broth by any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).

[0033] Before inoculation and fermentation, bacteria can be activated and seed cultured according to their storage status. Appropriate conditions and culture media can be selected according to conventional techniques in the art. For example, the seed culture medium can have the same composition as the fermentation medium, or it can be appropriately adjusted on this basis.

[0034] Other specific operation methods such as biotechnology involved can be implemented according to technical manuals, textbooks or literature reports that are easily available to those skilled in the art, and it is not necessary to describe the operation process in detail here. In addition, specific culture methods and culture media, etc. are selected in the following examples, but it does not mean that the purpose of the present invention can only be achieved through these specific selections, and the scope of the present invention cannot be limited to this. The essence and scope of the present invention are only limited by the claims.

[0035] The present invention is described in more detail below through specific examples.

[0036] Embodiment 1: This embodiment provides an evolutionary strain E. coli The construction method of ALE-HIS-C is as follows: 1.1 Test of tolerance of starting strain WHY2-3 to L-histidine The WHY2-3 strain stored in a -80℃ refrigerator was inoculated with 10 μL of bacterial solution into an LB shake tube and cultured for 12 h. 600 When the cell count reaches about 2-3, transfer to a round-bottom flask containing M9 medium with gradient concentrations of L-histidine (L-histidine concentration is 0-50 g / L), and then place the round-bottom flask in a shaker at 37°C and 220 rpm for 24 h. Samples are taken every 4 h to detect the OD value of the bacterial solution. 600 Finally, according to the bacterial solution OD 600 The data were used to draw the growth curve of the strain. The results showed that when the concentration of L-histidine reached 20 g / L, it would inhibit the growth of the strain.

[0037] 1.2 Adaptive evolution of the starting strain WHY2-3 The WHY2-3 strain stored in a -80℃ refrigerator was inoculated with 10 μL of bacterial solution into an LB shake tube and cultured for 12 h. 600 When the cell count reaches 2-3, transfer to a round-bottom flask containing M9 with 20 g / L L-histidine, and set up three round-bottom flasks as a parallel group, and then place in a shaker at 37℃ 220 rpm for 12-18 h for subculture, and store the bacterial solution during each subculture in a -80℃ refrigerator. The strain was cultured in M9 medium containing L-histidine concentration pressure, and its OD during growth was 600The process will show a gradual change from stress to tolerance, and then the concentration of L-histidine in the culture medium will be increased in time, and the culture will continue to be subcultured. Finally, in the above evolution process, an evolved bacterial solution that can tolerate 40 g / L L-histidine is obtained and preserved for the next step. Figure 1 shown.

[0038] 1.3 High-throughput screening Diazobenzenesulfonic acid can react with L-histidine to form an azo reaction, and the solution will appear orange-red, but there will be problems with unstable color development and cumbersome handling. Using p-aminobenzenesulfonic acid and sodium nitrite to replace diazobenzenesulfonic acid, sodium bicarbonate provides a weak alkalinity, and finally adding 20% ​​ethanol to the solution is conducive to maintaining the color of the color development reaction, and the maximum absorption value at 476 nm on the spectrophotometer is proportional to the concentration of L-histidine.

[0039] To screen the evolved bacterial solution using the above color reaction method, the color reaction reagent needs to be prepared first (Solution A: 0.45 g p-aminobenzenesulfonic acid, add water to dissolve, then add 4.5 mL concentrated hydrochloric acid, and dilute to 50 mL. When used, add an equal volume of 5% NaNO 2 Solution B: 10% sodium carbonate solution), separate single colonies from the evolved bacterial solution and inoculate them into a 96-well plate for 24 hours, dilute the culture solution 20 times, then take 100 μL of the sample and transfer it to a 1.5 mL EP tube, ice-bath solution A to zero degrees, then add 40 μL of solution A and mix for 15 minutes, then add 120 μL of solution B and mix for 10 seconds, finally add 400 μL of 10% ethanol solution, mix and let stand for 5 minutes, then add deionized water to 1 mL, and keep it away from light during the color reaction experiment. Take 200 μL of the color reaction sample into a 96-well plate, and use an enzyme reader to detect it at 476 nm on a spectrophotometer. According to the results, the sample with the highest value is a potential strain, and it will be further verified by shaking the bottle.

[0040] Finally, the evolved strain ALE-HIS-CL with excellent performance was screened from the mutant bacterial solution that tolerated 40 g / L L-histidine and was further identified as Escherichia coli ( Escherichia coli ) and preserve the strains.

[0041] 1.4 Comparative test of tolerance of evolved strains The strain tolerance comparison test was carried out in M9 medium containing high concentration of L-histidine (40 g / L). The strains involved in the test were: Evolved strains E. coli ALE-HIS-CL, starting strain E. coli WHY2-3, E. coliWHY3-1 (CN202010151245.X), E. coli HW6-3 (Wu H) and E. coli W3110. Each strain was first transferred from the -80℃ freezer to an LB shake tube, and then transferred to a triangular flask containing 30 mL of M9 medium at a 1% inoculation rate, and sealed with nine layers of gauze. After shaking and culturing at 37℃ and 220rpm for 12 h, samples were taken from the shake flask corresponding to each group of strains to measure the growth OD of the strain. 600 The growth curve is as follows Figure 2 As shown, samples were taken at 12 h, 16 h, 20 h and 24 h of growth to measure the growth OD of the strain. 600 And draw the growth curve of the corresponding strain, among which the evolved strain E. coli The slope of the growth curve of ALE-HIS-CL was significantly higher than that of other engineered strains. The results showed that compared with its starting strain and several other rationally modified production strains, E. coli ALE-HIS-CL showed remarkable growth tolerance to high concentration of L-histidine.

[0042] Embodiment 2: This embodiment provides a method of using strains E. coli The method for producing L-histidine by ALE-HIS-C fermentation is as follows: 2.1 Shake flask fermentation The strains (ALE-HIS-CL and WHY2-3) stored in a -80℃ refrigerator were transferred to LB shake tubes, and then placed in a 37℃ constant temperature incubator for 12 h. The strains were cultured and activated in the LB shake tubes for the first generation. The bacterial solution in the shake tube was transferred to a triangular flask containing 27 mL LB medium at an inoculation rate of 10%, and sealed with nine layers of gauze. The culture was shaken at 37℃ and 220 rpm for 8 h. The strains were cultured and activated in the shake flask for the second generation. The bacterial solution in the triangular flask was transferred to a triangular flask containing 26 mL fermentation medium at an inoculation rate of 10% (1 mL 60% (m / v) glucose solution was added as the initial culture carbon source), and sealed with nine layers of gauze. The culture was shaken at 37℃ and 220 rpm for 24 h. During the fermentation process, ammonia water was added through a micro syringe to maintain the pH at 7.0, and 60% (m / v) glucose solution was added to maintain the carbon source during the fermentation process.

[0043] The shake flask fermentation medium in this example was composed of 20 g / L glucose, 2 g / L yeast extract, 2 g / L peptone, and 2 g / L KH 2 PO 4 1 g / L, MgSO 4 7H 2 O 2 g / L, FeSO 4 7H2 O 10 mg / L, MnSO 4 7H 2 O 10 mg / L, VB1, VB3, VB5, VB12, VH 2 mg / L each, the rest is water, pH 7.0-7.2.

[0044] The results are attached Figure 3 As shown in the figure, after 24 h of shake flask fermentation, the production of L-histidine in the fermentation broth of the ALE-HIS-CL strain was 3.2 g / L. The production of L-histidine in the fermentation broth of the control group WHY2-3 strain was 2.0 g / L.

[0045] 2.2 Fermentation in 5 L fermenter Take the bacteria preservation tubes (ALE-HIS-CL and WHY2-3) stored in a -80℃ refrigerator and place them on an ice box to thaw naturally. Use an inoculation loop to take a ring of bacteria and streak them densely on the slant medium, and then place it in a 37℃ constant temperature incubator for 12 hours to obtain the first generation of seeds. Use an inoculation loop to scrape the bacteria on the slant medium and streak them densely in an eggplant-shaped bottle, and also place it in a 37℃ constant temperature incubator for 12 hours to obtain the second generation of seeds. Take one-third of the volume of the eggplant-shaped bottle and pour sterile water into the bottle. Resuspend the bacteria in the eggplant-shaped bottle and pour it into the seed fermentation tank for cultivation. During the cultivation process, the pH is stable at about 7.0, the temperature is constant at 37℃, and the dissolved oxygen is controlled at about 35% by adjusting the speed and ventilation volume. When the seed culture reaches the logarithmic growth phase of the cells, it is transferred to the fermentation culture. The seed culture solution is transferred to the fresh fermentation medium at an inoculation rate of 15%. Similarly, during the fermentation process, the pH is stable at about 7.0, the temperature is constant at 37℃, and the dissolved oxygen is controlled at about 35% by adjusting the speed and ventilation volume. When glucose is exhausted during the culture process, 80% (m / v) glucose solution needs to be fed to maintain the residual glucose concentration in the fermentation medium at 0.1-5 g / L.

[0046] The slant culture medium in this embodiment is composed of: 1 g / L glucose, 5 g / L peptone, 5 g / L beef extract, 1 g / L yeast powder, 1 g / L NaCl, 20 g / L agar, and the rest is water, pH 7.0-7.2.

[0047] The seed culture medium in this embodiment is composed of: glucose 15 g / L, yeast extract 5 g / L, peptone 5 g / L, KH 2 PO 4 5 g / L, MgSO 4 7HO 2 5 g / L, FeSO 4 7HO 2 10 mg / L, MnSO 4 HO 210 mg / L, VB1 2 mg / L, VH 1mg / L, 2 drops of defoamer, and the rest is water, pH 7.0-7.2.

[0048] The fermentation medium in this example is composed of 20 g / L glucose, 2 g / L yeast extract, 2 g / L peptone, and 2 g / L KH 2 PO 4 1 g / L, MgSO 4 7H 2 O 2 g / L, FeSO 4 7H 2 O 10 mg / L, MnSO 4 7H 2 O 10 mg / L, VB1, VB3, VB5, VB12, VH 2 mg / L each, the rest is water, pH 7.0-7.2.

[0049] The fermentation process curve is shown in the attached Figure 4A and 4B shown. E. coli ALE-HIS-CL strain ( Figure 4B ) After 44 hours of cultivation in a 5 L fermenter, the accumulation of L-histidine reached 60 g / L. After 52 hours of cultivation, the accumulation of L-histidine reached 68 g / L, and the conversion rate was 0.24 g L-histidine / g glucose. E. coli WHY2-3 strain ( Figure 4A ) After 44-52 h of cultivation in a 5 L fermenter, the accumulation of L-histidine reached 40-44 g / L, and the conversion rate was 0.2 g L-histidine / g glucose.

[0050] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. Escherichia coli ALE-HIS-CL strain, characterized in that: The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No. 31770.

2. The Escherichia coli ALE-HIS-CL strain according to claim 1, characterized in that: The strain tolerates L-histidine at a concentration of 40 g / L.

3. Use of the Escherichia coli ALE-HIS-CL strain according to claim 1 in fermenting and producing L-histidine.

4. A method for preparing L-histidine, comprising: The invention further comprises the steps of culturing the Escherichia coli ALE-HIS-CL strain according to claim 1 in a culture medium, and collecting L-histidine from the strain and / or the culture medium.

5. The method according to claim 4, characterized in that: The culture medium comprises: 20-30 g / L glucose, 2-5 g / L yeast extract, 2-4 g / L peptone, 1-3 g / L KH2PO4, 1-2 g / L MgSO4·7H2O, 5-20 mg / L FeSO4·7H2O, 5-20 mg / L MnSO4·7H2O, 1-3 mg / L each of VB1, VB3, VB5, VB12 and VH, and the rest is water, with a pH of 7.0-7.

2.

6. The method according to claim 4, characterized in that: The culture medium comprises: 20 g / L glucose, 2 g / L yeast extract, 2 g / L peptone, 1 g / L KH2PO4, 2 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, 2 mg / L each of VB1, VB3, VB5, VB12 and VH, and the rest is water, with a pH value of 7.0-7.

2.

7. The method according to claim 4, 5 or 6, characterized in that: The method further comprises: maintaining the culture temperature at a constant 37° C. and the pH at about 7.

0.

8. The method according to claim 7, characterized in that: The method also includes: maintaining the dissolved oxygen at about 35%.

9. The method according to claim 8, characterized in that: The method further comprises: during the culture process, when glucose is exhausted, a glucose solution of a certain concentration needs to be added to maintain the residual glucose concentration in the fermentation medium at 0.1-5 g / L.

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