Gamma-PGA high-yield strain mutation breeding method
阅读说明：本技术 一种γ-PGA高产菌株诱变选育方法 (Gamma-PGA high-yield strain mutation breeding method ) 是由 石元亮 张雷 王玲俐 于 2020-08-31 设计创作，主要内容包括：本发明公开了一种γ-PGA高效诱变和快速筛选的方法。首先通过硫酸二乙酯、亚硝基胍、紫外、5-溴尿嘧啶对出发菌株进行了复合诱变,通过透明圈法筛选初筛、24孔板复筛和传代稳定性试验后,获得了一株高产、稳定性强的菌株C2,该菌株聚谷氨酸产量达到21.3 g/L,比诱变前提高43.91%。将硫酸二乙酯、亚硝基胍、紫外、5-溴尿嘧啶等诱变剂结合使用,显著提高了菌株的突变幅度。结合多孔板技术,实现了聚谷氨酸突变株的快速筛选。(The invention discloses a method for efficient mutagenesis and rapid screening of gamma-PGA. Firstly, carrying out compound mutagenesis on an original strain through diethyl sulfate, nitrosoguanidine, ultraviolet and 5-bromouracil, and obtaining a high-yield and strong-stability strain C2 after primary screening, 24-pore plate secondary screening and passage stability test through a transparent circle method, wherein the yield of polyglutamic acid of the strain reaches 21.3 g/L and is improved by 43.91% compared with that before mutagenesis. The mutagens such as diethyl sulfate, nitrosoguanidine, ultraviolet, 5-bromouracil and the like are combined for use, so that the mutation amplitude of the strain is remarkably improved. And the rapid screening of the polyglutamic acid mutant strain is realized by combining a porous plate technology.)
1. A high-yield gamma-PGA strain mutation breeding method is characterized in that an original strain is subjected to multiple times of mutagenesis by using a UV-NTG-DSE-5' BrU compound mutagen, and a transparent circle method and a 6-hole/24-hole plate are assisted for breeding, so that a high-yield mutant strain is obtained.
2. The mutagenesis breeding method of high-yield gamma-PGA strain according to claim 1, characterized in that: after complex mutagenesis using UV-NTG-DSE, the resulting mixture was spread on a 5-BrU plate containing skim milk.
3. Wherein the UV-NTG-DSE complex mutagenesis can be performed in combination.
4. The mutation breeding method of high-yield γ -PGA strain according to any one of claims 1 to 2, wherein: the concentration of DSE is 0.5% -1.0%; NTG concentration is 0.02-0.06%, and incubation time is 20-40 min.
5. The mutation breeding method of high-yield γ -PGA strain according to any one of claims 1 to 2, wherein: UV irradiation time is 20-60s, power is 15-30W, distance is 15-40cm, and culturing for 6-12h in dark place after irradiation.
6. The mutation breeding method of high-yield γ -PGA strain according to any one of claims 1 to 2, wherein: the concentration of 5-BrU is 1.5-3.5%.
7. The mutation breeding method of high-yield γ -PGA strain according to any one of claims 2 to 3, wherein: the combination of NTG-DSE mutagenesis can be performed separately.
8. The mutation breeding method of high-yield γ -PGA strain according to any one of claims 2 to 4 and 6, wherein: the UV mutagenesis assay and the NTG-DSE mutagenesis assay can be performed in cycles.
9. The mutagenesis breeding method of high-yield gamma-PGA strain according to claim 1, characterized in that: strain fermentation was performed using 6-well/24-well plates.
Technical Field
The invention belongs to the field of microorganism breeding of bioengineering, and relates to a method for breeding a poly-gamma-glutamic acid (gamma-PGA) breeding mutant strain, according to which a fermentation strain with high yield of gamma-PGA can be quickly obtained.
Background
With the rise of bio-based polymer materials, γ -PGA has attracted much interest. gamma-PAG is mostly produced by bacillus and is an anionic heteromorphic peptide. The water-soluble chitosan/. According to the difference in molecular weight, γ -PGA can be used in various fields such as cosmetics, foods, hygiene, medical care, agriculture, etc.
There are three main approaches to the acquisition of γ -PGA: chemical synthesis, enzymatic synthesis and microbial fermentation. Among them, the chemical synthesis method and the enzymatic catalysis method are difficult to be applied in large scale due to the small weight average molecular weight of the produced γ -PGA molecule and the easy environmental pollution, and are gradually replaced by the biological fermentation method. The production of γ -PGA by microbial fermentation is the most commonly used method in the industry. However, the yield of the gamma-PGA fermentation strain is low, which always troubles the whole gamma-PGA industry.
The best way to realize high yield of the strain is to breed the high-yield strain. γ -PGA is a synthetic process of a multi-metabolic module, and genes involved in metabolism are distributed in the genome (FIG. 1). At present, a single mutagen is usually used for mutagenesis of gamma-PGA, and after multiple rounds of mutagenesis, the sensitivity of a strain to the mutagen is reduced, the mutation amplitude is reduced, and the negative mutation rate is increased; the mutagenized strain needs to be screened and obtained, and the screening flux is low at present, so that the acquisition of the gamma-PGA high-yield strain is influenced.
Different mutagens have different mutation hot spots, and Ultraviolet (UV) is the most commonly used physical mutagen at present, and can cause covalent connection between pyrimidines in a DNA chain to form pyrimidine dimers, thereby influencing the molecular structure of DNA. Diethyl Sulfate (DSE), Nitrosoguanidine (NTG) and common alkylating agents are used, wherein the former moves to a part with higher electron density (usually corresponding to a transcription region of a cell) through an electrophilic group such as alkyl and the like to perform electrophilic addition reaction to change the molecular structure of a basic group, and the latter mainly causes abnormal breakage of nucleic acid to cause SOS repair or homologous recombination repair of the cell, so as to achieve the purpose of gene mutation. 5-bromouracil (5-BrU) is a common chemical mutagen and, upon entry into cells, more readily pairs with guanine, converting the original base from A = T to G.ident.C. The research shows that: compared with single mutagenesis, the combination effect of multiple mutagens is better. The combination of different mutagens can improve the mutagenesis efficiency and the mutation amplitude, and improve the yield of the mutagenic strain.
Disclosure of Invention
The invention aims to provide a simple and practical breeding method with high mutation amplitude and high flux so as to solve the problem that the existing mutation breeding method for producing strains of Gamma-PGA has limited improvement on the yield of the strains.
In order to achieve the above object, the present invention provides a mutagenesis method of a high-yield γ -PGA strain, the technical scheme is: the original strain is subjected to compound mutagenesis by using four mutagens, namely DSE, NTG, UV and 5-BrU, and a mutant strain with high yield of gamma-PGA is screened by combining a transparent circle method. The method comprises the following specific steps.
(1) Activation of the strain: activating glycerol tube strain of gamma-PGA producing strain in LB culture medium plate at 30-37 deg.c for 18-24 hr.
(2) Preparing a bacterial liquid: picking a single colony in the step (1), inoculating the single colony into an LB liquid culture medium, culturing at the temperature of 35-37 ℃ for 18-24h at the temperature of 150-. Diluting the cultured bacterial liquid 1mL by 104-106Doubling the weight of the product for later use.
(3) And (3) carrying out strain mutagenesis.
1) Adding the bacterial liquid, DSE (0.5-1.0%) and NTG (0.02-0.06%) in the sterile test tube (2), shaking for 30min at the volume ratio of 1:2:2 and 30-37 ℃ and 180r/min, adding Na2S2O3Stopping the solution, centrifuging at 3000 r/min for 15min, discarding the supernatant, and adding equivalent physiological saline for heavy suspension to obtain mutagenic bacteria liquid A.
2) And (3) placing the bacterium liquid A in a 30W ultraviolet lamp for irradiating for 20-60s at a position of 30 cm, adding an equal volume of liquid fermentation medium, and incubating for 6-12h in dark in a dark place to obtain a bacterium liquid B.
3) And (3) taking the bacterium liquid B under a red light, adding 5-bromouracil with the same volume of 1.5-3.5%, uniformly mixing, and coating on a skimmed milk plate. Culturing at 30-37 deg.C for 12-18h to complete one round of mutagenesis. According to experience, more than 5 rounds of mutagenesis are recommended.
And (6) counting data.
Mutagenesis was performed in triplicate and colonies were counted. The lethality was calculated with the non-mutagenized strain as a reference. Selecting a plate with the lethality of 85% -95% for screening.
And (4) determining the lethality.
Lethality = (1-M/U). times.100%.
M: number of colonies in the plates after mutagenesis, number of colonies in the plates of the U control group.
(4) Strain screening: after the colony diameter and the transparent ring diameter are measured, strains with larger K value (the first 5-20%) are selected for multi-plate fermentation, and the content of gamma-PGA is measured.
In the step (4): k = D/D; d: the diameter of the colony; d: diameter of the transparent ring.
(5) And (3) testing the stability of the strain: and (3) subculturing the partial strains with high yield to 6 generations, detecting the content of gamma-PGA, and selecting the strains with relatively stable heredity.
(6) And (4) a culture medium.
LB medium (g/L): beef extract 5,
Seed medium (g/L): 25-35 parts of glucose, 8-14 parts of sodium glutamate, 8-12 parts of yeast extract powder and K2HPO4•3H2O 2-4，(NH4)2SO42-4，MgSO4•7H2O 0.1-0.2，MnSO4•H2O0.02-0.06, pH 7.0, and autoclaving at 115 deg.C for 20 min.
Fermentation medium (g/L): 30-45 parts of glucose, 30-45 parts of sodium glutamate and 8-12 parts of yeast extract powder, K2HPO4•3H2O2-4，(NH4)2SO42-4，MgSO4•7H2O 0.1-0.2，MnSO4•H2O0.02-0.06, pH 7.0, and autoclaving at 115 deg.C for 20 min.
Skim milk medium (g/L): 10-15 parts of skimmed milk powder, 15 parts of agar, and 115 ℃ for 20min by autoclaving.
(7) A method of culturing.
Seed culture: and (4) selecting the activated strains from the 1-2 rings, inoculating the strains into a seed culture medium, and culturing for 18-24 h.
Fermentation culture: inoculating the seed liquid into 6/24 pore plates (deep pore plates, liquid loading amount of 3-5 mL; 1-1.4 mL) at 5% inoculation amount, shaking at constant temperature of 30-37 deg.C and 220 r/min for 48-54 h.
Compared with the prior art, the invention has the following advantages.
Compared with other mutagenesis techniques, the method does not need expensive equipment, has simple mutagenesis operation, and can be operated by common technicians.
The invention combines four mutagens, has high mutagenesis efficiency and large mutation amplitude, and the screened strain has high stability, thereby solving the problem that the strain is insensitive to a single mutagen and avoiding the saturation effect of the single mutagen.
The screening of the strains after mutagenesis is carried out by combining a transparent ring method with an 6/24 pore plate, the target strains can be rapidly screened, the speed is at least improved by more than 10 times compared with the 250mL shaking flask fermentation screening, and the screening cost is saved.
Description of the drawings.
FIG. 1 is a flow chart of breeding of gamma-PGA high producing strain.
Fig. 2 is a schematic view of a γ -PGA transparent ring.
FIG. 3 shows the γ -PGA production by different fermentation strains.
FIG. 4 is a comparison of the yields after passage, first (colorless) and sixth (gray).
FIG. 5 shows the results of T-test between different strains in the sixth generation.
The present invention will be further described with reference to the following embodiments.
Example 1.
The method for mutagenizing a gamma-PGA-producing strain comprises the following steps.
And (4) preparing a mutagen.
NTG mother liquor: weighing NTG 0.4 g, adding a small amount of acetone to assist dissolution, transferring into a 10mL volumetric flask, fixing the volume, and refrigerating and storing in dark.
DES mother liquor: taking 0.5mL of DSE liquid, adding ethanol for assisting dissolution, using phosphate buffer solution for constant volume to 10mL, and refrigerating and storing in dark.
5-BrU mother liquor: 25mg of 5-BrU was weighed and 10mL of sterile water was added, and the mixture was stored under dark conditions.
When the above solution is taken out, it is sterilized by filtration through a 0.22 μm sterile filter, and diluted for use.
Phosphate Buffered Saline (PBS): liquid A (0.2 mol/L Na)2HPO4): weighing Na2HPO4.12H2O71.6 g, adding water, stirring for dissolving, adding water to a constant volume of 1L, and autoclaving at 121 deg.C for 20 min. Mother liquor B (0.2 mol/LNaH) is prepared2PO4): weighing NaH2PO4·2H2Adding water into O31.2 g, stirring for dissolving, adding water to a constant volume of 1L, autoclaving at 121 deg.C for 20min, and storing at room temperature. PBS working solution: 12.3mL of solution A was mixed with 87.7mL of solution B.
0.9% physiological saline: weighing 9 g of NaCl, dissolving in 500mL of sterile water, and fixing the volume to 1L; sterilizing at 121 deg.C under high pressure for 20min, and storing at 4 deg.C.
25% Na2S2O3Solution: 62.05g Na2S2O3·5H2Dissolving O in water, diluting to 1L, filtering with 0.22 μm sterile filter head for sterilization, and storing in dark.
(1) Activation of the strain: activating glycerol tube strain of gamma-PGA producing strain in LB culture medium plate at 30-37 deg.c for 18-24 hr.
(2) Preparing a bacterial liquid: picking a single colony in the step (1), inoculating the single colony into an LB liquid culture medium, culturing at the temperature of 35-37 ℃ for 18-24h at the temperature of 150-. Diluting the cultured bacterial liquid 1mL by 104Doubling the weight of the product for later use.
And (3) carrying out strain mutagenesis.
(1) Adding the bacteria solution, DSE and NTG (concentration shown in Table 1) in the (2) sterile test tube at a volume ratio of 1:2:2, 30-37 deg.C, shaking for 30min at 150-2S2O3Stopping the solution, centrifuging at 3000 r/min for 15min, discarding the supernatant, and adding equivalent physiological saline for heavy suspension to obtain the mutagenic bacteria solution.
(2) And (3) placing the bacterium liquid obtained in the step (1) in a 30W ultraviolet lamp for irradiation at a position of 30 cm (the irradiation time is shown in table 1), adding an equal volume of liquid fermentation medium, and incubating for 6-12h in a dark place to obtain a new mutagenic bacterium liquid.
(3) And (3) under a red light, adding 5-bromouracil (the concentration is shown in the table 1) with the same volume into the bacterial liquid obtained in the step (2), uniformly mixing, and coating on a skim milk plate (the numbers of different treatments are respectively 1-9). Culturing at 30-37 deg.C for 12-18 h.
TABLE 1 mutagen concentration and lethality in different plates
Treatment of
UV(t/s)
5-BrU(g/L)
NTG(g/L)
DES(%)
The lethality rate%
1
30
15
0.2
0.5
89.3
2
30
25
0.4
1
91.3
3
30
35
0.6
1.5
98.4
4
40
15
0.4
1.5
97.4
5
40
25
0.6
0.5
93.9
6
40
35
03
1
93.8
7
50
15
0.6
1
945
8
50
25
0.2
1.5
99.7
9
50
35
0.4
0.5
99.3
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