A microbial mutation breeding device and its application

Abstract

The present invention belongs to the technical field of microbial mutagenesis, and particularly relates to a microbial mutagenesis breeding device and its application. The microbial mutagenesis breeding device provided by the present invention includes a cube structure and a transmission structure. The cube structure is wrapped with light-impermeable materials around, and ultraviolet lamps are arranged inside. The transmission structure includes a single sorting area and a mutagenesis area. The mutagenesis area is inserted into the cube structure through the insertion opening of the cube structure and is arranged perpendicular to the ultraviolet lamps. Microorganisms can be sorted singly through the first horizontal spiral conveyor pipe in the single sorting area and transported to the second horizontal spiral conveyor pipe in the mutagenesis area, so that the time and intensity of the microorganisms being irradiated by ultraviolet light are uniform, significantly reducing the mutagenesis lethality rate of the microorganisms and significantly increasing the positive mutation rate. The operation is simple, the workload is small, and the cycle is short. Moreover, by treating the microorganisms with a protective agent having a specific composition before single sorting, the mutation uniformity and the positive mutation rate can be further improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial mutagenesis, and particularly relates to a microbial mutagenesis breeding device and its application. Background Art

[0002] Microbial mutagenesis technology is one of the main methods for microbial breeding. Chemical mutagenesis and physical mutagenesis play important roles in strain breeding and transformation. Ultraviolet light is one of the most commonly used physical mutagenic factors. By irradiating with ultraviolet light, dimers are formed between the two strands of DNA or between two adjacent thymine bases on the same strand, hindering the separation, replication of the double strand and the normal pairing of bases, thus causing mutations.

[0003] However, the existing ultraviolet mutagenesis methods usually directly act on the plates on which microorganisms grow or the prepared suspension, and it is impossible to ensure that each cell receives the same mutagenesis. Usually, the homogeneity after microbial mutagenesis is poor and the positive mutation rate is low, greatly increasing the workload and cycle of screening. Summary of the Invention

[0004] The purpose of the present invention is to provide a microbial mutagenesis breeding device and its application, which can improve the homogeneity and positive mutation rate after microbial mutagenesis, and is simple to operate, with small workload and short cycle.

[0005] To achieve the above purpose, the present invention provides a microbial mutagenesis breeding device. The microbial mutagenesis breeding device includes a cubic structure and a transmission structure. The cubic structure is wrapped with light-proof materials around its four sides and an ultraviolet lamp is arranged inside;

[0006] The transmission structure includes a single sorting area and a mutagenesis area. The mutagenesis area is inserted into the cubic structure through the insertion opening of the cubic structure and is arranged perpendicular to the ultraviolet lamp;

[0007] The single sorting area is provided with a first horizontal spiral conveying pipeline; the mutagenesis area is provided with a second horizontal spiral conveying pipeline;

[0008] The outlet of the first horizontal spiral conveying pipeline and the inlet of the second horizontal spiral conveying pipeline are connected through a horizontal pipeline; the first horizontal spiral conveying pipeline and the second horizontal spiral conveying pipeline are arranged in parallel;

[0009] The number of spirals of the first horizontal spiral conveying pipeline ≥ 10 turns, and the diameter of the pipeline is 20 - 30 μm;

[0010] The number of spirals of the second horizontal spiral conveying pipeline ≥ 10 turns, and the diameter of the pipeline is 20 - 30 μm.

[0011] Preferably, the distance between the center of the first horizontal spiral conveying pipe and the center of the second horizontal spiral conveying pipe is 0.8 cm.

[0012] Preferably, the inlet, outlet and center of the first horizontal spiral conveying pipe are on a straight line;

[0013] The inlet, outlet and center of the second horizontal spiral conveying pipe are on a straight line.

[0014] Preferably, the areas of the single sorting area and the mutagenesis area are 0.5 cm × 0.5 cm respectively.

[0015] Preferably, the materials of the first horizontal spiral conveying pipe, the second horizontal spiral conveying pipe and the horizontal pipe are silica respectively.

[0016] The present invention also provides an application of the microbial mutagenesis and breeding device described in the above technical solution in microbial mutagenesis and / or microbial breeding.

[0017] The present invention also provides a microbial mutagenesis method, which uses the microbial mutagenesis and breeding device described in the above technical solution to perform ultraviolet mutagenesis treatment on microorganisms, and includes the following steps:

[0018] Mix the solution containing microorganisms with a protective agent to obtain a microbial solution;

[0019] Pass the microbial solution into the first horizontal spiral conveying pipe of the microbial mutagenesis and breeding device for single sorting to obtain a single-sorted cell suspension;

[0020] When the single-sorted cell suspension enters the mutagenesis area of the microbial mutagenesis and breeding device, perform ultraviolet irradiation on the mutagenesis area until the single-sorted cell suspension passes through the outlet of the second horizontal spiral conveying pipe;

[0021] The protective agent includes calcium ions, trehalose and a solvent; the solvent is glycerol with a volume concentration of 50%.

[0022] Preferably, the concentration of calcium ions in the microbial solution is 0.1 - 1 mmol / L, the concentration of trehalose is 30 - 40 mmol / L, and the volume ratio of the solution containing microorganisms to the solvent is 1:(1 - 2);

[0023] The number of microorganisms in the solution containing microorganisms is 2×10 8 cells / mL.

[0024] Preferably, the rate of passing the microbial solution into the first horizontal spiral conveying pipe is 0.1 - 0.3 μL / min; the power of the ultraviolet irradiation is 0.9 - 1.5 W.

[0025] Preferably, after the ultraviolet irradiation, the method further includes: screening the cell suspension irradiated with ultraviolet light to obtain microorganisms with forward mutations.

[0026] Beneficial effects:

[0027] The present invention provides a microbial mutagenesis and breeding device. The microbial mutagenesis and breeding device includes a cubic structure and a transmission structure. The cubic structure is wrapped with light-impermeable materials around its perimeter and is internally provided with ultraviolet lamps. The transmission structure includes a single sorting area and a mutagenesis area. The mutagenesis area is inserted into the cubic structure through the insertion opening of the cubic structure and is arranged perpendicular to the ultraviolet lamps. The single sorting area is provided with a first horizontal spiral conveying pipeline. The mutagenesis area is provided with a second horizontal spiral conveying pipeline and ultraviolet lamps. The second horizontal spiral conveying pipeline and the ultraviolet lamps are arranged perpendicular to each other. The exterior of the mutagenesis area is wrapped with light-impermeable materials. The outlet of the first horizontal spiral conveying pipeline and the inlet of the second horizontal spiral conveying pipeline are connected through a horizontal pipeline. The first horizontal spiral conveying pipeline and the second horizontal spiral conveying pipeline are arranged in parallel. The number of spirals of the first horizontal spiral conveying pipeline is ≥10 turns, and the diameter of the pipeline is 20-30 μm. The number of spirals of the second horizontal spiral conveying pipeline is ≥10 turns, and the diameter of the pipeline is 20-30 μm. The microbial mutagenesis and breeding device provided by the present invention has a simple structure. By limiting the number of spirals and the diameter of the first horizontal spiral conveying pipeline, it can ensure that microorganisms are sorted singly through the first horizontal spiral conveying pipeline in the single sorting area. After entering the second horizontal spiral conveying pipeline in the mutagenesis area, it can ensure that the time and intensity of the microorganisms being irradiated by ultraviolet light are uniform, realizing precise mutagenesis of single-cell microorganisms. The results of the examples show that using the microbial mutagenesis and breeding device of the present invention to perform mutagenesis treatment on microorganisms, the lethality rate of the contemporary strains is significantly reduced, the forward mutation rate is significantly increased, and the mutation uniformity is high. The operation is simple, the workload is small, and the cycle is short.

[0028] The present invention mixes a solution containing microorganisms with a protective agent. By limiting the composition and concentration of the protective agent, the mutation effect can be further improved, and mutant microorganisms with high uniformity and high forward mutation rate can be obtained. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the literature, the following will briefly introduce the drawings required for use in the embodiments.

[0030] Figure 1 It is a top view of the transmission structure in the microbial mutagenesis and breeding device of Example 1;

[0031] Figure 2 It is a schematic diagram of the cubic structure in the microbial mutagenesis and breeding device of Example 1; wherein, 1 is the ultraviolet lamp, and 2 is the insertion opening;

[0032] Figure 3 Schematic diagram of the microbial mutagenesis and breeding device in Example 1; wherein, 1 is a cubic structure and 2 is a transmission structure;

[0033] Figure 4 Graph showing the relationship between the mutagenesis lethality rate and the forward mutation rate of Escherichia coli under different calcium ion concentrations;

[0034] Figure 5 Graph showing the relationship between the mutagenesis lethality rate and the forward mutation rate of Escherichia coli under different trehalose concentrations;

[0035] Figure 6 Graph showing the relationship between the mutagenesis lethality rate and the forward mutation rate of Escherichia coli under different proportions of 50% v / v glycerol;

[0036] Figure 7 Graph showing the relationship between the mutagenesis lethality rate and the forward mutation rate of Escherichia coli under different protectants;

[0037] Figure 8 Graph showing the proportion distribution of the mutation directions of Escherichia coli under different mutagenesis methods. Detailed implementation method

[0038] The present invention provides a microbial mutagenesis and breeding device, which includes a cubic structure and a transmission structure. The cubic structure is wrapped with light-proof materials around its perimeter and an ultraviolet lamp is arranged inside;

[0039] The transmission structure includes a single sorting area and a mutagenesis area. The mutagenesis area is inserted into the cubic structure through the insertion opening of the cubic structure and is arranged perpendicular to the ultraviolet lamp;

[0040] The single sorting area is provided with a first horizontal spiral conveyor pipe; the mutagenesis area is provided with a second horizontal spiral conveyor pipe and an ultraviolet lamp;

[0041] The outlet of the first horizontal spiral conveyor pipe and the inlet of the second horizontal spiral conveyor pipe are connected through a horizontal pipe; the first horizontal spiral conveyor pipe and the second horizontal spiral conveyor pipe are arranged in parallel;

[0042] The number of spirals of the first horizontal spiral conveyor pipe ≥ 10 turns, and the diameter of the pipe is 20 - 30 μm; the number of spirals of the second horizontal spiral conveyor pipe ≥ 10 turns, and the diameter of the pipe is 20 - 30 μm.

[0043] As an implementation manner, the length × width × height of the cube structure of the present invention is 0.5 cm × 0.5 cm × 5 cm. As an implementation manner, the ultraviolet lamp of the present invention is located at the top of the cube structure. As an implementation manner, an insertion port is provided on the side surface of the cube structure of the present invention, the distance from the insertion port to the ground is 1.2 mm, the mutagenesis area is inserted into the cube structure through the insertion port and is arranged perpendicular to the ultraviolet lamp. By defining the positions of the insertion port and the ultraviolet lamp, the present invention can reduce the variables of the mutagenesis parameters, that is, keep the distance of the ultraviolet lamp irradiation unchanged. Even if the vertical distance between the second horizontal spiral conveying pipe and the ultraviolet lamp is 5 cm, only the power parameter and the flow rate of the microbial solution need to be adjusted, which is convenient and fast.

[0044] As an implementation manner, the light-proof material of the present invention is a fully light-shielding black film. The present invention uses the light-proof material to wrap the four sides of the cube structure, so that when the ultraviolet lamp irradiates, the second horizontal spiral conveying pipe is in a dark environment, which has the effect of isolating the external light environment and avoiding the phenomenon of photoreactivation of microorganisms after ultraviolet irradiation. At the same time, it can avoid harm to the human body during the ultraviolet irradiation process.

[0045] As an implementation manner, the present invention randomly divides the glass slide into two areas. The first horizontal spiral conveying pipe is engraved on one area to form a single sorting area, and the second horizontal spiral conveying pipe is engraved on the other area to form a mutagenesis area.

[0046] As an implementation manner, the materials of the first horizontal spiral conveying pipe, the second horizontal spiral conveying pipe and the horizontal pipe of the present invention are respectively silicon dioxide. As an implementation manner, the connection line of the centers of the first horizontal spiral conveying pipe and the second horizontal spiral conveying pipe is parallel to the horizontal pipe. The present invention uses silicon dioxide as the material, and silicon dioxide does not absorb ultraviolet rays, which can improve the effect of ultraviolet mutagenesis.

[0047] As an implementation manner, the area of the single sorting area of the present invention is 0.5 cm × 0.5 cm. As an implementation manner, the area of the mutagenesis area of the present invention is 0.5 cm × 0.5 cm.

[0048] As an implementation manner, the distance between the center of the first horizontal spiral conveying pipe and the center of the second horizontal spiral conveying pipe of the present invention is 0.8 cm.

[0049] As an implementation manner, the number of turns of the first horizontal spiral conveying pipeline of the present invention is 10 turns. As an implementation manner, the size of the first horizontal spiral conveying pipeline of the present invention is 25 μm. By defining the number of turns and diameter of the first horizontal spiral conveying pipeline, the present invention can ensure that microorganisms complete a single sorting, and ensure that the time and intensity of ultraviolet light irradiation of the microorganisms entering the second horizontal spiral conveying pipeline in the mutagenesis area are uniform.

[0050] As an implementation manner, the distance between adjacent spirals of the first horizontal spiral conveying pipeline is 20-30 μm; as an implementation manner, the distance between adjacent spirals of the first horizontal spiral conveying pipeline is 25 μm.

[0051] As an implementation manner, the inlet, outlet and center of the first horizontal spiral conveying pipeline of the present invention are located on a straight line.

[0052] As an implementation manner, the number of turns of the second horizontal spiral conveying pipeline of the present invention is 10 turns. As an implementation manner, the diameter of the second horizontal spiral conveying pipeline of the present invention is 25 μm. By defining the number of turns and diameter of the second horizontal spiral conveying pipeline, the present invention can ensure that the time and intensity of ultraviolet light irradiation of microorganisms are uniform, and achieve precise mutagenesis of single-cell microorganisms.

[0053] As an implementation manner, the distance between adjacent spirals of the second horizontal spiral conveying pipeline is 20-30 μm; as an implementation manner, the distance between adjacent spirals of the second horizontal spiral conveying pipeline is 25 μm.

[0054] As an implementation manner, the inlet, outlet and center of the second horizontal spiral conveying pipeline of the present invention are located on a straight line.

[0055] The microbial mutagenesis and breeding device provided by the present invention has a simple structure. Microorganisms can form single-sorted cells through the first horizontal spiral conveying pipeline in the single sorting area. By setting the second horizontal spiral conveying pipeline in the mutagenesis area, precise mutagenesis of single-cell microorganisms can be achieved by adjusting the power of the ultraviolet lamp. The operation is simple, the workload is small, the cycle is short, the uniformity of the mutated microorganisms is high, and the positive mutation rate is high.

[0056] In view of the advantages of the microbial mutagenesis and breeding device of the present invention, the application of the microbial mutagenesis and breeding device in microbial mutagenesis and / or microbial breeding also belongs to the protection scope of the present invention. The present invention has no strict requirements on the type of the microorganisms, and conventional microorganisms in the art can be used, such as any one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus and Saccharomyces cerevisiae.

[0057] The present invention also provides a method for microbial mutagenesis. The microbial mutagenesis breeding device described in the above technical solution is used to perform ultraviolet mutagenesis treatment on microorganisms, including the following steps:

[0058] Mix a solution containing microorganisms with a protective agent to obtain a microbial solution;

[0059] Pass the microbial solution into the first horizontal spiral conveying pipeline of the microbial mutagenesis breeding device for single sorting to obtain a single-sorted cell suspension;

[0060] When the single-sorted cell suspension enters the mutagenesis area of the microbial mutagenesis breeding device, ultraviolet irradiation is performed on the mutagenesis area until the single-sorted cell suspension passes through the outlet of the second horizontal spiral conveying pipeline;

[0061] The protective agent includes calcium ions, trehalose and a solvent; the solvent is glycerol with a volume concentration of 50%.

[0062] In the present invention, a solution containing microorganisms is mixed with a protective agent to obtain a microbial solution; the protective agent includes calcium ions, trehalose and a solvent; the solvent is glycerol with a volume concentration of 50%. As an embodiment, the concentration of calcium ions in the microbial solution is 0.1 - 1 mmol / L, the concentration of trehalose is 30 - 40 mmol / L, and the volume ratio of the solution containing microorganisms to the solvent is 1:(1 - 2); as another embodiment, the concentration of calcium ions in the microbial solution is 1 mmol / L, the concentration of trehalose is 30 mmol / L, and the volume ratio of the solution containing microorganisms to the solvent is 1:2. By compounding calcium ions, trehalose and a solvent (glycerol with a volume concentration of 50%) and adjusting the dosages of calcium ions, trehalose and the solvent, the present invention can protect microorganisms, further improve the mutation effect of microorganisms, and obtain mutant strains with high homogeneity and high positive mutation rate.

[0063] As an embodiment, the number of microorganisms in the solution containing microorganisms is 2×10 8 cells / mL. The present invention has no strict requirements on the source of the solution containing microorganisms. The microorganisms can be cultured by conventional methods in the art to obtain the solution containing microorganisms.

[0064] After obtaining the microbial solution, the present invention passes the microbial solution into the first horizontal spiral conveying pipeline of the microbial mutagenesis breeding device for single sorting to obtain a single-sorted cell suspension. As an implementation manner, the rate of passing the microbial solution into the first horizontal spiral conveying pipeline is 0.1~0.3 μL / min. In the specific implementation process of the present invention, according to the specific type of microorganism, conventional selection can be carried out within the range of 0.1~0.3 μL / min. For example, when the microorganism is Escherichia coli, the flow rate can be 0.3 μL / min; when the microorganism is Corynebacterium glutamicum, the flow rate can be 0.3 μL / min; when the microorganism is Bacillus, the flow rate can be 0.1 μL / min; when the microorganism is yeast, the flow rate can be 0.2 μL / min.

[0065] When the single-sorted cell suspension enters the mutagenesis area of the microbial mutagenesis breeding device, ultraviolet irradiation is performed on the mutagenesis area. As an implementation manner, the power of the ultraviolet irradiation is 0.9~1.5 W. In the specific implementation process of the present invention, according to the specific type of microorganism, conventional selection can be carried out within the range of 0.9~1.5 W. For example, when the microorganism is Escherichia coli, the power of ultraviolet irradiation can be 0.9 W; when the microorganism is Corynebacterium glutamicum, the power of ultraviolet irradiation can be 1.2 W; when the microorganism is Bacillus, the power of ultraviolet irradiation can be 1.35 W; when the microorganism is yeast, the power of ultraviolet irradiation can be 1.5 W.

[0066] As an implementation manner, after the ultraviolet irradiation ends, the present invention screens the cell suspension irradiated by ultraviolet light to obtain microorganisms with positive mutations. As an implementation manner, flow cytometry is used for the screening. The present invention combines mutagenesis treatment and flow cytometry, which can further reduce the screening workload, shorten the screening cycle, and improve the mutagenesis and screening effects.

[0067] To further illustrate the present invention, the following describes in detail a microbial mutagenesis breeding device and its application provided by the present invention with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0068] The literature information referred to in the embodiments of the present invention is as follows:

[0069] Literature 1: Yang Cuiping. Research on Screening of High-yield L-lysine Strains Based on Artificial Rare Codons [D]. Qilu University of Technology, 2023.

[0070] Reference 2: Chen Y, Song W, Wang G, et al. Metabolic engineering of high L-lysine-producing Escherichia coli for de novo production of L-lysine-derived compounds[J]. ACS Synthetic Biology, 2024, 13(9): 2948-2959.

[0071] Reference 3: Cui Q, Liu H, Guo C, Wang J, Liu Y, Zhao Y, Wang R, Li P, Wang T, Wang J, Li N. Enhancing the erythritol production of Yarrowia lipolytica by high-throughput screening based on highly sensitive artificial sensor and anchor protein cwp2. J Ind Microbiol Biotechnol. 2024 Nov 23:kuae045.

[0072] Reference 4: Shi L, Lin Y, Song J, Li H, Gao Y, Lin Y, Huang X, Meng W, Qin W. Engineered Bacillus subtilis for the Production of Tetramethylpyrazine, (R,R)-2,3-Butanediol and Acetoin. Fermentation. 2023; 9(5):488.

[0073] Example 1

[0074] A microbial mutagenesis and breeding device consists of Figure 1 the shown transmission structure (slide) and Figure 2 the shown cubic structure. The length × width × height of the cubic structure is 0.5 cm × 0.5 cm × 5 cm, wrapped with light-impermeable materials (specifically, a fully light-shielding black film) around, and a UV lamp is set at the inner top ( Figure 1 shown as 1 in Figure 1as shown in Fig. 2; the transmission structure consists of a single sorting area and a mutagenesis area. The mutagenesis area is inserted into the cube structure through an insertion port and is arranged perpendicular to the ultraviolet lamp ( Figure 3 ), the area of the single sorting area is 0.5 cm × 0.5 cm, and the area of the mutagenesis area is 0.5 cm × 0.5 cm;

[0075] A first horizontal spiral conveying pipe is arranged inside the single sorting area. The number of spirals of the first horizontal spiral conveying pipe is 10 turns, the diameter of the pipe is 25 μm, and the inlet, outlet and center of the first horizontal spiral conveying pipe are on a straight line; the material of the first horizontal spiral conveying pipe is silica;

[0076] A second horizontal spiral conveying pipe and an ultraviolet lamp are arranged inside the mutagenesis area; the second horizontal spiral conveying pipe and the ultraviolet lamp are arranged perpendicular to each other, and the distance from the ultraviolet lamp to the microbial mutagenesis and breeding device is 5 cm; the number of spirals of the second horizontal spiral conveying pipe is 10 turns, the diameter of the pipe is 25 μm, the inlet, outlet and center of the second horizontal spiral conveying pipe; the material of the second horizontal spiral conveying pipe is silica;

[0077] The outlet of the first horizontal spiral conveying pipe and the inlet of the second horizontal spiral conveying pipe are connected through a horizontal pipe; the first horizontal spiral conveying pipe and the second horizontal spiral conveying pipe are arranged in parallel; the distance between the center of the first horizontal spiral conveying pipe and the center of the second horizontal spiral conveying pipe is 0.8 cm;

[0078] Among them, Figure 1 The left part is the single sorting area, the intersection point of the red arrows is the center position of the first horizontal spiral conveying pipe, and the blue arrow indicates the feeding direction; Figure 1 The right part is the mutagenesis area, and the blue arrow indicates the discharging direction.

[0079] Example 2

[0080] Effect of different calcium ion concentrations on the mutagenic lethality rate and positive mutation rate of Escherichia coli

[0081] 1. Preparation of recombinant bacteria

[0082] Referring to Reference 1, the fluorescently labeled plasmid was introduced into Escherichia coli W07 to obtain recombinant bacteria.

[0083] 2. Preparation of cell solution

[0084] Referring to Reference 2, the recombinant bacteria were cultured to an OD 600 of 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step 3 - 4 times to prepare a cell suspension with a concentration of 2×10 8CFU / mL. Calcium chloride was added to the cell suspension until the calcium ion reached the target concentration (0.1 mmol / L, 1 mmol / L, 10 mmol / L, 1000 mmol / L) to obtain a cell solution, and the cell mixture without added calcium ions was used as a control;

[0085] 3. Mutagenesis treatment

[0086] Using the power device, the cell solution was pumped into the first feed port of the microbial mutagenesis breeding device in Example 1 at a rate of 0.3 μL / min to turn the microorganisms in the cell solution into single cells; the power of the ultraviolet lamp was adjusted to 0.9 W, and the mutagenesis area in the microbial mutagenesis breeding device was irradiated with ultraviolet light until the cell solution flowed out through the outlet of the second horizontal spiral conveyor pipe.

[0087] 4. Detection of lethality rate and positive mutation rate

[0088] (1) Detection of lethality rate

[0089] Take 100 μL of the cell solution after mutagenesis in step 3 and coat it on the LB solid medium containing ampicillin resistance, and calculate the lethality rate according to the following formula. The results are shown in Table 1 and Figure 4 as shown.

[0090] ;

[0091] (2) Detection of positive mutation rate

[0092] Continue to culture the cell solution after mutagenesis in step 3 in the manner of referring to Document 2 until the OD 600 value is 2.0 - 2.3. Refer to Document 1 for flow cytometry sorting, and screen out the microbial cells with high fluorescence intensity into a 96-well plate. Refer to the method of Document 2 to ferment for 48 - 96 h to detect the yield of 5-hydroxyvaleric acid, and calculate the positive mutation rate according to the following formula. The results are shown in Table 1 and Figure 4 as shown;

[0093] ;

[0094] Among them, since according to the method of Document 2, after the end of the culture of Escherichia coli W07 in a 100 mL LB liquid shake flask containing ampicillin resistance, the yield of 5-hydroxyvaleric acid was detected by liquid chromatography to be 10.91 g / L. Therefore, strains with a 5-hydroxyvaleric acid yield less than 9 g / L were set as negative mutation strains, strains with a 5-hydroxyvaleric acid yield of 9 - 11 g / L were set as normal strains, and strains with a 5-hydroxyvaleric acid yield greater than 11 g / L were set as positive mutation strains.

[0095] Table 1 Lethality rate and positive mutation rate of Escherichia coli under different calcium ion concentrations

[0096]

[0097] According to Table 1 and Figure 4 it can be seen that when the calcium ion concentration in the bacterial cell solution is 1 mmol / L, the lethality of Escherichia coli is significantly reduced, significantly lower than that of other treatment concentrations; the forward mutation rate is significantly increased, significantly higher than that of other treatment concentrations; and when the calcium ion concentration in the bacterial cell solution is 1 mmol / L, the forward mutation rate increases from 0.0% to 15.0%, indicating that the mutagenesis direction and the ultraviolet mutagenesis intensity received by each cell are consistent, indicating good mutagenesis uniformity.

[0098] Example 3

[0099] Effect of different trehalose concentrations on the mutagenic lethality and forward mutation rate of Escherichia coli

[0100] 1. Referring to Document 2, the recombinant bacteria obtained in Example 2 were cultured to an OD 600 of 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step 3 to 4 times to prepare a bacterial cell suspension with a concentration of 2×10 8 cells / mL. Add trehalose to the bacterial cell suspension to the target concentrations (10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L) to obtain a bacterial cell solution, and use the bacterial mixture without added trehalose as a control.

[0101] 2. Perform mutagenesis treatment according to the steps of Example 2, and count the mutagenic lethality and forward mutation rate. The results are shown in Table 2 and Figure 5 as follows.

[0102] Table 2 Lethality and forward mutation rate of Escherichia coli at different trehalose concentrations

[0103]

[0104] According to Table 2 and Figure 5 it can be seen that when the trehalose concentration in the bacterial cell solution is 30 mmol / L, the lethality of Escherichia coli is significantly reduced, significantly lower than that of other treatment concentrations; the forward mutation rate is significantly increased, significantly higher than that of other treatment concentrations.

[0105] Example 4

[0106] Effect of different 50% v / v glycerol addition ratios on the mutagenic lethality and forward mutation rate of Escherichia coli

[0107] 1. Referring to Document 2, the recombinant bacteria obtained in Example 2 were cultured to an OD 600is 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step 3 - 4 times to prepare a bacterial cell suspension with a concentration of 2×10 8 cells / mL. Add 50% v / v glycerol to the bacterial cell suspension to the target ratios (bacterial cell suspension: 50% v / v glycerol is 1:0, 1:0.5, 1:1, 1:2) to obtain bacterial cell solutions, and use the bacterial mixture without adding 50% v / v glycerol as a control.

[0108] 2. Carry out mutagenesis treatment according to the steps of Example 2, and count the mutagenesis lethality rate and positive mutation rate. The results are shown in Table 3 and Figure 6 as follows.

[0109] Table 3 Lethality rate and positive mutation rate of Escherichia coli under different addition ratios of 50% v / v glycerol

[0110]

[0111] According to Table 3 and Figure 6 it can be seen that when the volume ratio of the bacterial cell suspension to 50% v / v glycerol in the bacterial cell solution is 1:2, the lethality rate of Escherichia coli decreases significantly, significantly lower than other treatment concentrations; the positive mutation rate increases significantly, significantly higher than other treatment concentrations.

[0112] Example 5

[0113] Effects of different protectants on mutagenesis lethality rate and positive mutation rate of Escherichia coli

[0114] 1. Referring to Document 2, culture the recombinant bacteria obtained in Example 2 until the OD 600 is 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step 3 - 4 times to prepare a bacterial cell suspension with a concentration of 2×10 8 cells / mL. Add different protectants (Protectant 1, 2, 3, 4, 5, 6, and 7) to the bacterial cell suspension to obtain bacterial cell solutions, and use the bacterial cell suspension without adding protectants as a control;

[0115] Among them, Protectant 1 is calcium chloride, and the final concentration of calcium ions in the bacterial cell solution is 1 mmol / L;

[0116] Protectant 2 is trehalose, and the final concentration in the bacterial cell solution is 30 mmol / L;

[0117] Protectant 3 is 50% v / v glycerol, and the volume ratio of the bacterial cell suspension to 50% v / v glycerol in the bacterial cell solution is 1:2;

[0118] The cryoprotectant 4 is calcium chloride and trehalose. The final concentration of calcium ions in the cell solution is 1 mmol / L, and the final concentration of trehalose is 30 mmol / L;

[0119] The cryoprotectant 5 is a composition of calcium chloride and glycerol with a volume concentration of 50%. The final concentration of calcium ions in the cell solution is 1 mmol / L, and the volume ratio of the cell suspension to glycerol with a volume concentration of 50% is 1:2;

[0120] The cryoprotectant 6 is a composition of trehalose and glycerol with a volume concentration of 50%. The final concentration of trehalose in the cell solution is 30 mmol / L, and the volume ratio of the cell suspension to glycerol with a volume concentration of 50% is 1:2;

[0121] The cryoprotectant 7 is a composition of calcium chloride, trehalose and glycerol with a volume concentration of 50%. The final concentration of calcium ions in the cell solution is 1 mmol / L, the final concentration of trehalose is 30 mmol / L, and the volume ratio of the cell suspension to glycerol with a volume concentration of 50% is 1:2.

[0122] 2. Perform mutagenesis treatment according to the steps of Example 2, and count the mutagenesis lethality rate and forward mutation rate. The results are shown in Table 4 and Figure 7 as follows.

[0123] Table 4 Lethality rate and forward mutation rate of Escherichia coli under different cryoprotectants

[0124]

[0125] According to Table 4 and Figure 7 it can be seen that through the combined experiments of different cryoprotectants at the optimal concentrations, the combination of calcium ions, trehalose and 50% v / v glycerol can achieve the best effect, and it is significantly higher than the additive effect of each item, that is: a calcium ion concentration of 1 mmol / L, a trehalose concentration of 30 mmol / L, and a volume ratio of the cell suspension to 50% v / v glycerol of 1:2 can reduce the lethality rate of ultraviolet mutagenesis of Escherichia coli to 70.4% and increase the forward mutation rate to 54.0%.

[0126] Comparative Example 1

[0127] Cultivate the recombinant bacteria obtained in Example 2 to an OD 600 of 0.7 with reference to Document 2. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat the washing 3 - 4 times to prepare a cell suspension with a concentration of 2×10 8 cells / mL. The distance from the ultraviolet lamp to the cell suspension is 5 cm, adjust the power of the ultraviolet lamp to 0.9 W, and perform ultraviolet irradiation for 45 s.

[0128] Comparative Example 2

[0129] (1) Referring to Document 2, the recombinant bacteria obtained in Example 2 were cultured until the OD 600 reached 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step 3 to 4 times to prepare a bacterial cell suspension with a concentration of 2×10 8 cells / mL.

[0130] (2) Using the power device, the bacterial cell suspension was pumped into the first feed port of the microbial mutagenesis breeding device in Example 1 at a rate of 0.3 μL / min to turn the microorganisms in the bacterial cell solution into single cells; the power of the ultraviolet lamp was adjusted to 0.9 W, and the mutagenesis area in the microbial mutagenesis breeding device was irradiated with ultraviolet light until the bacterial cell solution flowed out through the outlet of the second horizontal spiral conveyor pipe.

[0131] Example 6

[0132] (1) Referring to Document 2, the recombinant bacteria obtained in Example 2 were cultured until the OD 600 reached 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step 3 to 4 times to prepare a bacterial cell suspension with a concentration of 2×10 8 cells / mL. Protective agent 7 (calcium chloride, trehalose, and solvent (glycerol with a volume concentration of 50%)) was added to the bacterial cell suspension to obtain a bacterial cell solution; among them, the final concentration of calcium ions in the bacterial cell solution was 1 mmol / L, the final concentration of trehalose was 30 mmol / L, and the volume ratio of the bacterial cell suspension to the solvent was 1:2.

[0133] (2) Using the power device, the bacterial cell suspension was pumped into the first feed port of the microbial mutagenesis breeding device in Example 1 at a rate of 30 μL / min to turn the microorganisms in the bacterial cell solution into single cells; the power of the ultraviolet lamp was adjusted to 4.8 W, and the mutagenesis area in the microbial mutagenesis breeding device was irradiated with ultraviolet light until the bacterial cell solution flowed out through the outlet of the second horizontal spiral conveyor pipe.

[0134] Test Example 1

[0135] After the mutagenesis in Comparative Examples 1 to 2 and Example 6 was completed, the mutagenized bacterial cell solutions were continuously cultured until the OD 600 value reached 2.3. Flow cytometry sorting was carried out with reference to Document 1, and the microbial bacterial cells with high fluorescence intensity were screened into 96-well plates. According to the method in Document 2, the yield of 5-hydroxyvaleric acid was detected by fermentation for 48 to 96 h, and the percentages of negative mutant strains (the yield of 5-hydroxyvaleric acid was less than 9 g / L), normal strains (the yield of 5-hydroxyvaleric acid was 9 to 11 g / L), and positive mutant strains (the yield of 5-hydroxyvaleric acid was greater than 11 g / L) were counted. The results are shown in Table 5 and Figure 8 as follows; among them,Figure 8 *** represents P <0.005.

[0136] Table 5 Detection results of forward mutations of Escherichia coli under different mutagenesis methods

[0137]

[0138] According to Table 5 and Figure 8 It can be seen that the forward mutation rate of Example 6 is significantly higher than that of Comparative Example 1 and Comparative Example 2, and the forward mutation rate of Comparative Example 2 is significantly higher than that of Comparative Example 1. Moreover, in Example 6, with the microbial mutagenesis breeding device, the microorganisms in the cell solution form single cells before mutagenesis, and the ultraviolet irradiation intensity and time received by each cell are the same, with good uniformity. Compared with Comparative Example 1, the use of the microbial mutagenesis breeding device in Example 6 significantly increases the forward mutation rate.

[0139] Example 7

[0140] Ultraviolet mutagenesis of yeast

[0141] 1. Preparation of recombinant bacteria

[0142] Referring to Document 1, the fluorescently labeled plasmid was introduced into Y. lipolytica 5-14-E 6 Deyd1 to obtain recombinant bacteria.

[0143] 2. Preparation of cell solution

[0144] Referring to Document 3, the recombinant bacteria were cultured until the OD 600 reached 0.7. 10 mL of the sample was taken, shaken well, centrifuged at low speed, the supernatant was discarded, washed with sterile water, and this step was repeated 3 - 4 times to prepare a cell suspension with a concentration of 2×10 8 cells / mL. Protective agent 7 (calcium chloride, trehalose, and solvent (glycerol with a volume concentration of 50%)) was added to the cell suspension to obtain the cell solution; among them, the final concentration of calcium ions in the cell solution was 1 mmol / L, the final concentration of trehalose was 30 mmol / L, and the volume ratio of the cell suspension to the solvent was 1:2.

[0145] 3. Mutagenesis treatment

[0146] Using the power device, the cell suspension was pumped into the first feed port of the microbial mutagenesis breeding device of Example 1 at a rate of 0.2 μL / min to turn the microorganisms in the cell solution into single cells; the power of the ultraviolet lamp was adjusted to 1.5 W, and the mutagenesis area in the microbial mutagenesis breeding device was irradiated with ultraviolet light for 45 s.

[0147] Comparative Example 3

[0148] According to Reference 2, the recombinant bacteria obtained in Example 7 were cultured until the OD 600 reached 0.7. 10 mL of the sample was taken, shaken well, centrifuged at low speed, the supernatant was discarded, washed with sterile water, and this step was repeated 3 - 4 times to prepare a bacterial cell suspension with a concentration of 2×10 8 cells / mL. The distance from the ultraviolet lamp to the bacterial cell suspension was 5 cm, the power of the ultraviolet lamp was adjusted to 1.5 W, and ultraviolet irradiation was carried out until the single-sorted cell suspension passed through the outlet of the second horizontal spiral conveyor pipe.

[0149] Test Example 2

[0150] After the mutagenesis of Comparative Example 3 and Example 7, the mutagenized bacterial cell solutions were respectively continuously cultured until the OD 600 value reached 2.5. According to Reference 1, flow cytometry sorting was carried out, and the microbial cells with high fluorescence intensity were screened into a 96-well plate. According to the method of Reference 3, the erythritol yield was detected by fermentation for 48 - 96 h, and the number of negative mutant strains (erythritol yield less than 110 g / L), normal strains (5-hydroxyvaleric acid yield of 110 - 113 g / L), and positive mutant strains (erythritol yield greater than 113 g / L) was counted. According to the formula in Example 2 below, the positive mutation rate was calculated. The results showed that the positive mutation rate of Comparative Example 3 was 18%, and the positive mutation rate of Example 7 was 49%.

[0151] Example 8

[0152] Ultraviolet mutagenesis of Corynebacterium glutamicum

[0153] 1. Preparation of recombinant bacteria

[0154] According to Reference 1, the fluorescence-labeled plasmid was introduced into Corynebacterium glutamicum C.glutamicum-Pargw W2 to obtain recombinant bacteria.

[0155] 2. Preparation of bacterial cell solution

[0156] According to Reference 1, the recombinant bacteria were cultured until the OD 600 reached 0.7. 10 mL of the sample was taken, shaken well, centrifuged at low speed, the supernatant was discarded, washed with sterile water, and this step was repeated, washed 3 - 4 times to prepare a bacterial cell suspension with a concentration of 2×10 8 cells / mL. A protective agent 7 (calcium chloride, trehalose, and a solvent (glycerol with a volume concentration of 50%)) was added to the bacterial cell suspension to obtain a bacterial cell solution; among them, the final concentration of calcium ions in the bacterial cell solution was 1 mmol / L, the final concentration of trehalose was 30 mmol / L, and the volume ratio of the bacterial cell suspension to the solvent was 1:2.

[0157] 3. Mutagenesis treatment

[0158] Using a power device, the microbial suspension was pumped into the first feed port of the microbial mutagenesis breeding device in Example 1 at a rate of 0.3 μL / min, and the microorganisms in the microbial solution were turned into single cells; the power of the ultraviolet lamp was adjusted to 1.2 W, and the mutagenesis area in the microbial mutagenesis breeding device was irradiated with ultraviolet light for 45 s.

[0159] Comparative Example 4

[0160] Refer to Document 1 to culture the recombinant bacteria obtained in Example 8 until the OD 600 reached 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step 3 to 4 times to prepare a microbial suspension with a concentration of 2×10 8 cells / mL. The distance between the ultraviolet lamp and the microbial suspension was 5 cm, the power of the ultraviolet lamp was adjusted to 1.2 W, and ultraviolet irradiation was carried out for 45 s.

[0161] Test Example 3

[0162] After the mutagenesis of Comparative Example 4 and Example 8 was completed, the mutagenized microbial solutions were continuously cultured until the OD 600 value reached 2.3. Refer to Document 1 for flow cytometry sorting, and screen out the microbial cells with high fluorescence intensity into a 96-well plate. Refer to the method in Document 1 to ferment for 48 - 96 h to detect the lysine yield, and count the number of negative mutation strains (lysine yield less than 16.00 g / L), normal strains (lysine yield of 16.00 - 17.00 g / L), and positive mutation strains (erythritol yield greater than 7.00 g / L). Calculate the positive mutation rate according to the formula in Example 2 below. The results show that the positive mutation rate of Example 8 was 51%, compared with 23% of Comparative Example 4.

[0163] Example 9

[0164] Ultraviolet mutagenesis of Bacillus subtilis

[0165] 1. Preparation of recombinant bacteria

[0166] Refer to Document 1 to introduce the fluorescently labeled plasmid into Bacillus subtilis BS-ppb 11 to obtain recombinant bacteria.

[0167] 2. Preparation of microbial solution

[0168] Refer to Document 4 to culture the recombinant bacteria until the OD 600 reached 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step, wash 3 to 4 times, to prepare a microbial suspension with a concentration of 2×10 8CFU / mL. Protective agent 7 (calcium chloride, trehalose and a solvent (glycerol with a volume concentration of 50%)) was added to the cell suspension to obtain a cell solution; wherein, the final concentration of calcium ions in the cell solution was 1 mmol / L, the final concentration of trehalose was 30 mmol / L, and the volume ratio of the cell suspension to the solvent was 1:2.

[0169] 3. Mutagenesis treatment

[0170] Using a power device, the cell suspension was pumped into the first feed port of the microbial mutagenesis breeding device of Example 1 at a rate of 0.1 μL / min to turn the microorganisms in the cell solution into single cells; the power of the ultraviolet lamp was adjusted to 1.35 W, and the mutagenesis area in the microbial mutagenesis breeding device was irradiated with ultraviolet light for 45 s.

[0171] Comparative Example 5

[0172] Refer to Document 4 to culture the recombinant bacteria obtained in Example 8 until the OD 600 reached 0.7. Take 10 mL of the sample, shake it well, centrifuge it at low speed, discard the supernatant, wash it with sterile water, and repeat this step 3-4 times to prepare a cell suspension with a concentration of 2×10 8 CFU / mL. The distance from the ultraviolet lamp to the cell suspension was 5 cm, the power of the ultraviolet lamp was adjusted to 1.35 W, and ultraviolet irradiation was carried out until the single-sorted cell suspension passed through the outlet of the second horizontal spiral conveying pipe.

[0173] Test Example 4

[0174] After the mutagenesis in Comparative Example 5 and Example 9 was completed, the mutagenized cell solutions were continuously cultured until the OD 600 value reached 2.7. Refer to Document 1 for flow cytometry sorting, and screen out the microbial cells with high fluorescence intensity into a 96-well plate. Refer to the method in Document 4 to ferment for 48-96 h to detect the acetoin yield, and count the number of negative mutant strains (the acetoin yield is less than 50 g / L), normal strains (the acetoin yield is 50-55 g / L) and positive mutant strains (the acetoin yield is greater than 55 g / L). Calculate the positive mutation rate according to the formula in Example 2 below. The results show that the positive mutation rate of Comparative Example 5 was 16%, and the positive mutation rate of Example 9 was 39%.

[0175] It can be seen from the above content that the technical solution provided by the present invention can improve the mutation effect of microorganisms, significantly reduce the lethality of contemporary strains, increase the positive mutation rate, obtain mutant strains with high homogeneity and high positive mutation rate, and has simple operation, small workload and short cycle.

[0176] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for microbial mutagenesis, characterized in that, Using a microbial mutation breeding device to perform ultraviolet mutagenesis treatment on microorganisms, including the following steps: Mix a solution containing microorganisms with a protective agent to obtain a microbial solution; Pass the microbial solution through the first horizontal spiral conveying pipeline of the microbial mutation breeding device for single sorting to obtain a singly sorted cell suspension; When the singly sorted cell suspension enters the mutagenesis area of the microbial mutation breeding device, irradiate the mutagenesis area with ultraviolet light until the singly sorted cell suspension passes through the outlet of the second horizontal spiral conveying pipeline; The protective agent includes calcium ions, trehalose, and a solvent; the solvent is glycerol with a volume concentration of 50%; The concentration of calcium ions in the microbial solution is 1 mmol / L, the concentration of trehalose is 30 mmol / L, and the volume ratio of the solution containing microorganisms to the solvent is 1:2; The microbial mutation breeding device includes a cubic structure and a transmission structure. The cubic structure is wrapped with light-proof materials around, and ultraviolet lamps are arranged inside; The transmission structure includes a single sorting area and a mutagenesis area. The mutagenesis area is inserted into the cubic structure through the insertion opening of the cubic structure and is arranged perpendicular to the ultraviolet lamp; The single sorting area is provided with a first horizontal spiral conveying pipeline; the mutagenesis area is provided with a second horizontal spiral conveying pipeline; The outlet of the first horizontal spiral conveying pipeline and the inlet of the second horizontal spiral conveying pipeline are connected through a horizontal pipeline; the first horizontal spiral conveying pipeline and the second horizontal spiral conveying pipeline are arranged in parallel; The number of spirals of the first horizontal spiral conveying pipeline is ≥10 turns, and the diameter of the pipeline is 20 - 30 μm; The number of spirals of the second horizontal spiral conveying pipeline is ≥10 turns, and the diameter of the pipeline is 20 - 30 μm.

2. The microbial mutagenesis method according to claim 1, characterized in that, The distance between the center of the first horizontal spiral conveying pipeline and the center of the second horizontal spiral conveying pipeline is 0.8 cm.

3. The microbial mutagenesis method according to claim 1, characterized in that, The inlet, outlet, and center of the first horizontal spiral conveying pipeline are on a straight line; The inlet, outlet, and center of the second horizontal spiral conveying pipeline are on a straight line.

4. The microbial mutagenesis method according to claim 1, characterized in that, The areas of the single sorting area and the mutagenesis area are 0.5 cm × 0.5 cm respectively.

5. The microbial mutagenesis method according to any one of claims 1 to 4, characterized in that, The materials of the first horizontal spiral conveying pipeline, the second horizontal spiral conveying pipeline, and the horizontal pipeline are silica respectively.

6. The microbial mutagenesis method according to claim 1, characterized in that The number of microorganisms in the solution containing microorganisms is 2×10 8 CFU / mL.

7. The microbial mutagenesis method according to claim 1, characterized in that, The rate at which the microbial solution passes through the first horizontal spiral conveying pipeline is 0.1 - 0.3 μL / min; the power of the ultraviolet irradiation is 0.9 - 1.5 W.

8. The microbial mutagenesis method according to claim 1, characterized in that, After the ultraviolet irradiation, it further includes: screening the cell suspension that has undergone ultraviolet irradiation to obtain microorganisms with positive mutations.

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