Microorganism mutation breeding device and application thereof

Abstract

The invention belongs to the technical field of microbial mutagenesis, and particularly relates to a microbial mutagenesis breeding device and application thereof. The microorganism mutation breeding device comprises a cube structure and a transmission structure, the periphery of the cube structure is wrapped with a light-proof material, and an ultraviolet lamp is arranged in the cube structure; the transmission structure comprises a single sequencing area and a mutagenesis area, and the mutagenesis area is inserted into the cube structure through the insertion opening of the cube structure and is perpendicular to the ultraviolet lamp. Microorganisms can be singly sorted through the first horizontal spiral conveying pipeline in the single sorting area and conveyed to the second horizontal spiral conveying pipeline in the mutagenesis area, so that the time and the intensity of ultraviolet irradiation of the microorganisms are uniform, the mutagenesis fatality rate of the microorganisms is remarkably reduced, the forward mutation rate is remarkably increased, and the mutagenesis rate of the microorganisms is remarkably increased. Operation is simple, workload is small, and period is short. In addition, the microorganisms are treated by using a protective agent with specific composition before single sequencing, so that the mutation uniformity and the forward mutation rate can be further improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial mutagenesis, and in particular relates to a microbial mutagenesis breeding device and application thereof. Background Art

[0002] Microbial mutagenesis is one of the main methods of microbial breeding. Chemical mutagenesis and physical mutagenesis play an important role in strain selection and transformation. Ultraviolet light is one of the most commonly used physical mutagenic factors. Ultraviolet light irradiation causes the DNA double strands or two adjacent thymines on the same strand to form dimers, which hinders the separation, replication and normal pairing of double strands, thus causing mutations.

[0003] However, existing ultraviolet mutagenesis methods usually use ultraviolet rays to directly act on the plates where microorganisms grow or on the prepared suspensions. This cannot ensure that each cell is induced to undergo the same mutation, which usually results in poor uniformity of the microorganisms after mutagenesis and a low forward mutation rate, 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 uniformity and positive mutation rate of microorganisms after mutagenesis, and has simple operation, small workload and short cycle.

[0005] In order to achieve the above-mentioned object, the present invention provides a microorganism mutation breeding device, which comprises a cubic structure and a transmission structure. The cubic structure is surrounded by an opaque material and an ultraviolet lamp is arranged inside. The transmission structure includes a single sorting area and a mutagenesis area, and the mutagenesis area is inserted into the cube structure through the insertion port of the cube structure and is arranged vertically with 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, and the diameter of the pipeline is 20-30 μm; The number of spirals of the second horizontal spiral conveying pipeline is ≥10, and the diameter of the pipeline is 20-30 μm.

[0006] 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.

[0007] Preferably, the inlet, outlet and center of the first horizontal spiral conveying pipeline are located in a straight line; The inlet, outlet and center of the second horizontal spiral conveying pipeline are located in a straight line.

[0008] Preferably, the areas of the single sequencing region and the mutagenesis region are 0.5 cm×0.5 cm respectively.

[0009] Preferably, the first horizontal spiral conveying pipe, the second horizontal spiral conveying pipe and the horizontal pipe are respectively made of silicon dioxide.

[0010] The present invention also provides the use of the microorganism mutation breeding device described in the above technical solution in microorganism mutation and / or microorganism breeding.

[0011] The present invention also provides a microbial mutagenesis method, which uses the microbial mutagenesis breeding device described in the above technical solution to perform ultraviolet mutagenesis treatment on microorganisms, comprising the following steps: mixing a solution containing microorganisms with a protective agent to obtain a microorganism solution; Passing the microbial solution into the first horizontal spiral conveying pipe of the microbial mutation breeding device for single sorting to obtain a single sorted cell suspension; When the single-sorted cell suspension enters the mutagenesis zone of the microbial mutagenesis breeding device, ultraviolet irradiation is applied to the mutagenesis zone until the single-sorted cell suspension passes through the outlet of the second horizontal spiral conveying pipe; The protective agent comprises calcium ions, trehalose and a solvent; the solvent is glycerol with a volume concentration of 50%.

[0012] 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 the microorganisms to the solvent is 1:(1-2); The number of microorganisms in the solution containing microorganisms is 2×10 8 Pieces / mL.

[0013] Preferably, the rate at which the microbial solution is introduced into the first horizontal spiral conveying pipeline is 0.1-0.3 μL / min; and the power of the ultraviolet irradiation is 0.9-1.5 W.

[0014] Preferably, after the ultraviolet irradiation, the method further comprises: screening the ultraviolet irradiated cell suspension to obtain microorganisms with positive mutations.

[0015] Beneficial effects: The invention provides a microorganism mutation breeding device, which comprises a cube structure and a transmission structure. The cube structure is wrapped with opaque material on all sides and an ultraviolet lamp is arranged inside. The transmission structure comprises a single sorting area and a mutagenesis area. The mutagenesis area is inserted into the cube structure through an insertion port of the cube structure and is arranged vertically with 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 and an ultraviolet lamp. The second horizontal spiral conveying pipeline and the ultraviolet lamp are arranged vertically. The outside of the mutagenesis area is wrapped with opaque material. 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 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 the microorganisms are sorted in a single manner 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 is ensured that the time and intensity of ultraviolet light irradiation of the microorganisms are uniform and consistent, thereby achieving accurate mutagenesis of single-cell microorganisms. The results of the embodiment show that the lethality of 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 when the microorganisms are mutagenized using the microbial mutagenesis breeding device of the present invention.

[0016] The present invention mixes a solution containing microorganisms with a protective agent, and by limiting the composition and concentration of the protective agent, the mutation effect can be further improved to obtain mutant microorganisms with high uniformity and high positive mutation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the literature, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 It is a top view of the transmission structure in the microbial mutation breeding device of Example 1; Figure 2 This is a schematic diagram of the cubic structure of the microbial mutation breeding device in Example 1; wherein 1 is an ultraviolet lamp, and 2 is an insertion port; Figure 3 This is a schematic diagram of the microbial mutation breeding device of Example 1; wherein 1 is a cube structure, and 2 is a transmission structure; Figure 4 This is the relationship between the lethality and forward mutation rate of E. coli induced by different calcium ion concentrations; Figure 5This is the relationship between the lethality and forward mutation rate of E. coli induced by different trehalose concentrations; Figure 6 This is the relationship between the lethality and forward mutation rate of E. coli induced by different proportions of 50% v / v glycerol; Figure 7 This is a graph showing the relationship between the lethality and positive mutation rate of E. coli induced by different protective agents; Figure 8 This is the proportional distribution diagram of the mutation direction of Escherichia coli after different mutagenesis methods. DETAILED DESCRIPTION

[0019] The present invention provides a microorganism mutation breeding device, which comprises a cubic structure and a transmission structure. The cubic structure is surrounded by opaque materials and has an ultraviolet lamp arranged inside. The transmission structure includes a single sorting area and a mutagenesis area, and the mutagenesis area is inserted into the cube structure through the insertion port of the cube structure and is arranged vertically with 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 and an ultraviolet lamp; 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, and the diameter of the pipeline is 20-30 μm; the number of spirals of the second horizontal spiral conveying pipeline is ≥10, and the diameter of the pipeline is 20-30 μm.

[0020] As an embodiment, the length×width×height of the cube structure of the present invention is 0.5 cm×0.5 cm×5 cm. As an embodiment, the ultraviolet lamp of the present invention is located at the top of the cube structure. As an embodiment, an insertion port is set on the side of the cube structure of the present invention, and the distance from the insertion port to the ground is 1.2 mm. The mutagenesis zone is inserted into the cube structure through the insertion port and is arranged vertically with the ultraviolet lamp. By limiting the position 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 pipeline and the ultraviolet lamp is 5 cm, it is only necessary to adjust the power parameters and the flow rate of the microbial solution, which is convenient and quick.

[0021] As an implementation mode, 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 light is irradiated, the second horizontal spiral conveying pipeline is in a dark environment, which has the function of isolating the external light environment and preventing the microorganisms from photoreactivating after ultraviolet irradiation, and at the same time can avoid harm to the human body during ultraviolet irradiation.

[0022] As an embodiment, the present invention randomly divides the slide into two areas, engraves the first horizontal spiral conveying conduit on one area to form a single sorting area, and engraves the second horizontal spiral conveying conduit on the other area to form a mutagenesis area.

[0023] As an embodiment, the first horizontal spiral conveying pipe, the second horizontal spiral conveying pipe and the horizontal pipe of the present invention are made of silicon dioxide. As an embodiment, the line connecting the center points 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 a material, and silicon dioxide does not absorb ultraviolet rays, which can improve the effect of ultraviolet mutagenesis.

[0024] As an embodiment, the area of ​​the single sequencing region of the present invention is 0.5 cm×0.5 cm. As an embodiment, the area of ​​the mutagenesis region of the present invention is 0.5 cm×0.5 cm.

[0025] As an embodiment, 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.

[0026] As an embodiment, the number of spirals of the first horizontal spiral conveying pipeline of the present invention is 10. As an embodiment, the size of the first horizontal spiral conveying pipeline of the present invention is 25 μm. By limiting the number of spirals and the diameter of the first horizontal spiral conveying pipeline, the present invention can ensure that the microorganisms complete a single sorting and ensure that the time and intensity of ultraviolet irradiation of the microorganisms entering the second horizontal spiral conveying pipeline of the mutagenesis area are uniform.

[0027] As an implementation manner, the distance between adjacent spirals of the first horizontal spiral conveying pipe is 20-30 μm; as an implementation manner, the distance between adjacent spirals of the first horizontal spiral conveying pipe is 25 μm.

[0028] As an embodiment, the inlet, outlet and center of the first horizontal spiral conveying pipeline of the present invention are located on a straight line.

[0029] As an embodiment, the number of spirals of the second horizontal spiral conveying conduit of the present invention is 10. As an embodiment, the diameter of the second horizontal spiral conveying conduit of the present invention is 25 μm. By limiting the number of spirals and the diameter of the second horizontal spiral conveying conduit, the present invention can ensure that the time and intensity of ultraviolet light irradiation of microorganisms are uniform and consistent, thereby achieving precise mutagenesis of single-cell microorganisms.

[0030] As an implementation manner, the distance between adjacent spirals of the second horizontal spiral conveying conduit is 20-30 μm; as an implementation manner, the distance between adjacent spirals of the second horizontal spiral conveying conduit is 25 μm.

[0031] As an embodiment, the inlet, outlet and center of the second horizontal spiral conveying pipeline of the present invention are located on a straight line.

[0032] The microbial mutagenesis breeding device provided by the present invention has a simple structure. Microorganisms can form single-sorted cells by passing through the first horizontal spiral conveying pipeline in the single-sorting area. A second horizontal spiral conveying pipeline is arranged in the mutagenesis area, and 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 forward mutation rate is high.

[0033] In view of the advantages of the microorganism mutation breeding device of the present invention, the application of the microorganism mutation breeding device in microorganism mutation and / or microorganism breeding also belongs to the protection scope of the present invention. The present invention has no strict requirements on the type of the microorganism, and conventional microorganisms in the field can be used, for example, any one or more of Escherichia coli, Corynebacterium cerebri, Bacillus and yeast.

[0034] The present invention also provides a microbial mutagenesis method, which uses the microbial mutagenesis breeding device described in the above technical solution to perform ultraviolet mutagenesis treatment on microorganisms, comprising the following steps: mixing a solution containing microorganisms with a protective agent to obtain a microorganism solution; Passing the microbial solution into the first horizontal spiral conveying pipe of the microbial mutation breeding device for single sorting to obtain a single sorted cell suspension; When the single-sorted cell suspension enters the mutagenesis zone of the microbial mutagenesis breeding device, ultraviolet irradiation is applied to the mutagenesis zone until the single-sorted cell suspension passes through the outlet of the second horizontal spiral conveying pipe; The protective agent comprises calcium ions, trehalose and a solvent; the solvent is glycerol with a volume concentration of 50%.

[0035] The present invention mixes a solution containing microorganisms with a protective agent to obtain a microorganism 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 microorganism 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 microorganism solution is 1mmol / 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 present invention utilizes calcium ions, trehalose and solvent (glycerol with a volume concentration of 50%) to compound, adjust the dosage of calcium ions, trehalose and solvent, can protect microorganisms, further improve the mutation effect of microorganisms, and obtain mutant strains with high uniformity and high positive mutation rate.

[0036] As an embodiment, the number of microorganisms in the solution containing microorganisms is 2×10 8 The present invention has no strict requirements on the source of the solution containing microorganisms, and the solution containing microorganisms can be obtained by culturing microorganisms in a conventional manner in the art.

[0037] 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 embodiment, the rate at which the microbial solution is passed 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, a conventional selection can be made 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 cerebrospinal fluid, 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.

[0038] When the single-sorted cell suspension enters the mutagenesis zone of the microbial mutagenesis breeding device, the mutagenesis zone is subjected to ultraviolet irradiation. As an embodiment, the power of the ultraviolet irradiation is 0.9~1.5 W. In the specific implementation of the present invention, according to the specific type of microorganism, a conventional selection can be made 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.

[0039] As an embodiment, after the ultraviolet irradiation is completed, the present invention screens the cell suspension subjected to ultraviolet irradiation to obtain microorganisms with positive mutations. As an embodiment, the screening uses flow cytometry. The present invention combines mutagenesis treatment with flow cytometry, which can further reduce the workload of screening, shorten the screening cycle, and improve the effects of mutagenesis and screening.

[0040] In order to further illustrate the present invention, a microbial mutation breeding device and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] The literature information referenced by the embodiments of the present invention is as follows: Reference 1: Yang Cuiping. Research on screening of high-yield strains of L-lysine based on artificial rare codons[D]. Qilu University of Technology, 2023. Document 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-derivedcompounds[J]. ACS Synthetic Biology, 2024, 13(9): 2948-2959. Literature 3: Cui Q, Liu H, Guo C, Wang J, Liu Y, Zhao Y, Wang R, Li P, WangT, Wang J, Li N. Enhancing the erythritol production of Yarrowia lipolyticaby high-throughput screening based on highly sensitive artificial sensor andanchor protein cwp2. J Ind Microbiol Biotechnol. 2024 Nov 23:kuae045. Document 4: Shi L, Lin Y, Song J, Li H, Gao Y, Lin Y, Huang X, Meng W, QinW. Engineered Bacillus subtilis for the Production of Tetramethylpyrazine,(R,R)-2,3-Butanediol and Acetoin. Fermentation. 2023; 9(5):488. Example 1 A microbial mutation breeding device, comprising Figure 1 The transport structure shown (glass slide) and Figure 2 The cube structure is composed of a length × width × height of 0.5 cm × 0.5 cm × 5 cm, and is surrounded by opaque material (specifically, a full-light-shielding black film), and an ultraviolet lamp is set on the top of the interior ( Figure 1 As shown in Figure 1, an insertion port is set at 1.2 mm from the bottom on the side ( Figure 1 2); the transmission structure is composed of a single sorting region and a mutagenesis region, the mutagenesis region is inserted into the cubic structure through the insertion port, and is arranged vertically with 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; A first horizontal spiral conveying pipeline is arranged inside the single sorting area, the spiral number of the first horizontal spiral conveying pipeline is 10, the diameter of the pipeline is 25 μm, the inlet, outlet and center of the first horizontal spiral conveying pipeline are located in a straight line; the material of the first horizontal spiral conveying pipeline is silicon dioxide; A second horizontal spiral conveying pipeline and an ultraviolet lamp are arranged inside the mutagenesis zone; the second horizontal spiral conveying pipeline and the ultraviolet lamp are arranged vertically, and the distance from the ultraviolet lamp to the microbial mutagenesis breeding device is 5 cm; the number of spirals of the second horizontal spiral conveying pipeline is 10, the diameter of the pipeline is 25 μm, and the inlet, outlet and center of the second horizontal spiral conveying pipeline; the material of the second horizontal spiral conveying pipeline is silicon dioxide; 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 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; in, Figure 1 The left part is a single sorting area, the intersection of the red arrows is the center of the first horizontal spiral conveying pipeline, and the blue arrow indicates the feeding direction; Figure 1The right part is the mutagenesis area, and the blue arrow indicates the discharge direction.

[0042] Example 2 Effects of different calcium ion concentrations on lethality and forward mutation rate of Escherichia coli 1. Preparation of recombinant bacteria Referring to document 1, the fluorescent marker plasmid was introduced into Escherichia coli W07 to obtain recombinant bacteria.

[0043] 2. Preparation of bacterial solution Refer to reference 2 to culture the recombinant bacteria to OD 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 Calcium chloride was added to the bacterial suspension until the calcium ion concentration reached the target (0.1 mmol / L, 1 mmol / L, 10 mmol / L, 1000 mmol / L) to obtain a bacterial solution, and a bacterial mixture without calcium ion addition was used as a control; 3. Mutagenesis Using a power device, the bacterial 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 convert the microorganisms in the bacterial 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 solution flowed out through the outlet of the second horizontal spiral conveying pipe.

[0044] 4. Detection of lethality and positive mutation rates (1) Detection of mortality rate Take 100 μL of the mutagenic bacterial solution from step 3 and spread it on LB solid medium containing ampicillin resistance. The lethality rate was calculated according to the following formula. The results are shown in Table 1 and Figure 4 shown.

[0045] ; (2) Detection of positive mutation rate Continue to culture the mutagenized bacterial solution in step 3 according to the method of reference 2 until OD 600 The value was 2.0~2.3. Flow cytometry sorting was performed according to reference 1 to screen out microbial cells with high fluorescence intensity and put them into 96-well plates. According to the method of reference 2, the production of 5-hydroxyvaleric acid was detected after fermentation for 48~96 h. The forward mutation rate was calculated according to the following formula. The results are shown in Table 1 and Figure 4 As shown; ; Among them, according to the method of literature 2, after the Escherichia coli W07 was cultured in 100 mL LB liquid shake flask containing ampicillin resistance, the liquid chromatography detection showed that the production of 5-hydroxyvaleric acid was 10.91 g / L. Therefore, the strain with a production of 5-hydroxyvaleric acid less than 9 g / L was set as a negative mutant strain, the strain with a production of 5-hydroxyvaleric acid of 9~11 g / L was set as a normal strain, and the strain with a production of 5-hydroxyvaleric acid greater than 11 g / L was set as a positive mutant strain.

[0046] Table 1 The lethality and positive mutation rate of Escherichia coli at different calcium ion concentrations

[0047] According to Table 1 and Figure 4 It can be seen that when the calcium ion concentration in the bacterial solution is 1 mmol / L, the lethality of Escherichia coli is significantly reduced, significantly lower than other treatment concentrations; the forward mutation rate is significantly increased, significantly higher than other treatment concentrations; and when the calcium ion concentration in the bacterial solution is 1 mmol / L, the forward mutation rate increases from 0.0% to 15.0%, indicating that the direction of mutagenesis is consistent with the intensity of ultraviolet mutagenesis received by each cell, indicating that the uniformity of mutagenesis is good.

[0048] Example 3 Effects of different trehalose concentrations on lethality and positive mutation rate of Escherichia coli 1. Referring to Reference 2, the recombinant bacteria obtained in Example 2 were cultured to OD 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 Trehalose was added to the bacterial suspension to the target concentration (10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L) to obtain a bacterial solution, and a bacterial mixture without trehalose was used as a control.

[0049] 2. The mutagenesis treatment was carried out according to the steps of Example 2, and the mutagenesis lethality and forward mutation rate were statistically analyzed. The results are shown in Table 2 and Figure 5 shown.

[0050] Table 2 The lethality and positive mutation rate of Escherichia coli at different trehalose concentrations

[0051] According to Table 2 and Figure 5 It can be seen that when the trehalose concentration in the bacterial solution is 30 mmol / L, the lethality of Escherichia coli is significantly reduced, significantly lower than other treatment concentrations; the forward mutation rate is significantly increased, significantly higher than other treatment concentrations.

[0052] Example 4 Effects of different 50% v / v glycerol addition ratios on lethality and positive mutation rate of Escherichia coli 1. Referring to Reference 2, the recombinant bacteria obtained in Example 2 were cultured to OD 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 50% v / v glycerol was added to the bacterial suspension to the target ratio (bacterial suspension: 50% v / v glycerol was 1:0, 1:0.5, 1:1, 1:2) to obtain a bacterial solution, and a bacterial mixture without 50% v / v glycerol was used as a control.

[0053] 2. The mutagenesis treatment was carried out according to the steps of Example 2, and the mutagenesis lethality and forward mutation rate were statistically analyzed. The results are shown in Table 3 and Figure 6 shown.

[0054] Table 3 The lethality and positive mutation rate of Escherichia coli at different 50% v / v glycerol addition ratios

[0055] According to Table 3 and Figure 6 It can be seen that when the volume ratio of the bacterial suspension to 50% v / v glycerol in the bacterial solution is 1:2, the lethality of Escherichia coli is significantly reduced, significantly lower than other treatment concentrations; the forward mutation rate is significantly increased, significantly higher than other treatment concentrations.

[0056] Example 5 Effects of different protective agents on lethality and positive mutation rate of Escherichia coli 1. Referring to Reference 2, the recombinant bacteria obtained in Example 2 were cultured to OD 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 Different protective agents (protective agents 1, 2, 3, 4, 5, 6 and 7) were added to the bacterial suspension to obtain a bacterial solution, and a bacterial suspension without adding protective agents was used as a control; Among them, the protective agent 1 is calcium chloride, and the final concentration of calcium ions in the bacterial solution is 1 mmol / L; Protectant 2 was trehalose, with a final concentration of 30 mmol / L in the bacterial solution; Protective agent 3 is 50% v / v glycerol, and the volume ratio of bacterial suspension to 50% v / v glycerol in the bacterial solution is 1:2; Protective agent 4 is calcium chloride and trehalose, the final concentration of calcium ions in the bacterial solution is 1 mmol / L, and the final concentration of trehalose is 30 mmol / L; Protective agent 5 is a composition of calcium chloride and glycerol with a volume concentration of 50%, the final concentration of calcium ions in the bacterial solution is 1 mmol / L, and the volume ratio of the bacterial suspension to the glycerol with a volume concentration of 50% is 1:2; Protective agent 6 is a composition of trehalose and glycerol with a volume concentration of 50%, the final concentration of trehalose in the bacterial solution is 30 mmol / L, and the volume ratio of the bacterial suspension to the glycerol with a volume concentration of 50% is 1:2; Protective agent 7 is a composition of calcium chloride, trehalose and glycerol with a volume concentration of 50%. The final concentration of calcium ions in the bacterial solution is 1 mmol / L, the final concentration of trehalose is 30 mmol / L, and the volume ratio of the bacterial suspension to the glycerol with a volume concentration of 50% is 1:2.

[0057] 2. The mutagenesis treatment was carried out according to the steps of Example 2, and the mutagenesis lethality and forward mutation rate were statistically analyzed. The results are shown in Table 4 and Figure 7 shown.

[0058] Table 4 The lethality and positive mutation rate of Escherichia coli under different protective agents

[0059] According to Table 4 and Figure 7 It can be seen that after the combination experiment of different protective agents with optimal concentrations, the combination of calcium ions, trehalose and 50% v / v glycerol can achieve the best effect, and is significantly higher than the sum of each item, that is: 1 mmol / L calcium ion concentration, 30 mmol / L trehalose concentration, bacterial suspension and 50% v / v glycerol volume ratio of 1:2 can reduce the lethality of Escherichia coli induced by ultraviolet mutagenesis to 70.4%, and increase the forward mutation rate to 54.0%.

[0060] Comparative Example 1 Referring to document 2, the recombinant bacteria obtained in Example 2 were cultured to an OD of 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat washing 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 The distance between the UV lamp and the bacterial suspension was 5 cm, the UV lamp power was adjusted to 0.9 W, and UV irradiation was performed for 45 s.

[0061] Comparative Example 2 (1) Referring to Reference 2, the recombinant bacteria obtained in Example 2 were cultured to an OD of 600Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 Pieces / mL.

[0062] (2) Using a power device, the bacterial suspension is pumped into the first feed port of the microbial mutagenesis breeding device of Example 1 at a rate of 0.3 μL / min to convert the microorganisms in the bacterial solution into single cells; the power of the ultraviolet lamp is adjusted to 0.9 W to irradiate the mutagenesis area in the microbial mutagenesis breeding device with ultraviolet light until the bacterial solution flows out through the outlet of the second horizontal spiral conveying pipe.

[0063] Example 6 (1) Referring to Reference 2, the recombinant bacteria obtained in Example 2 were cultured to an OD of 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 Protective agent 7 (calcium chloride, trehalose and solvent (glycerol with a volume concentration of 50%)) was added to the bacterial suspension to obtain a bacterial solution; wherein the final concentration of calcium ions in the bacterial solution was 1 mmol / L, the final concentration of trehalose was 30 mmol / L, and the volume ratio of the bacterial suspension to the solvent was 1:2.

[0064] (2) Using a power device, the bacterial suspension is pumped into the first feed port of the microbial mutagenesis breeding device of Example 1 at a rate of 30 μL / min to convert the microorganisms in the bacterial solution into single cells; the power of the ultraviolet lamp is adjusted to 4.8 W, and the mutagenesis area in the microbial mutagenesis breeding device is irradiated with ultraviolet light until the bacterial solution flows out through the outlet of the second horizontal spiral conveying pipe.

[0065] Test Example 1 After the mutagenesis of Comparative Examples 1-2 and Example 6 was completed, the mutagenized bacterial solution was cultured until OD 600 The value was 2.3. Flow cytometry sorting was performed according to reference 1 to screen out microorganisms with high fluorescence intensity and put them into 96-well plates. According to the method of reference 2, the production of 5-hydroxyvaleric acid was detected after fermentation for 48-96 h. The percentages of negative mutant strains (5-hydroxyvaleric acid production less than 9 g / L), normal strains (5-hydroxyvaleric acid production of 9-11 g / L) and positive mutant strains (5-hydroxyvaleric acid production greater than 11 g / L) were counted. The results are shown in Tables 5 and 6. Figure 8 As shown; among them, Figure 8 In Chinese, *** means P <0.005.

[0066] Table 5 Forward mutation detection results of Escherichia coli under different mutagenesis methods

[0067] 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. In addition, Example 6 uses a microbial mutagenesis breeding device, and the microorganisms in the bacterial solution are induced to form a single cell, and the ultraviolet irradiation intensity and time received by each cell are the same, and the uniformity is good. Compared with Comparative Example 1, Example 6 uses a microbial mutagenesis breeding device, and the forward mutation rate is significantly improved.

[0068] Example 7 UV mutagenesis of yeast 1. Preparation of recombinant bacteria Referring to document 1, a fluorescent marker plasmid was introduced into the yeast Y. lipolytica 5-14-E 6 Deyd1 to obtain a recombinant bacterium.

[0069] 2. Preparation of bacterial solution Refer to reference 3 and culture the recombinant bacteria to OD 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step, wash 3 to 4 times, and prepare a bacterial suspension with a concentration of 2×10 8 Protective agent 7 (calcium chloride, trehalose and solvent (glycerol with a volume concentration of 50%)) was added to the bacterial suspension to obtain a bacterial solution; wherein the final concentration of calcium ions in the bacterial solution was 1 mmol / L, the final concentration of trehalose was 30 mmol / L, and the volume ratio of the bacterial suspension to the solvent was 1:2.

[0070] 3. Mutagenesis Using a power device, the bacterial suspension was pumped into the first feed port of the microbial mutagenesis breeding device in Example 1 at a rate of 0.2 μL / min to convert the microorganisms in the bacterial 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.

[0071] Comparative Example 3 Referring to document 2, the recombinant bacteria obtained in Example 7 were cultured to an OD of 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8The distance between the ultraviolet lamp and the bacterial suspension was 5 cm, the power of the ultraviolet lamp was adjusted to 1.5 W, and ultraviolet irradiation was performed until the single-sorted cell suspension passed through the outlet of the second horizontal spiral conveying pipe.

[0072] Test Example 2 After the mutagenesis in Comparative Example 3 and Example 7 was completed, the mutagenized bacterial solution was cultured until OD 600 The value was 2.5, and flow cytometry sorting was performed with reference to document 1, and microbial cells with high fluorescence intensity were screened into 96-well plates. The erythritol production was detected after fermentation for 48 to 96 h with reference to the method of document 3, and the number of negative mutant strains (erythritol production less than 110 g / L), normal strains (5-hydroxyvaleric acid production of 110 to 113 g / L) and positive mutant strains (erythritol production greater than 113 g / L) was counted. The forward mutation rate was calculated according to the formula of Example 2 below. The results showed that the forward mutation rate of Comparative Example 3 was 18%, and the forward mutation rate of Example 7 was 49%.

[0073] Example 8 UV mutagenesis of Corynebacterium glutamicum 1. Preparation of recombinant bacteria Referring to document 1, a fluorescent marker plasmid was introduced into C. glutamicum-Pargw W2 to obtain a recombinant bacterium.

[0074] 2. Preparation of bacterial solution Refer to reference 1 and culture the recombinant bacteria to OD 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step, wash 3 to 4 times, and prepare a bacterial suspension with a concentration of 2×10 8 Protective agent 7 (calcium chloride, trehalose and solvent (glycerol with a volume concentration of 50%)) was added to the bacterial suspension to obtain a bacterial solution; wherein the final concentration of calcium ions in the bacterial solution was 1 mmol / L, the final concentration of trehalose was 30 mmol / L, and the volume ratio of the bacterial suspension to the solvent was 1:2.

[0075] 3. Mutagenesis Using a power device, the bacterial 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 convert the microorganisms in the bacterial solution 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.

[0076] Comparative Example 4 Referring to document 1, the recombinant bacteria obtained in Example 8 were cultured to an OD of 600Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 The distance between the UV lamp and the bacterial suspension was 5 cm, the UV lamp power was adjusted to 1.2 W, and UV irradiation was performed for 45 s.

[0077] Test Example 3 After the mutagenesis in Comparative Example 4 and Example 8 was completed, the mutagenized bacterial solution was cultured until OD 600 The value was 2.3, and flow cytometry sorting was performed with reference to document 1, and microbial cells with high fluorescence intensity were screened into 96-well plates. The lysine production was detected after fermentation for 48 to 96 h with reference to the method of document 1, and the number of negative mutant strains (lysine production was less than 16.00 g / L), normal strains (lysine production was 16.00 to 17.00 g / L) and positive mutant strains (erythritol production was greater than 7.00 g / L) was counted. The positive mutation rate was calculated according to the formula of Example 2 below. The results showed that the positive mutation rate of Example 8 was 51% compared with that of Comparative Example 4, which was 23%.

[0078] Example 9 UV mutagenesis of Bacillus subtilis 1. Preparation of recombinant bacteria Referring to document 1, a fluorescent marker plasmid was introduced into Bacillus subtilis BS-ppb 11 to obtain a recombinant bacterium.

[0079] 2. Preparation of bacterial solution Refer to reference 4 to culture the recombinant bacteria to OD 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step, wash 3 to 4 times, and prepare a bacterial suspension with a concentration of 2×10 8 Protective agent 7 (calcium chloride, trehalose and solvent (glycerol with a volume concentration of 50%)) was added to the bacterial suspension to obtain a bacterial solution; wherein the final concentration of calcium ions in the bacterial solution was 1 mmol / L, the final concentration of trehalose was 30 mmol / L, and the volume ratio of the bacterial suspension to the solvent was 1:2.

[0080] 3. Mutagenesis Using a power device, the bacterial suspension was pumped into the first feed port of the microbial mutagenesis breeding device in Example 1 at a rate of 0.1 μL / min to convert the microorganisms in the bacterial 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.

[0081] Comparative Example 5 Referring to reference 4, the recombinant bacteria obtained in Example 8 were cultured to an OD of 600 Take 10 mL of sample, shake thoroughly, centrifuge at low speed, discard the supernatant, wash with sterile water, repeat this step 3 to 4 times to make a bacterial suspension with a concentration of 2×10 8 The distance between the ultraviolet lamp and the bacterial suspension was 5 cm, the power of the ultraviolet lamp was adjusted to 1.35 W, and ultraviolet irradiation was performed until the single-sorted cell suspension passed through the outlet of the second horizontal spiral conveying pipeline.

[0082] Test Example 4 After the mutagenesis in Comparative Example 5 and Example 9 was completed, the mutagenized bacterial solution was cultured until OD 600 The value was 2.7, and flow cytometry sorting was performed with reference to document 1, and microbial cells with high fluorescence intensity were screened out and placed in a 96-well plate. The method of document 4 was used to detect the acetoin production after fermentation for 48 to 96 h, and the number of negative mutant strains (acetoin production was less than 50 g / L), normal strains (acetoin production was 50 to 55 g / L) and positive mutant strains (acetoin production was greater than 55 g / L) was counted. The positive mutation rate was calculated according to the formula of Example 2 below. The results showed that the positive mutation rate of Comparative Example 5 was 16%, and the positive mutation rate of Example 9 was 39%.

[0083] Based on the above content, it can be seen 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 forward mutation rate, and obtain mutant strains with high uniformity and high forward mutation rate. It has simple operation, small workload and short cycle.

[0084] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A microbial mutation breeding device, characterized in that: The microbial mutation breeding device comprises a cubic structure and a transmission structure, wherein the cubic structure is surrounded by a light-proof material and an ultraviolet lamp is arranged inside; The transmission structure includes a single sorting area and a mutagenesis area, and the mutagenesis area is inserted into the cube structure through the insertion port of the cube structure and is arranged vertically 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, and the diameter of the pipeline is 20-30 μm; The number of spirals of the second horizontal spiral conveying pipeline is ≥10, and the diameter of the pipeline is 20-30 μm.

2. The microbial mutation breeding device according to claim 1, characterized in that: 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.

3. The microbial mutation breeding device according to claim 1, characterized in that: The inlet, outlet and center of the first horizontal spiral conveying pipeline are located in a straight line; The inlet, outlet and center of the second horizontal spiral conveying pipeline are located in a straight line.

4. The microbial mutation breeding device according to claim 1, characterized in that: The areas of the single sequencing area and the mutagenesis area are 0.5 cm×0.5 cm respectively.

5. The microbial mutation breeding device according to any one of claims 1 to 4, characterized in that: The first horizontal spiral conveying pipeline, the second horizontal spiral conveying pipeline and the horizontal pipeline are respectively made of silicon dioxide.

6. Use of the microorganism mutation breeding device according to any one of claims 1 to 5 in microorganism mutation and / or microorganism breeding.

7. A microbial mutagenesis method, characterized in that: The method of using the microbial mutagenesis breeding device according to any one of claims 1 to 5 to perform ultraviolet mutagenesis treatment on microorganisms comprises the following steps: mixing a solution containing microorganisms with a protective agent to obtain a microorganism solution; Passing the microbial solution into the first horizontal spiral conveying pipe of the microbial mutation breeding device for single sorting to obtain a single sorted cell suspension; When the single-sorted cell suspension enters the mutagenesis zone of the microbial mutagenesis breeding device, ultraviolet irradiation is applied to the mutagenesis zone until the single-sorted cell suspension passes through the outlet of the second horizontal spiral conveying pipe; The protective agent includes calcium ions, trehalose and a solvent; the solvent is glycerol with a volume concentration of 50%.

8. The microbial mutagenesis method according to claim 7, characterized in that: 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 the microorganisms to the solvent is 1:(1-2); The number of microorganisms in the solution containing microorganisms is 2×10 8 Pieces / mL.

9. The microbial mutagenesis method according to claim 8, characterized in that: The rate at which the microbial solution is introduced into 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.

10. The microbial mutagenesis method according to any one of claims 7 to 9, characterized in that: After the ultraviolet irradiation, the method further comprises: screening the cell suspension after ultraviolet irradiation to obtain microorganisms with positive mutations.

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