Method for screening high-yield butanol strain through cyclic compound mutation and application

Abstract

The invention belongs to the field of bioengineering, and relates to a method for screening high-yield butanol strains through cyclic compound mutation and application of the high-yield butanol strains, in order to increase the gene diversity of starting strains, the method comprises the following steps: equivalently mixing a plurality of clostridium acetobutylicum strains as the starting strains; taking compound mutagenesis of nitrosoguanidine-acridine orange-nitrosoguanidine-lithium chloride as a cycle, repeating for three cycles, and continuously domesticating the mutagenic flora by gradually increasing the butanol concentration of the culture medium, so as to obtain the mutant flora with high butanol tolerance. And carrying out single colony separation on the mutant flora with high butanol tolerance, measuring the butanol content through fermentation, and screening to obtain the high-yield butanol strain.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of bioengineering, and particularly relates to a method for screening a high butanol-producing strain through cyclic compound mutagenesis and application. BACKGROUND

[0002] Butanol is a renewable biofuel with great development potential and is widely used in chemical industry, medicine, petroleum industry and other industries. In recent years, under the pressure of rising crude oil prices and environmental degradation, the research on microbial fermentation production of butanol has attracted the attention of many researchers at home and abroad. At present, the strains used for fermentation production of butanol in industry mainly include Clostridium acetobutylicum (C. acetobutylicum), Clostridium beijerinckii (C. beijerinckii) and Clostridium saccharoperbutylacetonicum (C. saccharoperbutylacetonicum) and the like. Among them, C. acetobutylicum is widely used in industry. Clostridium acetobutylicum Clostridium beijerinckli Clostridium pasteurianum

[0003] Microbial fermentation production of butanol generally uses corn as raw material, and after pretreatment and liquefaction, a fermentation broth is obtained, and then a butanol-producing strain is inoculated in an anaerobic condition for fermentation to obtain a mixture of acetone, butanol and ethanol (Acetone-Butanol-Ethanol, ABE), and the product ratio is usually 3:6:1. The traditional acetone-butanol fermentation production mainly has the following restrictive factors: (1) The current production raw material of butanol is mainly corn, which not only has the social problem of "competing with people for food", but also has high raw material cost, which is not suitable for sustainable industrial production. (2) There is product inhibition, butanol has cytotoxicity, and when the butanol product accumulates to a certain concentration in the fermentation process, it will inhibit the growth of the strain, thereby reducing the butanol yield. Therefore, it is of great practical significance to find non-food crops or cheap substrates to produce butanol and to improve the ability of the strain to produce butanol and the ability to resist butanol toxicity.

[0004] Cassava is the fifth largest crop after rice, sweet potato, sugarcane and corn, and Guangxi is the largest cassava production area in China. Fresh cassava contains 70% of water, 24%-32% of starch, 1.1% of protein, and also contains various trace elements and growth factors, so it has great potential to be selected as a production substrate for butanol fermentation.

[0005] At present, the strain used for butanol fermentation is easily inhibited by the product butanol in the fermentation process, which leads to the strain being unable to produce more butanol. If a high butanol-tolerant strain can be obtained, the butanol concentration in the mature fermentation broth can be increased, the cost of butanol separation and purification can be reduced, and the economic competitiveness of fermentation production of butanol can be improved.

[0006] In the article "Butanol High-yield Strain Mutagenesis Breeding and Fermentation Condition Optimization Research" (China Brewing, 2011, (05): 84-86), Wang Fengqin reported the mutagenesis breeding of a facultative anaerobic butanol-producing Bacillus subtilis​​​(BaciLLus sp. )C2 strain complex mutagenesis, isolation and screening of 3 strains of butanol and total solvent production have significantly improved mutant strains, butanol and total solvent production is still relatively low, respectively, to 12.58g / L~13.77g / L and 21.25g / L~22.27g / L, the method using corn mash fermentation production, cost is still relatively high.

[0007] CN 101748114A discloses a method for obtaining butanol producing mutant strains, which uses Clostridium acetobutylicum SMB1 CGMCC No. 2287 as the starting strain, and adopts the method of alternating chemical mutagenesis and protoplast fusion, finally obtains mutant strain DGF4 with butanol tolerance of 19g / L and butanol yield of 10.43g / L.

[0008] CN 101864389A discloses a Clostridium acetobutylicum strain and its screening method and application. The method uses ion beam mutagenesis, xylose plate, 2-deoxy-D-glucose plate, bromo-chromocresol plate and resazurin plate screening, and finally obtains a strain capable of efficiently and rapidly utilizing xylose and starch to produce butanol by fermentation, with total solvent yield and butanol yield reaching 20.2g / L and 15.9g / L respectively.

[0009] The fermentation substrates used in the above patents are all semi-synthetic media, and the production cost is high. In addition, the butanol production capacity and butanol tolerance of the mutant strains obtained by mutagenesis are limited. SUMMARY

[0010] The application provides a method for screening high butanol-producing strains by cyclic complex mutagenesis and application. The method uses multiple Clostridium acetobutylicum strains as starting strains, and three kinds of mutagenic agents and three rounds of cyclic mutagenesis, which greatly improves the accumulation of mutant genes with butanol tolerance and butanol yield, facilitates the screening of strains with high butanol tolerance and high butanol yield, and reduces the cost of microbial fermentation for butanol production. The cyclic complex mutagenesis method greatly reduces the screening workload in the mutagenesis breeding process, improves the efficiency of mutagenesis breeding, and is particularly outstanding in the mutagenesis breeding of multiple genes controlling traits.

[0011] The technical scheme of the application is as follows: A method for screening high butanol-producing strains by cyclic complex mutagenesis, comprising the following contents: The starting strain, i.e. Clostridium acetobutylicum strain, is activated and cultured in stages. In different stages, nitrosoguanidine, acridine orange, nitrosoguanidine and lithium chloride are added for mutagenic culture as a cycle. After multiple cycles, the bacterial population is transferred to an environment containing butanol for culture, and the butanol concentration is gradually increased. The mutagenic bacterial population is continuously domesticated to obtain a mutant bacterial population with high butanol tolerance.

[0012] The aforementioned Clostridium acetone-butanol refers to Clostridium acetone-butanol. Clostridium acetobutylicum B19 , Clostridium acetobutylicum B30 Clostridium acetobutylicum Y20 and Clostridium acetobutylicum Y41.

[0013] The method for screening high-butanol-producing strains through cyclic compound mutagenesis comprises the following steps: (1) Activation of starting strains: Clostridium acetone-butanol strains B19, B30, Y20 and Y41 were inoculated into semi-synthetic medium and cultured statically at 37°C. (2) Cyclic compound mutagenesis: (a) Mix equal amounts of the strain from step (1) and centrifuge to collect the bacterial cells (about 0.1 g). Add 1 mL of phosphate buffer to resuspend the cells, add 0.1-0.3 mL of nitrosoguanidine solution and mix well. Let stand at 37°C, centrifuge and discard the supernatant. Add 1 mL of phosphate buffer to resuspend the bacterial cells and transfer them to a new semi-synthetic culture medium tube. Let stand at 37°C. (b) Add 0.3-2 mL of acridine orange solution to the test tube from step (a) after 24 h of growth, incubate statically at 37 °C, collect the cells by centrifugation, resuspend in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate statically at 37 °C. (c) Centrifuge the bacterial culture from step (b) to collect the bacterial cells, resuspend them in 1 mL of phosphate buffer, add 0.1-0.3 mL of nitrosoguanidine solution, incubate at 37°C, centrifuge and discard the supernatant, resuspend the bacterial cells in 1 mL of phosphate buffer, transfer to a new semi-synthetic culture medium tube, and incubate at 37°C. (d) Add 1-5 mL of lithium chloride solution to the test tube in step (c), incubate statically at 37°C, collect the bacterial cells by centrifugation, resuspend in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate statically at 37°C. (e) Repeat (a)-(d) multiple times; (3) Continuous acclimatization: The bacterial cells from the test tubes that had undergone multiple rounds of cyclic compound mutagenesis in step (e) were inoculated into a semi-synthetic medium containing 20 g / L butanol for growth. After centrifugation, the supernatant was discarded, and the bacterial cells were resuspended in 1 mL of phosphate buffer and transferred into a semi-synthetic medium containing 21 g / L butanol. This step was repeated by gradually increasing the butanol concentration in a 1 g / L gradient until the bacterial cells could no longer grow in the semi-synthetic medium containing butanol. (4) Single colony isolation: The mutant bacterial group with high butanol tolerance obtained in step (3) was diluted and plated to screen out single colony strains; (5) Fermentation screening: (a) Seed culture: Select single colonies on the plate and place them in seed culture medium and place them in static culture at 37°C; (b) Fermentation determination: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 10% and ferment at 37°C; high-butanol-producing strains were obtained by sampling and detecting butanol content.

[0014] Furthermore, the method for screening high-butanol-producing strains through cyclic compound mutagenesis includes the following steps: (1) Activation of starting strains: Clostridium acetone-butanol strains B19, B30, Y20 and Y41 were inoculated into semi-synthetic medium and cultured statically at 37°C for 24 h. (2) Cyclic compound mutagenesis: (a) Mix equal amounts of the strains grown in step (1) for 24 h, centrifuge to collect the bacterial cells, add 1 mL of phosphate buffer to resuspend, add 0.1-0.3 mL of nitrosoguanidine solution to mix, incubate at 37℃ for 10 min, centrifuge to discard the supernatant, add 1 mL of phosphate buffer to resuspend the bacterial cells, transfer to a new semi-synthetic culture medium test tube, and incubate at 37℃. (b) Add 0.3-2 mL of acridine orange solution to the test tube from step (a) after 24 h of growth, incubate statically at 37 °C, collect the cells by centrifugation after 6 h, resuspend in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate statically at 37 °C. (c) Centrifuge the bacterial culture grown in step (b) for 24 hours to collect the bacterial cells, resuspend them in 1 mL of phosphate buffer, add 0.1-0.3 mL of nitrosoguanidine solution, incubate at 37°C for 10 min, centrifuge and discard the supernatant, resuspend the bacterial cells in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate at 37°C. (d) Add 1-5 mL of lithium chloride solution to the test tube from step (c) after 24 h of growth, incubate statically at 37 °C, collect the cells by centrifugation after 6 h, resuspend in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate statically at 37 °C. (e) Repeat (a)-(d) for three rounds; (3) Continuous acclimatization: The test tubes that had undergone three rounds of compound mutagenesis in step (e) were inoculated into test tubes containing 20 g / L butanol semi-synthetic medium. After 24 h of growth, the supernatant was discarded by centrifugation, and the cells were resuspended in 1 mL of phosphate buffer and transferred into test tubes containing 21 g / L butanol semi-synthetic medium. This step was repeated by gradually increasing the butanol concentration in a 1 g / L gradient until the cells could no longer grow in test tubes containing butanol semi-synthetic medium. (4) Single colony isolation: The mutant bacterial group with high butanol tolerance obtained in step (3) was diluted and plated to screen out single colony strains; (5) Fermentation screening: (a) Seed culture: Select single colonies on the plate and place them in seed culture medium, and incubate at 37℃ for 24h. (b) Fermentation assay: The seed liquid was inoculated into the fermentation medium at a rate of 10%, and fermented statically at 37°C for 72 hours; high-butanol-producing strains were screened by sampling and detecting butanol content.

[0015] Furthermore, the semi-synthetic culture medium is composed of: 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, and distilled water to a final volume of 100mL, pH 6.8-7.0, sterilized at 121℃ for 30min.

[0016] The centrifugation conditions were 12000 r / min for 5 min.

[0017] The phosphate buffer solution comprises: 10.5 g disodium hydrogen phosphate, 3.12 g sodium dihydrogen phosphate, and distilled water to a final volume of 1000 mL, sterilized at 121°C for 30 min.

[0018] The nitrosoguanidine solution comprises: 10 mg nitrosoguanidine dissolved in 1.5 mL acetone, and diluted to 5 mL with distilled water; the acridine orange solution comprises: 10 mg acridine orange, diluted to 10 mL with distilled water; the lithium chloride solution comprises: 3 g anhydrous lithium chloride, diluted to 10 mL with distilled water, and sterilized at 121°C for 30 min.

[0019] The test tube preparation of the semi-synthetic culture medium containing butanol is as follows: (1) 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, distilled water to 100mL, pH adjusted to 6.8-7.0, evenly dispensed into 5 test tubes, sterilized at 121℃ for 30min.

[0020] (2) On a sterile laminar flow hood, add 506 uL, 532 uL, 558 uL and 585 uL of 99.5% butanol solution to test tubes containing 20 mL of semi-synthetic culture medium, respectively, so that the butanol concentrations in the test tubes are 20 g / L, 21 g / L, 22 g / L and 23 g / L, respectively.

[0021] The single colony isolation specifically includes the following: (1) Preparation of culture medium: 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, 2g agar powder, distilled water to 100mL, adjust pH to 6.8-7.0, sterilize at 121℃ for 30min.

[0022] (2) Pour the sterilized culture medium into a sterile petri dish on a sterile laminar flow hood and let it cool to make a sterile plate; dilute the high butanol-tolerant mutant bacterial group obtained in step (3) to a suitable gradient and spread it on the surface of the plate; place the plate in a desiccator with added oxygen remover and incubate for 48 hours; the oxygen remover ratio is: 5g sodium bicarbonate, 5g sodium dithionite, 1g iron powder and 2.1mL water per 1L container, and prepare it fresh for use.

[0023] The seed culture medium comprises: 8g cassava flour, 1g soybean meal (or peanut bran), 0.001% amylase, and tap water. It is heated at 80-100℃ for 30 minutes, then brought to a final volume of 100 mL and sterilized at 121℃ for 30 minutes. The fermentation culture medium comprises: 770g cassava flour, 70g soybean meal (or peanut bran), 0.001% amylase, and tap water. It is heated at 80-100℃ for 30 minutes, then brought to a final volume of 7000 mL and sterilized at 121℃ for 30 minutes.

[0024] Application of the butanol strain prepared using the method described in this invention in the preparation of butanol.

[0025] Compared with the prior art, the beneficial effects of this invention are as follows: This invention is the first to utilize the cyclical compound mutagenesis method of nitrosoguanidine-acridine orange-nitrosoguanidine-lithium chloride to obtain four mutant strains with strong butanol production capacity and tolerance to butanol. The four mutant strains were fermented in 10L fermentation bottles with cassava flour as the sole carbon source. The mutant strain with the highest butanol production and total solvent production reached 1.96 g / L and 3.08 g / L, respectively, which are 39.01% and 40.00% higher than the starting strain, respectively. This will greatly improve the utilization of raw materials and enhance the economic efficiency of microbial fermentation for butanol production. Detailed Implementation

[0026] The following detailed description of specific embodiments of the present invention is provided through examples. It should be understood that the specific embodiments described are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0027] Example 1

[0028] A method for screening high-butanol-producing strains through cyclic combined mutagenesis specifically includes the following steps: (1) Activation of the starting strain: Clostridium acetone-butanol B19, B30, Y20 and Y41 were inoculated into semi-synthetic medium for activation; (2) Composite mutagenesis: (a) The bacterial cells of the strain grown in step (1) for 24 h were collected by centrifugation, resuspended in 1 mL phosphate buffer, mixed with 0.1 mL nitrosoguanidine solution, and incubated at 37 °C for 10 min. After centrifugation, the supernatant was discarded, the bacterial cells were resuspended in 1 mL phosphate buffer, transferred to a new semi-synthetic culture medium tube, and cultured at 37 °C. (b) Add 0.3 mL of acridine orange solution to the test tube from step (a) after 24 h of growth and incubate statically at 37 °C. After 6 h, centrifuge the test tube to collect the bacterial cells, resuspend them in 1 mL of phosphate buffer, transfer them to a new semi-synthetic medium test tube, and incubate statically at 37 °C. (c) Centrifuge the bacterial culture grown in step (b) for 24 hours to collect the bacterial cells, resuspend them in 1 mL of phosphate buffer, add 0.1 mL of nitrosoguanidine solution, incubate at 37°C for 10 min, centrifuge and discard the supernatant, resuspend the bacterial cells in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate at 37°C. (d) Add 1 mL of lithium chloride solution to the test tube in step (2) after 24 h of growth, and culture statically at 37 °C. After 6 h, centrifuge the test tube to collect the bacterial cells, add 1 mL of phosphate buffer to resuspend, transfer to a new synthetic medium test tube, and culture statically at 37 °C. (e) Repeat (a)-(d) for three rounds.

[0029] (3) Continuous acclimatization: The test tubes that had undergone three rounds of compound mutagenesis in step (e) were inoculated into a semi-synthetic medium with a butanol concentration of 20 g / L. After growing for 24 h, the supernatant was discarded by centrifugation, the cells were resuspended in 1 mL of phosphate buffer, and transferred to a semi-synthetic medium containing 21 g / L butanol. After growing for 24 h, the supernatant was discarded by centrifugation, the cells were resuspended in 1 mL of phosphate buffer, and transferred to a semi-synthetic medium containing 22 g / L butanol. After growing for 24 h, the supernatant was discarded by centrifugation, the cells were resuspended in 1 mL of phosphate buffer, and transferred to a semi-synthetic medium containing 23 g / L butanol.

[0030] (4) Strain screening: Dilute and spread the strain grown in step (3) on a plate to screen for single colony strains. Clostridium acetobutylicum M1.

[0031] (5) The M1 strain and the initial strain were inoculated into a 10L fermentation bottle at an inoculation rate of 10%, and fermented at 37℃ for 72h. After fermentation, the butanol and total solvent contents were measured respectively, and the results are shown in Table 1.

[0032] Table 1 Comparison of butanol and total solvent content between strain M1 and the initial strain Strain Acetone (g / L) Ethanol (g / L) Butanol (g / L) Total solvent (g / L) M1 0.82 0.11 1.62 2.55 B19 0.76 0.02 1.50 2.28 B30 0.74 0.02 1.47 2.23 Y20 0.70 0.23 1.46 2.39 Y41 0.77 0.02 1.41 2.20 As can be seen from the experimental results in Table 1, after using cyclic compound mutagenesis, the butanol and total solvent contents of the mutant strain increased by 8.00%-14.89% and 6.69%-15.91%, respectively.

[0033] Example 2

[0034] The cyclic compound mutagenesis and strain screening conditions were the same as in Example 1, except that the amount of nitrosoguanidine solution added was 0.15 mL, and single-colony strains were screened. Clostridium acetobutylicum M2. The M2 strain and the initial strain were inoculated into 10L fermentation flasks at an inoculation rate of 10%, and fermented at 37℃ for 72h. After fermentation, the butanol and total solvent contents were measured, and the results are shown in Table 2.

[0035] Table 2 Comparison of butanol and total solvent content between M2 strain and the initial strain Strain Acetone (g / L) Ethanol (g / L) Butanol (g / L) Total solvent (g / L) M2 0.84 0.09 1.71 2.64 B19 0.76 0.02 1.50 2.28 B30 0.74 0.02 1.47 2.23 Y20 0.70 0.23 1.46 2.39 Y41 0.77 0.02 1.41 2.20 As can be seen from the experimental results in Table 2, after using cyclic compound mutagenesis, the butanol and total solvent contents of the mutant strain increased by 14.00%-21.28% and 10.46%-20.00%, respectively.

[0036] Example 3

[0037] The cyclic compound mutagenesis and strain screening conditions were the same as in Example 1, except that the amount of acridine orange solution added was 0.6 mL, and single-colony strains were screened. Clostridium acetobutylicum M3. The M3 strain and the initial strain were inoculated into 10L fermentation flasks at an inoculation rate of 10%, and fermented at 37℃ for 72h. After fermentation, the butanol and total solvent contents were measured, and the results are shown in Table 3.

[0038] Table 3 Comparison of butanol and total solvent content between M3 strain and the initial strain Strain Acetone (g / L) Ethanol (g / L) Butanol (g / L) Total solvent (g / L) M3 0.88 0.10 1.82 2.80 B19 0.76 0.02 1.50 2.28 B30 0.74 0.02 1.47 2.23 Y20 0.70 0.23 1.46 2.39 Y41 0.77 0.02 1.41 2.20 As can be seen from the experimental results in Table 3, after using cyclic compound mutagenesis, the butanol and total solvent contents of the mutant strain increased by 17.15%-27.27% and 21.33%-29.08%, respectively.

[0039] Example 4

[0040] The cyclic combined mutagenesis and strain screening conditions were the same as in Example 1, except that the amount of lithium chloride solution added was 2 mL, and single-colony strains were screened. Clostridium acetobutylicum M4. The M4 strain and the initial strain were inoculated into 10L fermentation flasks at an inoculation rate of 10%, and fermented at 37℃ for 72h. After fermentation, the butanol and total solvent contents were measured, and the results are shown in Table 4.

[0041] Table 4 Comparison of butanol and total solvent content between M4 strain and the initial strain Strain Acetone (g / L) Ethanol (g / L) Butanol (g / L) Total solvent (g / L) Clostridium acetobutylicum Strain Acetone (g / L) Ethanol (g / L) Butanol (g / L) Total solvent (g / L) M4 1.02 0.10 1.96 3.08 B19 0.76 0.02 1.50 2.28 B30 0.74 0.02 1.47 2.23 Y20 0.70 0.23 1.46 2.39 Y41 0.77 0.02 1.41 2.20 As can be seen from the experimental results in Table 4, after using cyclic compound mutagenesis, the butanol and total solvent contents of the mutant strain increased by 28.87%-40.00% and 30.67%-39.01%, respectively.

[0042] The results above show that the butanol and total solvent content of the mutant strains obtained by the cyclic compound mutagenesis method were significantly improved.

[0043] Example 5

[0044] The method for screening high-butanol-producing strains by cyclic compound mutagenesis of the present invention includes the following steps: (1) Activation of starting strains: Clostridium acetone-butanol strains B19, B30, Y20 and Y41 were inoculated into semi-synthetic medium and cultured at 37°C for 24 hours.

[0045] (2) Composite mutagenesis: (a) Mix the strain grown in step (1) for 24 h, centrifuge to collect the bacterial cells (about 0.1 g), add 1 mL of phosphate buffer to resuspend, add 0.1-0.3 mL of nitrosoguanidine solution to mix, incubate at 37°C for 10 min, centrifuge and discard the supernatant, add 1 mL of phosphate buffer to resuspend the bacterial cells, transfer to a new semi-synthetic medium test tube, and incubate at 37°C. (b) Add 0.3-2.0 mL of acridine orange solution to the test tube from step (a) after 24 h of growth and culture at 37 °C. After 6 h, centrifuge the test tube to collect the bacterial cells, add 1 mL of phosphate buffer to resuspend, transfer to a new synthetic medium test tube, and culture at 37 °C. (c) Centrifuge the bacterial culture grown in step (b) for 24 hours to collect the bacterial cells, resuspend them in 1 mL of phosphate buffer, add 0.1-0.3 mL of nitrosoguanidine solution, incubate at 37°C for 10 min, centrifuge and discard the supernatant, resuspend the bacterial cells in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate at 37°C. (d) Add 1-3 mL of lithium chloride solution to the test tube from step (c) after 24 h of growth, and incubate statically at 37 °C. After 6 h, centrifuge the test tube to collect the bacterial cells, resuspend them in 1 mL of phosphate buffer, transfer them to a new semi-synthetic medium test tube, and incubate statically at 37 °C. (e) Repeat (a)-(d) for three rounds.

[0046] (3) Continuous acclimatization: The test tubes that had undergone three rounds of compound mutagenesis in step (e) were inoculated into test tubes containing 20 g / L butanol semi-synthetic medium. After 24 h of growth, the cells were centrifuged, the supernatant was discarded, and the cells were resuspended in 1 mL of phosphate buffer and transferred into test tubes containing 21 g / L butanol semi-synthetic medium. This step was repeated by gradually increasing the butanol concentration in increments of 1 g / L until the cells could no longer grow in test tubes containing butanol semi-synthetic medium.

[0047] (4) Single colony isolation: The mutant bacterial group with high butanol tolerance obtained in step (3) was diluted and plated to screen out single colony strains.

[0048] (5) Fermentation screening: (a) Seed culture: Select single colonies on the plate and place them in seed culture medium, and incubate at 37°C for 24 hours.

[0049] (b) Fermentation assay: The seed liquid was inoculated into the fermentation medium at a rate of 10%, and fermented statically at 37°C for 72 hours; high-butanol-producing strains were screened by sampling and detecting butanol content.

[0050] The semi-synthetic culture medium consists of: 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, and distilled water to a final volume of 100mL. The pH is 6.8-7.0, and the medium is sterilized at 121℃ for 30min.

[0051] The centrifugation conditions were 12000 r / min for 5 min.

[0052] The phosphate buffer solution comprises: 10.5 g disodium hydrogen phosphate, 3.12 g sodium dihydrogen phosphate, and distilled water to a final volume of 1000 mL, sterilized at 121°C for 30 min.

[0053] The nitrosoguanidine solution comprises: 10 mg nitrosoguanidine dissolved in 1.5 mL acetone, and diluted to 5 mL with distilled water.

[0054] The acridine orange solution comprises: 10 mg acridine orange, diluted to 10 mL with distilled water.

[0055] The lithium chloride solution comprises: 3 g of anhydrous lithium chloride, diluted to 10 mL with distilled water, and sterilized at 121°C for 30 min.

[0056] The test tube containing butanol with a concentration of 20 g / L-23 g / L was prepared as follows: (1) 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, distilled water to 100mL, pH adjusted to 6.8-7.0, evenly dispensed into 5 test tubes, sterilized at 121℃ for 30min.

[0057] (2) On a sterile laminar flow hood, add 506uL, 532uL, 558uL and 585uL of butanol solution with a concentration of ≥99.5% to test tubes with a concentration of 20g / L, 21g / L, 22g / L and 23g / L respectively.

[0058] The single colony screening method is as follows: (1) Preparation of culture medium: 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, 2g agar powder, distilled water to 100mL, adjust pH to 6.8-7.0, sterilize at 121℃ for 30min.

[0059] (2) Pour the sterilized culture medium into a sterile petri dish on a sterile laminar flow hood and let it cool to make a sterile plate; dilute the mutant bacteria obtained in step (3) to a suitable gradient and spread it on the surface of the plate; place the plate in a desiccator with added oxygen remover and incubate for 48 hours; the oxygen remover ratio is: 5g sodium bicarbonate, 5g sodium dithionite, 1g iron powder and 2.1mL water per 1L container, and prepare it fresh for use.

[0060] In the above method, the seed culture medium includes: 8g cassava flour, 1g soybean meal (or peanut bran), 0.001% amylase, and tap water. The mixture is heated at 80~100℃ for 30min, brought to a final volume of 100mL, and sterilized at 121℃ for 30min.

[0061] The fermentation medium consists of 770g cassava flour, 70g soybean meal, 0.001% amylase, and tap water. It is heated at 80-100℃ for 30 minutes, brought to a final volume of 7000 mL, and then sterilized at 121℃ for 90 minutes.

[0062] The butanol content was determined by gas chromatography.

[0063] The prepared butanol strain was used for butanol production.

Claims

1. A method for screening high-butanol-producing strains through cyclic compound mutagenesis, characterized in that, Includes the following: The starting strain, Clostridium acetone-butanol strain, was activated and cultured in stages. In different stages, nitrosoguanidine, acridine orange, nitrosoguanidine, and lithium chloride were added to induce mutagenesis as a cycle. After multiple cycles, the bacterial population was transferred to an environment containing butanol and the butanol concentration was gradually increased. The mutant bacterial population was continuously acclimatized to obtain a mutant bacterial population with high butanol tolerance.

2. The method for screening high-butanol-producing strains using cyclic compound mutagenesis according to claim 1, characterized in that, The aforementioned Clostridium acetone-butanol refers to Clostridium acetone-butanol. Clostridium acetobutyLicum B19 Clostridium acetobutylicum B30 Clostridium acetobutylicum Y20 and Clostridium acetobutylicum Y41 。 3. The method for screening high-butanol-producing strains using cyclic compound mutagenesis according to claim 1, characterized in that, The specific steps are as follows: (1) Activation of starting strains: Clostridium acetone-butanol strains B19, B30, Y20 and Y41 were inoculated into semi-synthetic medium and cultured statically at 37°C. (2) Cyclic compound mutagenesis: (a) Mix equal amounts of the strain from step (1) and centrifuge to collect the bacterial cells. Add 1 mL of phosphate buffer to resuspend the cells, add 0.1-0.3 mL of nitrosoguanidine solution and mix well. Let stand at 37°C, centrifuge and discard the supernatant. Add 1 mL of phosphate buffer to resuspend the bacterial cells and transfer them to a new semi-synthetic culture medium tube. Let stand at 37°C. (b) Add 0.3-2 mL of acridine orange solution to the test tube from step (a) after 24 h of growth, incubate statically at 37 °C, collect the cells by centrifugation, resuspend in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate statically at 37 °C. (c) Centrifuge the bacterial culture from step (b) to collect the bacterial cells, resuspend them in 1 mL of phosphate buffer, add 0.1-0.3 mL of nitrosoguanidine solution, incubate at 37°C, centrifuge and discard the supernatant, resuspend the bacterial cells in 1 mL of phosphate buffer, transfer to a new semi-synthetic culture medium tube, and incubate at 37°C. (d) Add 1-5 mL of lithium chloride solution to the test tube in step (c), incubate statically at 37°C, collect the bacterial cells by centrifugation, resuspend in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate statically at 37°C. (e) Repeat (a)-(d) multiple times; (3) Continuous acclimatization: The bacterial cells from the test tubes that had undergone multiple rounds of cyclic compound mutagenesis in step (e) were inoculated into a semi-synthetic medium containing 20 g / L butanol for growth. After centrifugation, the supernatant was discarded, and the bacterial cells were resuspended in 1 mL of phosphate buffer and transferred into a semi-synthetic medium containing 21 g / L butanol. This step was repeated by gradually increasing the butanol concentration in a 1 g / L gradient until the bacterial cells could no longer grow in the semi-synthetic medium containing butanol. (4) Single colony isolation: The mutant bacterial group with high butanol tolerance obtained in step (3) was diluted and plated to screen out single colony strains; (5) Fermentation screening: (a) Seed culture: Select single colonies on the plate and place them in seed culture medium and place them in static culture at 37°C; (b) Fermentation determination: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 10% and ferment at 37°C; high-butanol-producing strains were obtained by sampling and detecting butanol content.

4. The method for screening high-butanol-producing strains by cyclic compound mutagenesis according to claim 3, characterized in that, The steps include the following: (1) Activation of starting strains: Clostridium acetone-butanol strains B19, B30, Y20 and Y41 were inoculated into semi-synthetic medium and cultured statically at 37°C for 24 h. (2) Cyclic compound mutagenesis: (a) Mix equal amounts of the strains grown in step (1) for 24 h, centrifuge to collect the bacterial cells, add 1 mL of phosphate buffer to resuspend, add 0.1-0.3 mL of nitrosoguanidine solution to mix, incubate at 37℃ for 10 min, centrifuge to discard the supernatant, add 1 mL of phosphate buffer to resuspend the bacterial cells, transfer to a new semi-synthetic culture medium test tube, and incubate at 37℃. (b) Add 0.3-2 mL of acridine orange solution to the test tube from step (a) after 24 h of growth, incubate statically at 37 °C, collect the cells by centrifugation after 6 h, resuspend in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate statically at 37 °C. (c) Centrifuge the bacterial culture grown in step (b) for 24 hours to collect the bacterial cells, resuspend them in 1 mL of phosphate buffer, add 0.1-0.3 mL of nitrosoguanidine solution, incubate at 37°C for 10 min, centrifuge and discard the supernatant, resuspend the bacterial cells in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate at 37°C. (d) Add 1-5 mL of lithium chloride solution to the test tube from step (c) after 24 h of growth, incubate statically at 37 °C, collect the cells by centrifugation after 6 h, resuspend in 1 mL of phosphate buffer, transfer to a new semi-synthetic medium test tube, and incubate statically at 37 °C. (e) Repeat (a)-(d) for three rounds; (3) Continuous acclimatization: The test tubes that had undergone three rounds of compound mutagenesis in step (e) were inoculated into test tubes containing 20 g / L butanol semi-synthetic medium. After 24 h of growth, the supernatant was discarded by centrifugation, and the cells were resuspended in 1 mL of phosphate buffer and transferred into test tubes containing 21 g / L butanol semi-synthetic medium. This step was repeated by gradually increasing the butanol concentration in a 1 g / L gradient until the cells could no longer grow in test tubes containing butanol semi-synthetic medium. (4) Single colony isolation: The mutant bacterial group with high butanol tolerance obtained in step (3) was diluted and plated to screen out single colony strains; (5) Fermentation screening: (a) Seed culture: Select single colonies on the plate and place them in seed culture medium, and incubate at 37℃ for 24h. (b) Fermentation assay: The seed liquid was inoculated into the fermentation medium at a rate of 10%, and fermented statically at 37°C for 72 hours. High-butanol-producing strains were screened by sampling and detecting butanol content.

5. The method for screening high-butanol-producing strains by cyclic compound mutagenesis according to claim 3 or 4, characterized in that, The semi-synthetic culture medium comprises: 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, and distilled water to a final volume of 100mL, pH 6.8-7.0, sterilized at 121℃ for 30min; the phosphate buffer comprises: 10.5g disodium hydrogen phosphate, 3.12g sodium dihydrogen phosphate, and distilled water to a final volume of 1000mL, sterilized at 121℃ for 30min; the seed culture medium comprises: 8g cassava flour, 1g soybean meal (or peanut bran), 0.001% amylase, and tap water, heated at 80-100℃ for 30min, and diluted to a final volume of 100mL, sterilized at 121℃ for 30min; the fermentation culture medium comprises: 770g cassava flour, 70g soybean meal (or peanut bran), 0.001% amylase, and tap water, heated at 80-100℃ for 30min, and diluted to a final volume of 7000mL. mL, sterilize at 121℃ for 30 min.

6. The method according to claim 3 or 4, characterized in that, The centrifugation conditions were 12000 r / min for 5 min.

7. The method for screening high-butanol-producing strains by cyclic compound mutagenesis according to claim 3 or 4, characterized in that, The nitrosoguanidine solution comprises: 10 mg nitrosoguanidine dissolved in 1.5 mL acetone, and diluted to 5 mL with distilled water; the acridine orange solution comprises: 10 mg acridine orange, diluted to 10 mL with distilled water; the lithium chloride solution comprises: 3 g anhydrous lithium chloride, diluted to 10 mL with distilled water, and sterilized at 121°C for 30 min.

8. The method for screening high-butanol-producing strains by cyclic compound mutagenesis according to claim 3 or 4, characterized in that, The test tube preparation of the semi-synthetic culture medium containing butanol is as follows: (1) 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, distilled water to a final volume of 100mL, pH adjusted to 6.8-7.0, evenly dispensed into 5 test tubes, sterilized at 121℃ for 30min; (2) On a sterile laminar flow hood, add 506 uL, 532 uL, 558 uL and 585 uL of 99.5% butanol solution to test tubes containing 20 mL of semi-synthetic culture medium, respectively, so that the butanol concentrations in the test tubes are 20 g / L, 21 g / L, 22 g / L and 23 g / L, respectively.

9. A method for screening high-butanol-producing strains using cyclic compound mutagenesis according to claim 3 or 4, characterized in that, The single colony isolation specifically includes the following: (1) Preparation of culture medium: 2g glucose, 1g peptone, 0.5g yeast extract, 0.1g ascorbic acid, 2g agar powder, distilled water to 100mL, adjust pH to 6.8-7.0, sterilize at 121℃ for 30min; (2) Pour the sterilized culture medium into a sterile petri dish on a sterile laminar flow hood and let it cool to make a sterile plate; dilute the high butanol-tolerant mutant bacterial group obtained in step (3) to a suitable gradient and spread it on the surface of the plate; place the plate in a desiccator with added oxygen remover and incubate for 48 hours; the oxygen remover ratio is: 5g sodium bicarbonate, 5g sodium dithionite, 1g iron powder and 2.1mL water per 1L container, and prepare it fresh for use.

10. The application of the butanol strain prepared by the method according to claims 1-10 in the preparation of butanol.