Schizochytrium limacinum mutant strain for producing eicosapentaenoic acid as well as mutagenesis method and application of schizochytrium limacinum mutant strain

Abstract

The invention provides a schizochytrium limacinum mutant strain for producing eicosapentaenoic acid as well as a mutagenesis method and application of the schizochytrium limacinum mutant strain, and belongs to the technical field of microorganisms. In order to culture a new EPA high-yield microbial strain, the method comprises the following steps: firstly, carrying out high-throughput screening based on Nile red staining on schizochytrium limacinum by utilizing a method of combining normal-pressure and room-temperature plasma (ARTP) mutagenesis and sethoxydim pressure, so as to obtain a high-fat-content mutant strain; then the high-fat-content mutant strain is used for diethyl sulfate (DES) mutation breeding, a triclos-2, 2-bipyridine composite plate screening technology, shake flask fermentation and GC quantitative analysis to breed an EPA high-yield strain, and finally the schizochytrium limacinum high-yield mutant strain DBT-64 is obtained. The mutant strain DBT-64 is subjected to passage for many times, the character is stable, the yield of EPA is increased by 490.33%, the economic effect is remarkable, and the mutant strain DBT-64 can be used for commercial production of EPA.

Description

Technical Field

[0001] The invention relates to a Schizochytrium mutant strain producing eicosapentaenoic acid and a mutagenesis method and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid (PUFAs) that is very important to the human body. It has broad commercial prospects in the health food, pharmaceutical, feed and other industries. It has important physiological functions in the prevention and treatment of cardiovascular diseases, the treatment of schizophrenia and depression, and anti-inflammatory and anti-cancer effects. At present, the main dietary source of EPA is fish oil, but due to the increasing depletion of marine resources and environmental pollution such as nuclear leaks, the supply of high-quality fish oil has dropped significantly. Marine microalgae are primary producers of PUFAs. Their safe and controllable production process and lack of arable land requirements have made them an alternative source of PUFAs and have attracted widespread attention. At present, common microalgae that produce EPA, such as Phaeodactylum tricornutum and Nannochloropsis sp., may not be the best commercial EPA producers due to their low final biomass. Researchers are still looking for suitable EPA producers.

[0003] Schizochytrium sp. is a heterotrophic marine fungus with the advantages of high PUFAs content, fast growth rate and low fermentation cost. It has become a candidate for commercial production of PUFAs. There are two fatty acid synthesis pathways in Schizochytrium. The fatty acid synthase (FAS) pathway mainly synthesizes short-chain saturated fatty acids, and the polyketide synthase pathway (PKS) synthesizes PUFAs. In recent years, research on Schizochytrium at home and abroad has mainly focused on mutagenesis breeding, genetic engineering and fermentation process optimization to improve oil content and docosahexaenoic acid production. Research on EPA production still needs to be improved.

[0004] Current research has been conducted to increase the EPA content in Schizochytrium by adding certain organic reagents and special process conditions such as controlling the CO2 in the environment. For example, by adding fluridone during the culture of Schizochytrium, the percentage of EPA in the total fatty acids was increased to 0.65%; or by adding inorganic salts during the fed-batch fermentation process, the percentage of EPA in the total fatty acids was increased to 0.98%.

[0005] However, due to the toxicity of exogenous additives and the increase in fermentation costs, it is difficult to further increase the production of EPA using traditional fermentation optimization strategies. Therefore, it is particularly important to cultivate new strains of high-EPA-producing microorganisms. Summary of the invention

[0006] In order to cultivate a new EPA high-yielding microbial strain, the present invention uses Schizochytrium ATCC20888 as the starting strain, and screens out a EPA high-yielding strain DBT-64 through ARTP-DES composite mutagenesis technology, multiple resistance screening technology, Nile red staining primary screening, shake flask fermentation and GC quantitative analysis technology. The specific technical scheme of the present invention is as follows:

[0007] The first object of the present invention is to provide a mutant strain of Schizochytrium sp. producing eicosapentaenoic acid, which is classified and named Schizochytrium sp. DBT-64, and was deposited in the China Center for Type Culture Collection on June 19, 2023, with a deposit number of CCTCC M 20231041, and the deposit address is Wuhan University, Wuhan, China.

[0008] The second object of the present invention is to provide a method for inducing mutation breeding of the above-mentioned Schizochytrium mutant strain, which specifically comprises the following steps:

[0009] S1: ARTP mutagenesis and sethoxydim plate screening were performed on Schizochytrium ATCC20888 to construct a high-throughput screening library, and a high-lipid content mutant strain A-32 was obtained based on Nile red staining primary screening and shake flask fermentation secondary screening;

[0010] S2: The mutant strain A-32 obtained in S1 was subjected to DES mutagenesis, and the EPA production was detected by triclosan-2,2-diapyridine composite screening plate screening and shake flask fermentation culture;

[0011] S3: Screening of Schizochytrium mutant strains with high EPA production based on EPA production.

[0012] In one embodiment of the present invention, the S1 specifically includes the following steps:

[0013] S11: Cultivate Schizochytrium ATCC20888 to the logarithmic phase and dilute with sterile phosphate-buffered saline (PBS) buffer to an OD 595 =0.6-0.7 to obtain a bacterial suspension, treat the bacterial suspension with ARTP mutagenesis for 40-60s, obtain the mutated bacterial suspension by shaking and eluting, draw 100 μL of the mutated bacterial suspension and spread it on a screening plate containing 30-50 mg / L sethoxydim, and culture in the dark.

[0014] S12: Select the larger colonies screened by the thiamethoxam screening plate in S11, inoculate them in a 48-well cell culture plate containing 1 ml of fermentation medium, and incubate them in a constant temperature shaker. Take 200 μL of the culture solution and inoculate it in a 96-well transparent plate. Measure the OD 595The culture medium was then transferred to a black 96-well plate, and 50 μL of 20% dimethyl sulfoxide and 50 μL of 100 μg / mL Nile red dye were added. After incubation at 25°C in the dark, the fluorescence intensity was detected with an ELISA reader at an excitation wavelength of 485±20 nm and an emission wavelength of 595±20 nm. The FI / OD ratio of each culture plate was calculated. 595 The mutants with the top 5 values ​​were screened. 595 The mutant strains with the top 5 values ​​were inoculated into the culture plate again and the FI / OD was measured. 595 Value, FI / OD 595 The strain with the larger value was transferred and preserved to obtain the mutant strain A-32 with high lipid content.

[0015] In one embodiment of the present invention, the S2 specifically includes the following steps:

[0016] S21: The mutant strain A-32 was cultured to the logarithmic phase and diluted with sterile phosphate-buffered saline (PBS) buffer to an OD 595 =0.6-0.7 to obtain a bacterial suspension, add 50-150 μL of DES solution (20%, v / v), and induce at 25-30°C, 200 rpm for 10-30 min. After the mutagenesis is completed, add sodium thiosulfate solution (25%, w / v) to terminate the reaction, resuspend the solution after mutagenesis with sterile PBS buffer, and draw 100 μL of the resuspended solution and evenly apply it on a triclosan-2,2-bipyridine composite screening plate containing 5-15 mg / L, and culture in the dark. The volume ratio of the bacterial suspension to the DES solution is 19:1, and the volume ratio of the sodium thiosulfate solution to the DES solution is 6:1.

[0017] S22: Select large single colonies obtained from the triclosan-2,2-bipyridine composite screening plate and inoculate them into the seed culture medium. Subculture once every 48 hours for three consecutive times, and then inoculate into the fermentation medium for continuous culture.

[0018] In one embodiment of the present invention, the PBS buffer is prepared as follows: 8.74 g of sodium dihydrogen phosphate dihydrate is weighed, 20.44 g of sodium dihydrogen phosphate is dissolved in ultrapure water, and the volume is fixed to 1 L to prepare a 0.2 M, pH 7.2 buffer.

[0019] In one embodiment of the present invention, the dark-proof culture in S11 and S21 is carried out at 25-30° C. for 4 days.

[0020] In one embodiment of the present invention, the constant temperature shaking incubation in S12 is 25-30°C, 160-220 rpm, and the culture is carried out for 5 days.

[0021] In one embodiment of the present invention, the culture medium used for the sethoxydim screening plate described in S11 is sethoxydim solid culture medium: 40 mg / L sethoxydim, 5 g / L glucose, 1 g / L trypsin, 1 g / L yeast powder, 17.5 g / L sea crystal, 20 g / L agar, the remainder is water, and sterilized at 121°C for 20 min.

[0022] In one embodiment of the present invention, the culture medium used for the triclosan-2,2-diapyridine composite screening plate described in S21 is a triclosan-2,2-diapyridine solid culture medium: 10 mg / L triclosan-2,2-diapyridine, 5 g / L glucose, 1 g / L trypsin, 1 g / L yeast powder, 17.5 g / L sea crystal, 20 g / L agar, the remainder is water, and sterilized at 121°C for 20 min.

[0023] In one embodiment of the present invention, the seed culture medium includes 30 g / L glucose, 10 g / L tryptone, 5 g / L yeast powder, 15 g / L sea crystal, 0.05 g / L VB1, 0.05 g / L VB6, 0.05 g / L VB 12 0.0005g / L, the balance is water, sterilize at 115℃ for 20min.

[0024] In one embodiment of the present invention, the fermentation medium includes: 100 g / L glucose, 17.5 g / L sea crystal, 20 g / L L-glutamate, monohydrate, 2.5 g / L potassium dihydrogen phosphate, 7.2 g / L magnesium sulfate, 12.8 g / L sodium sulfate, 0.4 g / L calcium chloride, 0.1 g / L B1, 0.1 g / L B6, 0.001 g / L B 12 .

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a method for mutagenesis of a Schizochytrium mutant strain with high EPA production. First, the Schizochytrium ATCC20888 strain is subjected to high-throughput screening based on Nile red staining by ARTP mutagenesis and sethoxydim plate screening, thereby obtaining a high-fat content mutant strain A-32. Next, the obtained high-fat content mutant strain is used for DES mutagenesis and triclosan-2,2-bipyridine composite screening, thereby obtaining a Schizochytrium mutant strain DBT-64 with high EPA production. The mutagenesis breeding method of the present invention has many advantages, such as simple operation, wide application range, strong purpose, and high screening efficiency.

[0027] After ARTP mutagenesis for 50s, 40mg / L pyrimidine screening plates, and high-throughput screening based on Nile red staining, the total oil and EPA production of mutant strain A-32 were improved compared with wild-type Schizochytrium ATCC20888. Mutant strain A-32 was mutagenized with DES for 15min, and after triclosan-2,2-bipyridine composite screening, the total oil and EPA production of mutant strain DBT-64 obtained after shake flask fermentation increased by 44.85% and 490.33% respectively compared with wild-type Schizochytrium ATCC20888, and the EPA production reached 904.21mg / L. After multiple passages, the mutant strain DBT-64 has stable traits and increased EPA production by 490.33%, with significant economic effects, and can be used for commercial production of EPA. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A flow chart of the method for mutagenesis breeding of Schizochytrium mutant strains with high EPA production;

[0029] Figure 2 is the lethality of wild-type Schizochytrium ATCC20888 under the addition of different concentrations of sethoxydim;

[0030] Figure 3 is the lethality of wild-type Schizochytrium ATCC20888 at different treatment times in the ARTP mutagen;

[0031] Figure 4 is the FI / OD of the mutants obtained after ARTP mutagenesis and sethoxydim screening. 595 value;

[0032] Figure 5 The biomass and oil yield of wild-type Schizochytrium ATCC20888 and mutant strain A-32;

[0033] Figure 6 is the lethality of mutant strain A-32 when adding different concentrations of triclosan;

[0034] Figure 7 The lethality rate of mutant strain A-32 when adding different concentrations of 2,2-diapyridine;

[0035] Figure 8 is the lethality of mutant strain A-32 when different concentrations of triclosan-2,2-diapyridine complex are added;

[0036] Fig. 9 is the lethality rate of mutant strain A-32 at different times of DES mutagenesis;

[0037] Fig.10 The biomass, oil yield and EPA yield of wild-type Schizochytrium ATCC20888 and mutant strain DBT-64 are shown in Figure 2 . DETAILED DESCRIPTION

[0038] The technical solutions described in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1: Mutation breeding of Schizochytrium mutant strains with high EPA production

[0040] The flowchart of the mutagenesis breeding method of the Schizochytrium mutant strain with high EPA production used in the present invention is shown in Figure 1 .

[0041] S1. Perform ARTP mutagenesis and sethoxydim plate screening on Schizochytrium ATCC20888 to construct a high-throughput screening library, and obtain mutants with high lipid content based on Nile red staining primary screening and shake flask fermentation secondary screening. The specific steps are as follows:

[0042] (1) Seed solution preparation: In a sterile clean bench, the starting strain Schizochytrium ATCC20888 was inoculated into the seed culture medium and cultured in a shaking incubator at 28°C and 200 rpm for 48 h;

[0043] (2) Preparation of bacterial suspension: Take an appropriate amount of the above seed solution, dilute it with 0.2 M sterile PBS buffer (pH = 7.2), and adjust the cell OD 595 =0.6-0.7, used for ARTP mutagenesis treatment;

[0044] (3) Determine the concentration of the sethoxydim screening plate: Take 100 μL of the bacterial suspension obtained in step (2) and spread it on the sethoxydim screening plates with sethoxydim concentrations of 0, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, and 60 mg / L, respectively. After incubation at 28°C for 4 days, count the colonies on the plates and calculate the lethality (e.g. Figure 2 As shown). The mass concentration of 40 mg / L of thiamethoxam with a lethality of about 90% was selected as the basis for subsequent screening;

[0045] (4) ARTP mutagenesis treatment: Take 10 μL of the bacterial suspension obtained in step (2) and evenly spread it on a sterile ARTP-specific metal slide, place the metal slide in the groove of the ARTP mutagenizer, and fix an EP tube containing 500 μL of sterile PBS buffer on the fixing seat below the groove to receive the slide. The helium flow rate of the ARTP mutagenizer is set to 10 L / min and the power is 100 W. The treatment times are 0, 10s, 20s, 30s, 40s, 50s, and 60s, respectively. After the mutagenesis is completed, take out the EP tube containing the metal carrier, shake to resuspend the cells in the EP tube, aspirate 100 μL of the resuspension and spread it on a solid plate, and place it in a 28°C incubator in the dark for incubation for 4 days. Count the number of single colonies grown, and calculate the lethality of the ARTP mutagenizer at different treatment times (see Figure 3 ), select the treatment time (50s) with a lethality of about 90% for mutagenesis. Dilute the bacterial suspension after mutagenesis with sterile PBS by a certain multiple, take 100 μL of the bacterial suspension and spread it on the sethoxydim selection plate containing 40 mg / L sethoxydim, and then culture it at 28°C in the dark for 4 days;

[0046] (5) High-throughput screening: Select the mutants with larger morphology screened by the oxadiazine screening plate in step (4) and inoculate them into a 48-well cell culture plate containing 1 mL of fermentation medium. Culture them at 28°C, 200 rpm for 4 days. Take 200 μL of the culture solution and place it in a 96-well transparent plate to measure the OD 595 The culture medium was then transferred to a black 96-well plate, and 50 μL of 20% dimethyl sulfoxide and 5 μL of 100 μg / mL Nile red dye were added. After incubation at 25°C in the dark for 20 min, the fluorescence intensity at an excitation wavelength of 485±20 nm and an emission wavelength of 595±20 nm was detected using a microplate reader. The FI / OD value of each plate was calculated. 595 The mutant strains with the top 5 values ​​were screened (see Figure 4 ), each board FI / OD 595 The mutant strains with the top 5 values ​​were inoculated into the culture plate again and the FI / OD was measured. 595 Value, FI / OD 595 The strains with large values ​​are transferred and preserved;

[0047] (6) Fermentation culture: The FI / OD screened in step (5) 595 The 10 mutant strains with higher values ​​were inoculated into seed culture medium, subcultured into new seed culture medium every 48 hours for three times, and then 10 ml of seed solution was inoculated into fermentation medium for continuous culture for 5 days. The biomass, EPA production and oil production of the strains were measured, and the mutant strain A-32 with high lipid content was obtained. The biomass, oil production and EPA production of wild-type Schizochytrium ATCC20888 and mutant strain A-32 (see Figure 5 ).

[0048] The solutions and culture medium used in S1 are as follows:

[0049] The PBS buffer solution was prepared as follows: 8.74 g of sodium dihydrogen phosphate dihydrate was weighed, 20.44 g of sodium dihydrogen phosphate was dissolved in ultrapure water, and the volume was adjusted to 1 L to prepare a 0.2 M, pH 7.2 buffer solution.

[0050] The culture medium used for the sethoxydim screening plate is sethoxydim solid culture medium: 40 mg / L sethoxydim, 5 g / L glucose, 1 g / L tryptone, 1 g / L yeast powder, 17.5 g / L sea crystal, 20 g / L agar, the balance is water, sterilized at 121°C for 20 min.

[0051] Seed culture medium: glucose 30g / L, tryptone 10g / L, yeast powder 5g / L, sea crystal 15g / L, VB10.05g / L, VB6 0.05g / L, VB 12 0.0005g / L, the balance is water, sterilize at 115℃ for 20min.

[0052] Fermentation medium: 100g / L glucose, 17.5g / L sea crystal, 20g / L L-glutamate monosodium water, 2.5g / L potassium dihydrogen phosphate, 7.2g / L magnesium sulfate, 12.8g / L sodium sulfate, 0.4g / L calcium chloride, 0.1g / LVB1, 0.1g / LVB6, 0.001g / LVB 12 .

[0053] The culture medium used for the solid plate: glucose 5 g / L, tryptone 1 g / L, yeast powder 1 g / L, sea crystal 17.5 g / L, agar 20 g / L, sterilized at 121°C for 20 min.

[0054] The culture conditions used in S1 are as follows:

[0055] Seed culture: Take the seed solution and inoculate it into the seed culture medium at an inoculum rate of 10% (v / v), and culture it at 28°C and 200 rpm for 48 h.

[0056] Fermentation culture: Take the seed liquid and inoculate it into the fermentation medium at an inoculum rate of 10% (v / v), and culture it at 28° C. and 200 rpm for 5 days.

[0057] The detection method used in S1 is as follows:

[0058] Strain biomass determination: Take 30 ml of fermentation liquid, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the cells twice with deionized water to obtain wet cells of Schizochytrium, freeze-dry the wet cells in a vacuum freeze dryer, and weigh the dry cells after freeze-drying.

[0059] Determination of fatty acid content: Acid-heat cell wall breaking and n-hexane extraction were used. The specific method was as follows: 1 g of freeze-dried bacteria was transferred to a 50 mL centrifuge tube, 10 mL of concentrated hydrochloric acid and 5 mL of deionized water were added respectively, and the mixture was incubated at 70-80 °C for 1 h, with shaking every 10 min; after cooling, 4 mL of anhydrous ethanol and 10 mL of n-hexane were added, and the mixture was thoroughly shaken and centrifuged at 3000 r / min. -1 Centrifuge for 2 minutes under the same conditions to extract the oil, collect the upper n-hexane layer and place it in a rotating bottle of known weight, repeat 3 times, and rotary evaporation in a vacuum water bath at 65°C for 10 minutes until the n-hexane is completely evaporated, and finally weigh the weight of the fatty acids.

[0060] EPA content determination: The gas chromatograph used an Agilent HP-INNOWAX column with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm; the program temperature was set at 130 °C and then at 10 °C min after 2 min. -1 The temperature was raised to 180℃ at a rate of 4℃min -1 The temperature was raised to 240℃ and maintained for 10min. The vaporization chamber temperature was 250℃, the detector temperature was 250℃, and the nitrogen flow rate was set to 30mL·min -1 , hydrogen flow rate is 40mL·min -1， , air flow rate is 400mL·min -1 The injection volume was 1 μL, and splitless injection was used.

[0061] S2. The mutant strain A-32 obtained in S1 was subjected to DES mutagenesis, screened by triclosan-2,2-bipyridine composite screening plate, and then cultured by shake flask fermentation to detect EPA production to obtain a high-yield EPA mutant strain. The specific steps are as follows:

[0062] (1) Seed solution preparation: In a sterile clean bench, inoculate Schizochytrium A-32 into the seed culture medium and culture it in a shaker at 28°C and 200 rpm for 48 h;

[0063] (2) Preparation of bacterial suspension: Take an appropriate amount of the seed solution obtained in step (1), dilute it with 0.2 M sterile PBS buffer (pH = 7.2), and adjust the cell OD 595 =0.6-0.7;

[0064] (3) Determine the concentration of triclosan plate: Take 100 μL of the bacterial suspension obtained in step (2) and spread it on triclosan plates with concentrations of 0, 1.5 mg / L, 3 mg / L, 4.5 mg / L, 6 mg / L, 7.5 mg / L, and 9 mg / L, respectively. After incubation at 28°C for 4 days, count the colonies on the plates and calculate the lethality (see Figure 6 ). The mass concentration of triclosan (4.5 mg / L) with a lethality rate of about 90% was selected as the basis for subsequent screening;

[0065] (4) Determine the concentration of 2,2-bipyridine plate: Take 100 μL of the bacterial suspension in step (2) and spread it on 2,2-bipyridine plates with concentrations of 0, 1.5 mg / L, 3 mg / L, 4.5 mg / L, 6 mg / L, 7.5 mg / L, 9 mg / L, and 10.5 mg / L, respectively. After incubation at 28°C for 4 days, count the colonies on the plates and calculate the lethality (see Figure 7 ). The mass concentration of 2,2-bipyridine (7.5 mg / L) with a lethality rate of about 90% was selected as the basis for subsequent screening;

[0066] (5) Determine the concentration of triclosan-2,2-diapyridine composite screening plate: Take 100 μL of the bacterial suspension in step (2) and spread it on triclosan-2,2-diapyridine plates with concentrations of 0, 1.5 mg / L, 3 mg / L, 4.5 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, 12 mg / L, and 14 mg / L (the concentration ratio of triclosan to 2,2-diapyridine is 3:5), respectively. After incubation at 28°C for 4 days, count the colonies on the plates and calculate the lethality (e.g. Figure 8 As shown). The mass concentration of triclosan-2,2-bipyridine (10 mg / L) with a lethality rate of about 90% was selected as the basis for subsequent screening;

[0067] (6) DES mutagenesis: Take 1.9 ml of the bacterial suspension obtained in step (2) and place it in a sterile EP tube. Add 100 μL of DES solution (20%, v / v). Place the EP tube at 28°C and 200 rpm for mutagenesis for 0, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, respectively. After each mutagenesis, add 600 μL of sodium thiosulfate solution (25%, w / v) to the EP tube to terminate the reaction. Resuspend the mutagenized solution with sterile PBS buffer, take 100 μL of the resuspended solution and evenly spread it on a solid plate. Place it in a 28°C incubator and incubate it in the dark for 4 days. Count the number of single colonies grown and calculate the lethality at different DES mutagenesis times (see Fig. 9 ), select the mutagenesis time (15 min) with a lethality of about 90% for mutagenesis treatment. Take 100 μL of the resuspension and evenly spread it on a composite screening plate with 10 mg / L triclosan-2,2-bipyridine, and then culture it at 28°C for 4 days.

[0068] (7) Fermentation culture: Select single colonies with large morphology and high yield obtained by triclosan-2,2-bipyridine composite screening plate and inoculate them into seed culture medium. Subculture them into new seed culture medium every 48 hours for three consecutive subcultures. Then take 10 ml of seed liquid and inoculate it into fermentation culture medium for continuous culture for 5 days. The biomass, EPA production and oil production of the strain were measured respectively. Fig.10As shown, after EPA yield detection, a high-yield EPA mutant strain DBT-64 with an EPA yield of 904.21 mg / L was obtained. The EPA yield of the mutant strain was 490.33% higher than that of the wild-type Schizochytrium ATCC20888, and the oil content was increased by 44.85%. After multiple passages, the mutant strain DBT-64 has stable properties and significant economic effects, and can be used for commercial production of EPA.

[0069] The culture medium formula involved in S2 is as follows:

[0070] The culture medium for triclosan plate: triclosan 4.5 mg / L, glucose 5 g / L, trypsin 1 g / L, yeast powder 1 g / L, sea crystal 17.5 g / L, agar 20 g / L, sterilized at 121℃ for 20 min.

[0071] The culture medium for 2,2-diapyridine plate: 2,2-diapyridine 7.5 mg / L, glucose 5 g / L, trypsin 1 g / L, yeast powder 1 g / L, sea crystal 17.5 g / L, agar 20 g / L, sterilized at 121°C for 20 min.

[0072] The culture medium of the triclosan-2,2-diapyridine composite screening plate: triclosan-2,2-diapyridine 10 mg / L (the concentration ratio of triclosan and 2,2-diapyridine is 3:5), glucose 5 g / L, trypsin 1 g / L, yeast powder 1 g / L, sea crystal 17.5 g / L, agar 20 g / L, sterilized at 121°C for 20 min.

[0073] The seed culture medium, solid culture medium and fermentation medium, seed culture and fermentation culture conditions are the same as those of S1.

[0074] The bacterial biomass determination method, fatty acid content determination method and EPA content determination method involved in S2 are the same as those in S1.

[0075] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A mutant strain of Schizochytrium sp. that produces eicosapentaenoic acid, which is classified and named Schizochytrium sp. DBT-64, and was deposited in the China Center for Type Culture Collection on June 19, 2023, with the deposit number: CCTCCM20231041.

2. The method for inducing mutation breeding of the Schizochytrium mutant strain DBT-64 according to claim 1, characterized in that: The steps include: S1: ARTP mutagenesis and sethoxydim plate screening were performed on Schizochytrium ATCC20888 to construct a high-throughput screening library, and a high-lipid content mutant strain A-32 was obtained based on Nile red staining primary screening and shake flask fermentation secondary screening; S2: The mutant strain A-32 obtained in S1 was subjected to DES mutagenesis, screened on triclosan-2,2-diapyridine composite screening plates, and then cultured by shake flask fermentation to detect EPA production; S3: Screening of Schizochytrium mutant strains with high EPA production based on EPA production.

3. The mutation breeding method according to claim 2, characterized in that: The S1 specifically includes the following steps: S11: Cultivate Schizochytrium ATCC20888 to the logarithmic phase and dilute with PBS buffer to OD 595 =0.6-0.7 to obtain a bacterial suspension, treat the bacterial suspension with ARTP mutagenesis for 40-60s, take 100 μL of the mutagenic bacterial suspension and spread it on a screening plate containing 30-50 mg / L sethoxydim, and culture in the dark; S12: Select the larger colonies screened by the thiamethoxam screening plate in S11, inoculate them in a 48-well cell culture plate containing 1 ml of fermentation medium, and incubate them in a constant temperature shaker. Take 200 μL of the culture solution and inoculate it in a 96-well transparent plate. Measure the OD 595 The culture medium was then transferred to a black 96-well plate, and 50 μL of 20% dimethyl sulfoxide and 5 μL of 100 μg / mL Nile red dye were added. After incubation at 25°C in the dark, the fluorescence intensity was detected by a microplate reader with an excitation wavelength of 485±20 nm and an emission wavelength of 595±20 nm. The FI / OD value of each plate was calculated. 595 The mutants with the top 5 values ​​were screened and the FI / OD 595 The mutant strains with the top 5 values ​​were inoculated into the culture plate again and the FI / OD was measured. 595 Value, FI / OD 595 The strain with the larger value was transferred and preserved to obtain the mutant strain A-32 with high lipid content.

4. The mutation breeding method according to claim 3, characterized in that: The culture medium used for the thiabendazole screening plate described in S11 is the sethofoxadazole solid culture medium: 40 mg / L sethofoxadazole, 5 g / L glucose, 1 g / L trypsin, 1 g / L yeast powder, 17.5 g / L sea crystal, 20 g / L agar, and the remainder is water, sterilized at 121°C for 20 min.

5. The mutation breeding method according to claim 2, characterized in that: The S2 specifically includes the following steps: S21: The mutant strain A-32 was cultured to the logarithmic phase and diluted with PBS buffer to OD 595 =0.6-0.7 to obtain a bacterial suspension, add 50-150 μL of a 20% DES solution by volume, and induce at 25-30°C, 200 rpm for 10-30 min. After the mutagenesis is completed, add a 25% sodium thiosulfate solution by mass to terminate the reaction, resuspend the solution after mutagenesis with sterile PBS buffer, and evenly apply 100 μL of the resuspended solution on a triclosan-2,2-bipyridine composite screening plate containing 5-15 mg / L, and culture in the dark; S22: Select large single colonies obtained from the triclosan-2,2-bipyridine composite screening plate and inoculate them into the seed culture medium. Subculture once every 48 hours for three consecutive times, and then inoculate into the fermentation medium for continuous culture.

6. The mutation breeding method according to claim 5, characterized in that: The culture medium used for the triclosan-2,2-diapyridine composite screening plate described in S21 is triclosan-2,2-diapyridine solid culture medium: 10 mg / L triclosan-2,2-diapyridine, 5 g / L glucose, 1 g / L trypsin, 1 g / L yeast powder, 17.5 g / L sea crystal, 20 g / L agar, the remainder is water, and sterilized at 121°C for 20 min.

7. Use of the Schizochytrium mutant strain DBT-64 according to claim 1 in producing eicosapentaenoic acid.

8. The use according to claim 7, characterized in that: The application is to inoculate the Schizochytrium mutant strain DBT-64 into a seed culture medium, subculture once every 48 hours, and continuously subculture for three times, and then inoculate into a fermentation culture medium for continuous culture for 5 days.

9. The use according to claim 8, characterized in that: The formula of the seed culture medium is glucose 30g / L, tryptone 10g / L, yeast powder 5g / L, sea crystal 15g / L, VB1 0.05g / L, VB6 0.05g / L, VB 12 0.0005g / L, the remainder is water.

10. The use according to claim 8, characterized in that: The formula of the fermentation medium is 100g / L glucose, 17.5g / L sea crystal, 20g / L L-glutamate monosodium water, 2.5g / L potassium dihydrogen phosphate, 7.2g / L magnesium sulfate, 12.8g / L sodium sulfate, 0.4g / L calcium chloride, 0.1g / L LVB1, 0.1g / L LVB6, 0.001g / L LVB 12 , the balance is water.

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