Chlorella mutant strain and application thereof in production of EPA and DHA

Abstract

The invention provides a chlorella mutant strain and application thereof in production of EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), and belongs to the technical field of microalgae biology. The Chlorella sp. Mutant strain M41 provided by the invention is preserved in the China General Microbiological Culture Collection Center on October 18, 2024, the preservation date is October 18, 2024, and the preservation number is CGMCC No. 46154. The Chlorella sp. Mutant strain M41 provided by the invention has the advantages that the preservation number is CGMCC No. 46154; the invention further provides a culture medium and a production method suitable for the chlorella mutant strain M41, and high yield of EPA and DHA is achieved.

Description

Technical Field

[0001] The invention belongs to the field of microalgae biotechnology, and specifically relates to a chlorella mutant and application thereof in the production of EPA and DHA. Background Art

[0002] Fatty acids are an important source of energy for animals and support other activities of the body, especially ω-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have a significant impact on the growth, development, reproduction and other processes of animals. At the same time, they can make up for the animal's own immune deficiency, reduce harmful immune responses, and also have the effects of improving blood circulation, lowering blood pressure, improving blood lipids, and anti-inflammatory. At present, most EPA and DHA are only obtained from the fat of deep-sea fish, which has problems such as low yield and high price. Therefore, how to achieve high yield of EPA and DHA has become an urgent problem to be solved.

[0003] Microalgae cultivation is a cutting-edge and sustainable production method. Microalgae can produce oils through photosynthesis. At the same time, microalgae have the characteristics of fast growth and environmental friendliness, which meet the requirements of modern society for sustainable development. Chlorella sp. is a common microalgae with small individuals and an algal cell diameter of about 2 to 10 μm. It is one of the microalgae currently used to produce oils. However, due to the competitive relationship between the precursors of starch and oil synthesis in algal cells, and the negative correlation between oil and starch accumulation, the ability of Chlorella to produce oil is inhibited. On this basis, the concept of starch synthesis defective mutants is proposed in this field, which restricts the starch synthesis pathway of Chlorella so that its photosynthetic fixed carbon can be synthesized into lipids as much as possible through central carbon metabolism. However, starch synthesis defective mutants of Chlorella generally have low biomass. The accumulation of starch in Chlorella is usually an important part of cell growth and metabolism. When this ability is impaired, the overall biomass of Chlorella will be low, and the low biomass further affects the accumulation of photosynthetic pigments and lipids. Therefore, providing a new starch synthesis-deficient mutant strain of Chlorella and a photosynthetic microalgae biological method for Chlorella to efficiently synthesize EPA and DHA is one of the key issues urgently needed in this field. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the object of the present invention is to provide a Chlorella sp. mutant strain M41, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No46154.

[0005] The present invention also aims to provide a culture medium for increasing the EPA and / or DHA production of the Chlorella mutant strain M41 and a method for efficiently synthesizing EPA and / or DHA, wherein the EPA and DHA contents can reach 3.37 μg / g and 0.86 μg / g, respectively.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Chlorella sp. mutant strain M41, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of October 18, 2024 and a deposit number of CGMCC No 46154.

[0008] The present invention also provides the use of the Chlorella mutant strain M41 in producing EPA and / or DHA.

[0009] The present invention also provides a culture medium for improving the EPA and / or DHA production of the above-mentioned Chlorella mutant strain M41, wherein the culture medium uses a nitrogen-limited culture medium as a basic culture medium, acetic acid is added to the basic culture medium, and the added amount of the acetic acid is 200-1000 μL / L.

[0010] Preferably, the nitrogen-limited culture medium is a nitrogen-limited BG11 culture medium, and the content of NaNO3 in the nitrogen-limited BG11 culture medium is 0.08-0.1 g / L.

[0011] Preferably, the pH of the culture medium is 7-8.

[0012] The present invention also provides a method for producing EPA and / or DHA using the above-mentioned Chlorella mutant strain M41, comprising the following steps: inoculating the above-mentioned Chlorella into the above-mentioned culture medium for cultivation, and collecting EPA and / or DHA from the culture solution.

[0013] Preferably, the inoculation amount of Chlorella is 1×10 6 cell / mL~4×10 6 cell / mL.

[0014] Preferably, the culture temperature is 24-26° C., the light intensity is 1400-1600 Lux, the illumination time is 11-13 h / d, and the culture time is 7-9 d.

[0015] Preferably, the pH value is maintained at 8.5-9.5 during the culturing process.

[0016] The present invention also provides application of the culture medium or the method in producing EPA and / or DHA.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0018] The Chlorella sp. mutant strain M41 provided by the present invention exhibits a smaller cell volume in structure and has limited starch synthesis ability. It was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 18, 2024, with a deposit number of CGMCC No 46154.

[0019] The present invention makes the carbon source (carbon fixed by photosynthesis and acetic acid) obtained from the outside by the starch synthesis defective mutant strain be efficiently converted into oil (EPA and DHA) by additionally supplementing acetic acid in the nitrogen-limited culture medium. At the same time, acetic acid is acidic and has the function of regulating pH while being added as an additional carbon source, neutralizing OH- ions released by algae during photosynthesis during growth, so that the pH of the culture medium is maintained within a suitable range. The present invention cultivates the starch synthesis defective Chlorella by the improved culture medium, and can effectively increase the EPA and / or DHA yield of Chlorella M41, so that the EPA and DHA contents can reach 3.37 μg / g and 0.86 μg / g, respectively, which are 485.24% and 160.70% higher than the wild type, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flowchart and phenotypic identification obtained for mutant strain Chlorella vulgaris M41;

[0021] Figure 2 are the cell density, specific growth rate and dry weight content of WT and M41;

[0022] Figure 3 is the photosynthetic pigment content of WT and M41;

[0023] Figure 4 The yields of polyunsaturated fatty acids EPA and DHA in WT and M41.

[0024] Chlorella M41, classified and named as Chlorella sp., deposited at the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short), deposited at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No: 46154, deposit date October 18, 2024. DETAILED DESCRIPTION

[0025] The present invention uses wild-type Chlorella vulgaris from Erhai Lake, Yunnan as the original algae, and obtains a Chlorella vulgaris mutant by chemical mutagenesis and iodine fumigation screening, which shows a smaller cell volume in structure and has limited starch synthesis ability. The Chlorella vulgaris mutant of the present invention is named M41, and is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit date of October 18, 2024, and a deposit number of CGMCC No 46154.

[0026] The invention provides the use of a chlorella mutant strain M41 in producing EPA and / or DHA, and proposes a culture medium and a culture method for improving the EPA and / or DHA yield of the chlorella mutant strain M41.

[0027] The culture medium for increasing the EPA and / or DHA production of the Chlorella mutant strain M41 of the present invention is a modified culture medium, which is a nitrogen-limited culture medium and is obtained by adding acetic acid to the basic culture medium. The amount of acetic acid added to the culture medium of the present invention is 200 to 1000 μL / L, preferably 250 to 400 μL / L, and more preferably 300 to 350 μL / L. The pH of the culture medium of the present invention is 7 to 8, preferably 7.5.

[0028] As an organic acid, acetic acid can be added to the culture medium as an additional carbon source for Chlorella. Compared with glucose, acetic acid is less likely to cause microbial contamination. At the same time, the weak acidity of acetic acid can buffer the pH value in the cell and maintain a stable growth environment. Finally, acetic acid can be directly converted into Acetyl-CoA in one step, further improving the synthesis efficiency of lipids. Using starch synthesis defective Chlorella as an experimental subject, the present invention adds acetic acid as an additional carbon source in the nitrogen-limited culture medium, which can significantly improve the cell density, photosynthetic pigment content, and dry weight content of starch synthesis defective Chlorella, ensuring that Chlorella efficiently synthesizes EPA and DHA.

[0029] The nitrogen-limited culture medium of the present invention is preferably a nitrogen-limited BG11 culture medium, and the content of NaNO3 in the nitrogen-limited BG11 culture medium is 0.08-0.1 g / L. The formula of the nitrogen-limited BG11 culture medium of the present invention is: NaNO3 0.08-0.1 g / L, K2HPO4 0.03-0.05 g / L, MgSO4·7H2O 0.07-0.08 g / L, CaCl2·2H2O 0.03-0.04 g / L, citric acid 0.005-0.007 g / L, ammonium ferric citrate 0.005-0.007 g / L, Na2EDTA is 0.0005-0.0015 g / L, Na2CO3 is 0.01-0.03 g / L, and A5 trace element mixture is 0.5-1.5 mL / L. Preferably, NaNO3 0.09-0.95 g / L, K2HPO4 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ammonium ferric citrate 0.006 g / L, Na2EDTA 0.001 g / L, Na2CO3 0.02 g / L, A5 trace element mixture 1 mL / L. The A5 trace element mixture of the present invention includes H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, Co(NO3)2·6H2O. Preferably, it includes: H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.22 g / L, Na2MoO4·2H2O 0.39 g / L, CuSO4·5H2O 0.08 g / L, and Co(NO3)2·6H2O 0.05 g / L.

[0030] The present invention also provides a method for producing EPA and / or DHA by a mutant strain of Chlorella, comprising the following steps: inoculating Chlorella into a culture medium for cultivation, and collecting EPA and / or DHA from the culture medium. The inoculation amount of the Chlorella in the present invention is 1×10 6 cell / mL~4×10 6 cell / mL, preferably 2×10 6 cell / mL~2.5×10 6 The temperature of the chlorella culture of the present invention is 24-26°C, preferably 25°C, the light intensity of the culture is 1400-1600 Lux, preferably 1500 Lux, the light time of the culture is 11-13 h / d, preferably 12 h / d, and the culture time is 7-9 d, preferably 8 d.

[0031] The present invention preferably regulates the pH of the culture system during the culturing stage of Chlorella, so that the pH value of the culture is 8.5 to 9.5, and the pH value is preferably 9 to 9.4. As an optional embodiment, during the culturing process, the culture bottle is shaken regularly every day, and the pH is regulated by an acetic acid reagent to maintain it at 8.5 to 9.5. By regulating the pH during the culturing of Chlorella, the present invention can further maintain a stable growth environment, increase the cell density, photosynthetic pigment content, dry weight content, and biological yield of EPA and DHA of Chlorella.

[0032] The present invention collects EPA and DHA from the culture solution, and the collection method can be a conventional method in the art.

[0033] The present invention also provides application of the culture medium or the method in producing EPA and / or DHA.

[0034] The technical solutions 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 a part of the embodiments of the present invention, not all of them. 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.

[0035] In a specific embodiment of the present invention, the formula and dosage of the BG11 culture medium used are as follows: the concentrations of the prepared mother solutions are: NaNO3 150 g / L, K2HPO4 40 g / L, MgSO4·7H2O 75 g / L, CaCl2·2H2O 36 g / L, citric acid 6 g / L, ammonium ferric citrate 6 g / L, Na2EDTA 1 g / L, Na2CO3 20 g / L, A5 trace element mixture (H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.22 g / L, Na2MoO4·2H2O 0.39 g / L, CuSO4·5H2O 0.08 g / L, Co(NO3)2·6H2O 0.05 g / L). Except for NaNO3 added at 10 mL / L, the other components were added at 1 mL / L. The final BG11 medium was: NaNO3 1.5 g / L, K2HPO4 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ammonium ferric citrate 0.006 g / L, Na2EDTA 0.001 g / L, Na2CO3 0.02 g / L, A5 trace element mixture 1 mL / L.

[0036] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example 1

[0039] Wild-type Chlorella (WT) was isolated and purified from water samples collected from Erhai Lake and stored in the Water Ecology Research Laboratory of the College of Agriculture and Biological Sciences of Dali University. The starch mutant (M41) was obtained by chemical mutagenesis and iodine fumigation. The specific method is as follows:

[0040] After concentrating 30L of Erhai water sample to 500mL using a No. 25 plankton collection net, prepare solid BG11 culture medium and pour it into several culture dishes. After solidification, spread 100μL of concentrated water sample on each solid plate. After the algae grow on the plate, pick out wild Chlorella by streaking method and transfer it to new solid culture medium. Repeat the above operation until pure wild Chlorella is obtained.

[0041] Take 1mL of the solution with a concentration of 2×10 6 The supernatant was removed by centrifugation at 1500×g, 1% ethyl methanesulfonate (EMS) solution was added, and the cells were wrapped with tin foil to avoid induction for 2 h. The reaction was terminated with 10% Na2S2O3, and washed twice with 0.2 M PBS and diluted to 10 4 Finally, at 50 μmol photons m -2 s -1 At 25°C, 100 μL of algae solution was applied to each plate and cultured for 5 days. After the 10th day, the light intensity was 150 μmolphotons m -2 s -1 Use a 10 μL pipette tip to transfer the monoclonal algae to a 96-well cell culture dish, and transfer another 10 μL to 1 / 16N nitrogen-limited solid medium for high light (300 μmol photons m -2 s -1) was cultured for 5 days, and then the position of the mutant was determined by iodine fumigation (iodine particles volatilize and encounter synthetic starch, and the Chlorella will turn gray and black, while the mutant without synthetic starch will not change color). Then the position of the target mutant in the 96-well cell culture dish was determined according to its position, so as to obtain the mutant. Then, the mutant was obtained and subcultured 1000 times under the stress of adding acetic acid at a concentration of 0.1% (1000μL / L) using the strategy of adaptive laboratory evolution until the mutant was fully adapted to the growth under high concentration of acetic acid. The mutant with the best growth performance was selected and named M41. Single colonies of WT and M41 were extracted from the agar plate and inoculated into 50mL conical flasks containing 20mL BG11 medium respectively. Cultured in a constant temperature incubator with a set value of 25℃, a light intensity of 800Lux, and a light time of 12h (LFR type, Longfei Seed Equipment, China). Shake twice a day and culture two algae strains to the logarithmic growth phase.

[0042] Wild-type Chlorella and M41 were observed by optical microscopy.

[0043] The starch content of wild-type Chlorella and M41 was semi-quantitatively determined: 1 mL of algae solution was centrifuged at 12,000 rpm for 10 min (TGL-16B, Shanghai Anting Scientific Instruments, China), and the supernatant was discarded. 1 mL of methanol was added to the precipitate and mixed, and the sample precipitate after centrifugation was dissolved in methanol for further starch determination. The iodine-starch colorimetric method was used for semi-quantitative determination. 2.5 μL of Lugo reagent (composed of iodine and potassium iodide) was added to the precipitate, the reagent and the precipitate were fully mixed, and the precipitate was allowed to stand in the dark to observe the color reaction formed by the precipitate and the reagent.

[0044] Flow chart of obtaining mutant strain Chlorella M41 and phenotypic identification Figure 1 As shown in the figure, A is the screening flow chart, B is the cell morphology of wild-type Chlorella and M41, and C is the semi-quantitative detection result of starch content of wild-type Chlorella and M41. WT is wild-type Chlorella, and Ac is acetic acid. The results show that the volume of a single cell of M41 is slightly smaller than that of WT. The starch-rich WT cells are stained dark black by iodine solution, while the color of M41 cells is only the color of iodine solution, indicating that the starch synthesis pathway of M41 is obviously restricted and starch cannot be synthesized, which is a starch synthesis-deficient Chlorella.

[0045] The mutant strain M41 exhibits a smaller cell volume in structure and has limited starch synthesis ability. The mutant strain M41 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 18, 2024, with the deposit number CGMCC No 46154.

[0046] Example 2

[0047] Culture medium for improving EPA and DHA production in mutant strains of Chlorella vulgaris:

[0048] The nitrogen-limited BG11 medium was used as the basic medium, acetic acid was added at 300 μL / L, and the pH was adjusted to 7.5 using an acetic acid reagent.

[0049] Preparation of nitrogen-limited BG11 medium: The concentrations of the prepared mother solution are: NaNO3 150 g / L, K2HPO4 40 g / L, MgSO4·7H2O 75 g / L, CaCl2·2H2O 36 g / L, citric acid 6 g / L, ammonium ferric citrate 6 g / L, Na2EDTA 1 g / L, Na2CO3 20 g / L, A5 trace element mixture (H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.22 g / L, Na2MoO4·2H2O 0.39 g / L, CuSO4·5H2O 0.08 g / L, Co(NO3)2·6H2O 0.05 g / L). Except for NaNO3 added at 625 μL / L, the other components were added at 1 mL / L. The final nitrogen-limited BG11 culture medium was: NaNO3 0.09375 g / L, K2HPO4 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ammonium ferric citrate 0.006 g / L, Na2EDTA 0.001 g / L, Na2CO3 0.02 g / L, and A5 trace element mixture 1 mL / L.

[0050] Example 3

[0051] Culture medium for improving EPA and DHA production in mutant strains of Chlorella vulgaris:

[0052] The nitrogen-limited BG11 medium was used as the basal medium, acetic acid was added at 200 μL / L, and the pH was adjusted to 7 using an acetic acid reagent. The nitrogen-limited BG11 medium was prepared as in Example 2.

[0053] Example 4

[0054] Culture medium for improving EPA and DHA production in mutant strains of Chlorella vulgaris:

[0055] The nitrogen-limited BG11 medium was used as the basal medium, acetic acid was added at 400 μL / L, and the pH was adjusted to 8 using an acetic acid reagent. The nitrogen-limited BG11 medium was prepared as in Example 2.

[0056] Example 5

[0057] Method for efficiently synthesizing EPA and DHA by the Chlorella M41 mutant (using the culture medium described in Example 2):

[0058] Chlorella M41 expansion culture:

[0059] The initial cell density was 2 × 10 6 The inoculation volume of Chlorella M41 was 2×10 6 cell / mL was inoculated into a 250 mL conical flask containing 150 mL of culture medium and cultured for 8 days in a constant temperature incubator (LFR type, Longfei Seed Equipment, China) with a temperature of 25 °C, a light intensity of 1500 Lux, and a lighting time of 12 h to obtain a culture solution rich in EPA and DHA.

[0060] Example 6

[0061] Method for efficiently synthesizing EPA and DHA by the Chlorella M41 mutant (using the culture medium described in Example 2):

[0062] Chlorella M41 expansion culture:

[0063] The initial cell density was 4 × 10 6 The inoculation volume of Chlorella M41 was 4×10 6 cell / mL was inoculated into a 250 mL conical flask containing 150 mL of culture medium and cultured for 7 days in a constant temperature incubator (LFR type, Longfei Seed Equipment, China) with a temperature of 24 °C, a light intensity of 1600 Lux, and a lighting time of 12 h to obtain a culture solution rich in EPA and DHA.

[0064] Example 7

[0065] Method for efficiently synthesizing EPA and DHA by the Chlorella M41 mutant (using the culture medium described in Example 2):

[0066] Chlorella M41 expansion culture:

[0067] The initial cell density was 2 × 10 6 The inoculation volume of Chlorella M41 was 2×10 6 cell / mL was inoculated into a 250mL conical flask containing 150mL of culture medium and cultured for 8 days in a constant temperature incubator (LFR type, Longfei Seed Equipment, China) at a temperature of 25°C, a light intensity of 1500Lux, and a light duration of 12h. During the culture process, the conical flask was shaken regularly every day and the pH was regulated to maintain it at 9.5. After the culture, a culture solution rich in EPA and DHA was obtained.

[0068] Test Example 1

[0069] Acetate group: Initial cell density was 2 × 10 6cell / mL of Chlorella M41 and wild-type Chlorella (WT) were inoculated at 2×10 6 cell / mL was inoculated into a 250mL conical flask containing 150mL of culture medium (the culture medium described in Example 2), and cultured for 8 days in a constant temperature incubator (LFR type, Longfei Seed Equipment, China) at a temperature of 25°C, a light intensity of 1500Lux, and a light duration of 12h. During the culture process, the conical flask was shaken regularly every day, and the pH was regulated to maintain it at 9.5. After the culture was completed, a culture solution rich in EPA and DHA was obtained.

[0070] Group without acetic acid addition: Initial cell density was 2×10 6 cell / mL of Chlorella M41 and wild-type Chlorella (WT) were inoculated at 2×10 6 cell / mL was inoculated into a 250mL conical flask containing 150mL of culture medium (nitrogen-limited BG11 culture medium, the preparation method is the same as that of the nitrogen-limited BG11 culture medium in Example 2), and cultured for 8 days in a constant temperature incubator (LFR type, Longfei Seed Equipment, China) at a temperature of 25°C, a light intensity of 1500Lux, and a light duration of 12h. During the culture process, the conical flask was shaken regularly every day, and the pH was regulated to maintain it at 9.5. After the culture was completed, a culture solution rich in EPA and DHA was obtained.

[0071] During the culture period, the algae liquid was counted every 2 days. After the 8th day of culture, an appropriate amount of algae liquid sample was taken to measure the chlorophyll content, dry weight, starch content, monosaccharide content, and fatty acid content in the algae cells (sent to Nomi Metabolism Co., Ltd. (China) for testing):

[0072] (1) The chlorophyll measurement method is as follows: take 1 mL of algae liquid, centrifuge it at 12000 rpm for 10 min (TGL-16B, Shanghai Anting Scientific Instrument, China), and discard the supernatant. Add 1 mL of methanol to each precipitate and mix well, then incubate in the dark at 60°C for 15 min. Centrifuge it again at 12000 rpm for 10 min. The obtained supernatant is measured by a spectrophotometer (V-5100, Shanghai Yuanxi Instrument, China) to determine the pigment content. Photosynthetic pigments are measured using the following three formulas.

[0073] Chlorophyll a=13.9(A 665 -A 750 );

[0074] Chlorophyll b = 21.43A 644 -4.65A 662 ;

[0075] Carotenoids = 4.7A 440 -(1.38A 662 +5.48A 644 )

[0076] (2) The dry weight measurement method is as follows: first, place the glass fiber filter membrane (water system 50 mm, Tianjin Jinteng Experimental Equipment, China) in a constant temperature oven (65°C) (101-1BS, Shaoxing Supo Instrument, China) for 12 h, then take it out and weigh it, record it as We, then take out 50 mL of algae solution cultured to the logarithmic phase (volume record it as V) through the filter membrane and wash it twice with 0.5 M ammonium bicarbonate, using 10 mL. After the filtered filter paper is placed in an oven to dry for 12 h, take it out and weigh the dry weight, record it as Ws, and the dry weight is calculated using the following formula.

[0077] DW(g / L)=(Ws-We) / V

[0078] (3) The semi-quantitative method for starch content is the same as in Example 1.

[0079] (4) The monosaccharide measurement method is as follows: take 50 mg of sample, add 500 μL 50% methanol water, add 4 2 mm steel beads; put it into a high-throughput tissue grinder (TISSUELYSER-64, Shanghai Jingxin Technology, China), oscillate at 30 Hz for 20 s, let it stand for 10 s, cycle 8 times, then add 500 μL 50% methanol water, vortex (MS3 basic, IKA, Germany) for 1 min, and then sonicate for 5 min; centrifuge at 12000 rpm (EPPENDORF, Edelweiss, Germany) for 2 min, take 400 μL of the supernatant solution and add it to a new 2 mL The sample was concentrated to dryness in an EP tube using a vacuum concentrator (8-10 h), and the remaining supernatant was placed in a -80°C refrigerator (BIO-MEDICAL, HAIER, China) for backup; 80 μL of 20 mg / mL methoxyamine pyridine solution was added to the concentrated sample for re-dissolution, vortexed for 30 s, and incubated at 60°C for 60 min; finally, 100 μL of BSTFA-TMCS (99:1) derivatization reagent was added, vortexed for 30 s, incubated at 70°C for 90 min, centrifuged at 14000 rpm for 3 min, and 90-100 μL of the supernatant was added to the detection bottle; gas chromatography-mass spectrometry (gas chromatograph: 7890, Agilent, USA; mass spectrometer: LECO BT time-of-flight mass spectrometer, LECO, USA) detection was completed within 24 h.

[0080] (5) The fatty acid content was measured as follows: accurately weigh an appropriate amount of sample into a 2 mL centrifuge tube, accurately add 1 mL of chloroform-methanol (2:1) solution, add 100 mg of glass beads, immerse the sample tube in liquid nitrogen, freeze for 2 min, wait for the reagent in the sample tube to thaw, then put the sample tube into a tissue grinder (MB-96S, Meibi, China) and shake at 60 Hz for 1 min, repeat twice, centrifuge at 12000 rpm and 4 °C (H2050R, Xiangyi, China) for 5 min, take all the supernatant into a 15 mL centrifuge tube; accurately add 2 mL of 1% sulfuric acid-methanol solution, mix thoroughly and shake for 1 min; esterify in a 80 °C water bath (HWS-26, Yiheng, China) for 30 min; take out and cool, accurately add 1 mL of n-hexane for extraction, shake and mix (MV54, SCRC, China) for 30 s, let stand for 5 min, then add 5 mL of H2O (4 °C) for washing, and centrifuge at 3500 rpm. Centrifuge at 4℃ for 10min; accurately pipette 700μL supernatant into a 2mL centrifuge tube, add 100mg anhydrous sodium sulfate powder to remove excess water, shake and mix for 30s, and centrifuge at 12000rpm for 5min; after the sample is diluted 5 times, accurately pipette 300μL dilution into a 2mL centrifuge tube, add 15μL 500ppm methyl salicylate as internal standard, shake and mix for 10s, and accurately pipette 200μL supernatant into the test bottle. Then refer to the method of Hoving and Beccaria et al. (Beccaria et al., 2018; Hoving, Heijink, van Harmelen, van Dijk, & Giera, 2018) for gas chromatography-mass spectrometry (gas chromatograph: TRACE 1300, Thermo, USA; mass spectrometer: ISQ 7000, Thermo, USA) analysis of long-chain fatty acids.

[0081] Cell density, specific growth rate and dry weight content of WT and M41 Figure 2 As shown. In the figure, A shows the change of WT and M41 cell density without acetic acid addition; B shows the change of WT and M41 cell density under acetic acid addition; C shows the comparison of M41 specific growth rate before and after acetic acid addition; D shows the dry weight of WT and M41 without acetic acid addition; E shows the dry weight of WT and M41 under acetic acid addition. +Ac means acetic acid addition, -Ac means no acetic acid addition. The difference between M41 and WT was statistically significant (P<0.05), marked by letters a and b. Values ​​are expressed as mean ± standard deviation (n=3). The results showed that the cell density of M41 was significantly higher than that of WT, especially under acetic acid addition, when the cell density of M41 reached 6.5×10 on the 8th day. 7 cell / L, compared with 3.9×10 7cell / L, the cell density of M41 increased by 66.67%. At the same time, compared with the case without adding acetic acid, the specific growth rate of M41 increased significantly, with a year-on-year increase of 22.22%. The results of the dry weight content of M41 and WT under the two culture modes showed that on the 8th day of culture, the dry weight of M41 was only 0.45 mg / L, while that of WT was 0.55 mg / L. The dry weight content of M41 was 18.35% lower than that of WT. After adding acetic acid, the dry weight of M41 reached 0.79 mg / L, and the dry weight increased by 33% compared with WT cultured with the same addition of acetic acid.

[0082] The photosynthetic pigment contents of WT and M41 are as follows Figure 3 As shown. In the figure, A is the photosynthetic pigment content of WT and M41 without the addition of acetic acid; B is the photosynthetic pigment content of WT and M41 with the addition of acetic acid; +Ac is the addition of acetic acid, -Ac is the absence of acetic acid, Chlorophyll a is chlorophyll a, Chlorophyll b is chlorophyll b, and Carotenoid is carotenoid. The differences between M41 and WT were statistically significant (P<0.05), marked by letters a, b, c, d, and e. In addition, in the absence of Ac, the levels of chlorophyll a and carotenoid changed less compared with WT (P<0.05), and the chlorophyll b content did not change significantly (P>0.05). However, when Ac was added, these photosynthetic pigments in M41 increased significantly (P < 0.05), with chlorophyll a, chlorophyll b and carotenoids reaching 12.48 mg / L, 3.11 mg / L and 5.52 mg / L, respectively, representing increases of 131.19%, 113.21% and 101.99%, respectively.

[0083] The polyunsaturated fatty acid EPA and DHA production results of WT and M41 are shown in Figure 4As shown. In the figure, A is the chemical structure of EPA; B is the chemical structure of DHA; C is the content of EPA and DHA; D is the content of EPA and DHA in a single cell; E is the yield of EPA and DHA; F is the yield of EPA and DHA. WT is wild-type Chlorella, and M41 is starch synthesis-deficient Chlorella. The difference between M41 and WT is statistically significant (P<0.05), marked by letters a and b. The values ​​are expressed as mean ± standard deviation (n=3). The results showed that after adding acetic acid to ω-3 fatty acids C20:5 (EPA) and C22:6 (DHA) under nitrogen-limited conditions, the contents of EPA and DHA in M41 reached 3.37μg / g and 0.86μg / g, respectively, which increased by 485.24% and 160.70% respectively compared with WT. The EPA and DHA content in a single cell of M41 also increased significantly (P < 0.05), reaching 0.52pg / Cell and 0.13pg / Cell, respectively, an increase of 439.58% and 60.58% compared with WT, respectively. The EPA and DHA production of M41 reached 4.27mg / L and 1.46μg / L, respectively, an increase of 484.93% and 160.71% compared with WT, respectively (P < 0.05). The EPA and DHA yields of M41 reached 0.53mg / L / d and 0.18mg / L / d, respectively, an increase of 484.93% and 160.71% compared with WT, respectively (p < 0.05).

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chlorella sp. mutant strain M41, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is October 18, 2024, and the deposit number is CGMCC No 46154.

2. Use of the Chlorella mutant strain according to claim 1 in producing EPA and / or DHA.

3. A culture medium for improving the EPA and / or DHA production of the Chlorella mutant strain M41 according to claim 1, characterized in that: The culture medium uses nitrogen-limited culture medium as the basic culture medium, and acetic acid is added to the basic culture medium, and the added amount of the acetic acid is 200-1000 μL / L.

4. The culture medium according to claim 3, characterized in that The nitrogen-limited culture medium is a nitrogen-limited BG11 culture medium, and the content of NaNO3 in the nitrogen-limited BG11 culture medium is 0.08-0.1 g / L.

5. The culture medium according to claim 3, characterized in that The pH of the culture medium is 7-8.

6. A method for producing EPA and / or DHA using the Chlorella mutant M41 according to claim 1, characterized in that: The method comprises the following steps: the chlorella described in claim 1 is inoculated into the culture medium described in any one of claims 3 to 5 for cultivation, and EPA and / or DHA are collected from the culture solution.

7. The method according to claim 6, characterized in that The inoculation amount of Chlorella is 1×10 6 ~4×10 6 cell / L.

8. The method according to claim 6, characterized in that The culture temperature is 24-26° C., the light intensity is 1400-1600 Lux, the light time is 11-13 h / d, and the culture time is 7-9 d.

9. The method according to claim 6, characterized in that The pH value is maintained at 8.5-9.5 during the culture process.

10. Use of the culture medium according to any one of claims 3 to 5 or the method according to any one of claims 6 to 9 in producing EPA and / or DHA.

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