Schizochytrium limacinum mutagenesis strain A1-11 and application thereof in synthesis of odd carbon chain fatty acid

Abstract

The invention belongs to the technical field of microbial engineering and synthetic biology, and particularly relates to a schizochytrium limacinum mutant strain A1-11 and application thereof in synthesis of odd-carbon-chain fatty acid. According to the invention, ARTP plasma mutagenesis is carried out on wild-type Aurantiochytrium sp. Sdu080, a mutagenic strain A1-11 with genetic stability is successfully screened, and compared with an original strain, the total proportion of C15: 0 and C17: 0 of the mutagenic strain under a conventional shake-flask culture condition is increased from 7.17% to 33.13%. Furthermore, after 20 mM of L-valine is added into the fermentation liquid culture medium, the proportion of odd fatty acid of A1-11 can be further increased to 52.14%. The schizochytrium limacinum A1-11 provided by the invention not only remarkably breaks through the limitation of the odd fatty acid yield and proportion in the prior art, but also has the advantages of simple process, easiness in large-scale fermentation and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering and synthetic biology technology, specifically relating to a Schizochytrium mutant strain A1-11 and its application in the synthesis of odd-chain fatty acids. Background Technology

[0002] Fatty acids are naturally found in bacteria, fungi, algae, higher plants, and animals. Most fatty acids, such as caprylic acid (8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), and stearic acid (18:0), contain an even number of carbon atoms, hence they are called even-number fatty acids. In many studies, odd-number fatty acids, such as pentadecanoic acid (C15:0) and margarine (C17:0), have been shown to be positively correlated with health. Serum levels of odd-chain fatty acids are negatively correlated with the risk of cardiovascular disease. Higher levels of odd-chain fatty acids in the blood can alleviate type 2 diabetes, improve fatty acid metabolism, and enhance anti-inflammatory capabilities (Zhang LS, Liang S, Zong MH, et al. Microbial synthesis of functional odd-chain fatty acids: a review[J]. World Journal of Microbiology and Biotechnology, 2020, 36(3).DOI:10.1007 / s11274-020-02814-5.).

[0003] Odd-number fatty acids are mainly obtained through chemical synthesis or extraction from natural products, which have drawbacks such as pollution or unsustainability. Microbial fermentation has become one of the most promising methods for the industrial production of odd-number fatty acids. Schizochytrium sp., an oil-containing microorganism, can not only efficiently synthesize docosahexaenoic acid (DHA, C22:6n-3) via the polyketide synthase (PKS) pathway, but also accumulate odd-number fatty acids via the fatty acid synthase (FAS) pathway (Li Z, Meng T, Ling X, et al. Overexpression of Malonyl-CoA: ACP Transacylase in Schizochytrium sp. to Improve Polyunsaturated Fatty Acid Production[J]. Journal of Agricultural and Food Chemistry, 2018.DOI:10.1021 / acs.jafc.8b01026.). Based on substrate feeding strategies and metabolic engineering, the strain was able to synthesize 2.34 g / L of odd-number fatty acids, but the titer and proportion of odd-number fatty acids still cannot meet the needs of industrial production. Therefore, strains that produce high levels of odd-number fatty acids are needed to achieve industrial-scale production. Summary of the Invention

[0004] The purpose of this invention is to address existing problems by providing a composite material based on bio-straw powder and polypropylene, and a method for preparing the same.

[0005] This invention is achieved through the following technical solution: A Schizochytrium mutant strain A1-11, the preservation name of which is... Aurantiochytrium sp. sdu080 A1-11 was deposited at the China Center for Type Culture Collection on August 22, 2025, with accession number CCTCC NO: M 20251884.

[0006] Furthermore, the Schizochytrium mutant strain A1-11 was derived from the starting strain Aurantiochytrium sp. sdu080 was obtained by ambient pressure room temperature plasma (ARTP) mutagenesis.

[0007] Furthermore, the specific conditions for the ambient pressure room temperature plasma (ARTP) mutagenesis are as follows: the starting strain... Aurantiochytrium After preparing a bacterial suspension of sp. sdu080 with an OD600 of 0.6-0.8, it was placed on the sample stage of an ARTP mutagen and subjected to mutagenesis treatment with plasma irradiation for 20-60 s.

[0008] A method for producing odd-numbered carbon chain fatty acids, the method comprising the following steps: 1) Seed culture: The above-mentioned Schizochytrium mutant strain A1-11 was inoculated into seed liquid culture medium and cultured overnight at 25°C with shaking at 600 rpm to obtain seed liquid; The seed liquid culture medium consists of: 10 g / L glucose, 20 g / L yeast extract, and 15 g / L sea salt. 2) Fermentation culture: At an inoculum rate of 2%, the seed culture obtained in step 1) was transferred to the fermentation liquid culture medium and cultured at 25°C and 600 rpm for 4 days with shaking. The fermentation broth containing the cells was then collected. The fermentation liquid culture medium consists of: 60 g / L glucose, 20 g / L yeast extract, and 15 g / L sea salt. 3) Oil extraction and detection: Extract the microbial oil from the fermentation broth in step 2) to obtain oil containing odd-numbered carbon chain fatty acids.

[0009] Furthermore, the odd-numbered carbon chain fatty acids include pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0).

[0010] Furthermore, the fermentation liquid culture medium contains precursor substances that promote the synthesis of odd-chain fatty acids.

[0011] Furthermore, the precursor is propionate or L-valine, and the concentration of the precursor in the fermentation liquid culture medium is: propionate 5-20 mM, or L-valine 10-30 mM.

[0012] Furthermore, the propionate is selected from one or more of sodium propionate, potassium propionate, and calcium propionate.

[0013] Furthermore, the specific steps of extracting bacterial cell oil and performing methyl esterification treatment on the oil include: a) Take 2 mL of fermentation broth containing bacteria into a pre-weighed 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, and discard the supernatant; centrifuge at 500 rpm for 5 s to allow the liquid droplets on the tube wall to settle, open the centrifuge tube cap, and pre-freeze at -80℃ for at least 30 min; b) Transfer the pre-frozen sample to a freeze dryer and freeze-dry overnight under vacuum ≤10 Pa and temperature ≤-50℃. Determine the drying quality of the freeze-dried cells. c) Weigh 40-50 mg of lyophilized bacterial cells, add 600 μL of 1 M HCl solution, vortex at 2000 rpm for 30 s, and let stand at room temperature for 30 min; then perform boiling water bath for 5 min and freezing at -80℃ for 5 min in sequence, and repeat this temperature cycle once. d) Once the sample has recovered to 25±2℃, add 900 μL of chloroform-methanol mixture (2:1, v / v), vortex at 2000 rpm for 30 s, centrifuge at 12000 rpm for 3 min, and transfer the lower organic phase; add 600-750 μL of saturated sodium chloride solution, vortex, and centrifuge at 12000 rpm for 3 min. e) Transfer the lower oil phase to a pre-weighed 10 mL glass bottle and vacuum dry at 55 °C for 2-2.5 h to obtain bacterial oil.

[0014] Furthermore, the procedure also includes the steps of methylation and detection of the bacterial cell oil obtained in step f): 1 mL of chloroform and 2.5 mL of sulfuric acid-methanol mixture (sulfuric acid volume fraction 2%) are added to the dried bacterial cell oil, and the mixture is capped and methylated at 85°C for 1-2.5 h; after returning to room temperature, the cap is opened, 3 mL of n-hexane and 3 mL of saturated sodium chloride are added, and the mixture is vigorously mixed and extracted until the layers are completely separated; 1 mL of the upper organic phase is taken and filtered, and the content of odd-chain fatty acids is detected by GC-MS analysis.

[0015] The present invention has the following advantages over the prior art: This invention utilizes wild-type Schizochytrium fungi Aurantiochytrium ARTP plasma mutagenesis was performed on sp. sdu080, and a genetically stable mutant strain A1-11 was successfully screened. Compared with the starting strain, the total proportion of odd fatty acids (C15:0 and C17:0) in this mutant strain increased from 7.17% to 33.13% under conventional shake-flask culture conditions, with C15:0 monomers accounting for as high as 23.04%. Further addition of 20 mM L-valine to the fermentation liquid medium further increased the proportion of odd fatty acids in A1-11 to 52.14%, with C15:0 accounting for 44.38%, which is the highest level reported to date.

[0016] In the scale-up experiment in a 5L fermenter, the biomass of A1-11 reached 154.35 g / L, the total oil yield was 94.91 g / L, and the oil accounted for 61.49% of the cell dry weight. The total yield of odd-chain fatty acids was 53.26 g / L, accounting for 56.12% of the total lipids, of which C15:0 accounted for 48.80% and C17:0 accounted for 7.32%. At the same time, DHA (C22:6n-3) maintained a high proportion of 30.04%, achieving synergistic high yield of odd-chain fatty acids and ω-3 polyunsaturated fatty acids.

[0017] Therefore, the Schizochytrium A1-11 provided by this invention not only significantly breaks through the limitations of odd fatty acid yield and proportion in the prior art, but also has advantages such as simple process, low substrate cost, and easy large-scale fermentation, providing a green, sustainable, and high-value-added new way of producing functional oils for the food, medicine, and health product industries. Attached Figure Description

[0018] Figure 1 The fatty acid composition accumulated by the mutant strain A1-11; Figure 2 The effects of propionate and L-valine on fatty acids in the A1-11 mutant strain. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 In this invention: The Latin name of Schizochytrium Aurantiochytrium sp. sdu080 was isolated from beach soil in Shenzhen Bay. Moist soil samples were collected from 1-3 cm below the surface. Under aseptic conditions, 10 g of soil was added to an Erlenmeyer flask containing artificial seawater. After shaking and incubation at 30°C, the supernatant was diluted and spread onto seed culture medium containing kanamycin. Colony morphology was observed after incubation. Colonies that were white, pale yellow, round, smooth, raised, and viscous were selected for purification culture, ultimately yielding the strain. Aurantiochytrium sp. sdu080.

[0021] from Aurantiochytrium Starting with sp. sdu080, ARTP mutagenesis yielded a genetically stable Schizochytrium mutant strain A1-11. This genetically stable Schizochytrium mutant strain A1-11 was deposited on August 22, 2025, at the China Center for Type Culture Collection (CCTCC; address: No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China), under the name […]. Aurantiochytrium sp. sdu080 A1-11, accession number CCTCC NO: M 20251884.

[0022] The culture medium involved in this invention is as follows: Seed liquid culture medium: 10 g / L glucose, 20 g / L yeast extract, 15 g / L sea salt.

[0023] Solid culture medium: 10 g / L glucose, 20 g / L yeast extract, 10 g / L sea salt, 10 g / L agar.

[0024] Fermentation liquid culture medium: 60 g / L glucose, 20 g / L yeast extract, 15 g / L sea salt.

[0025] The oil extraction process and detection method involved in the embodiments of the present invention are as follows: 1) Take 2 mL of the culture medium (containing the cell suspension) after the fermentation of Schizochytrium is completed and put it into a 2 mL centrifuge tube that has been weighed. Centrifuge at 12000 rpm for 1 min and discard the supernatant. After a short centrifugation (500 rpm, 5 s) to allow the liquid droplets on the tube wall to settle, open the centrifuge tube cap and place it at -80℃ for pre-freezing for more than 30 min. 2) Transfer the pre-frozen sample to a freeze dryer for overnight freeze-drying; 3) Take out the freeze-dried Schizochytrium mycelium, determine its dry weight and calculate the dry weight of the mycelium (DCW). 4) Accurately weigh 40-50 mg of lyophilized bacterial sample, add 600 μL of 1 M HCl solution, vortex to mix (2000 rpm, 30 s), and let stand at room temperature for 30 min; then perform the following temperature cycle: ① boil water bath treatment for 5 min, ② immediately transfer to -80℃ and freeze for 5 min; repeat this temperature cycle once. 5) After the sample temperature has naturally equilibrated to room temperature (25±2℃), add 900 μL of chloroform-methanol mixture (2:1, v / v), vortex to mix (2000 rpm, 30 s); centrifuge at 12000 rpm for 3 min, transfer the lower organic phase to a new 2 mL centrifuge tube; add 600-750 μL of saturated sodium chloride solution, vortex to mix, and centrifuge at 12000 rpm for 3 min. 6) Use a 200 μL pipette tip to transfer the lower oil phase into a 10 mL glass bottle (weigh the empty bottle beforehand), and dry under vacuum at 55℃ for 2-2.5 h; 7) After the temperature returns to room temperature, weigh and calculate the total amount of oil, add 1 mL of chloroform and 2.5 mL of sulfuric acid-methanol mixture (2% sulfuric acid), cap and methyl esterify at 85℃ for 1-2.5 h; 8) After returning to room temperature, open the lid, add 3 mL of n-hexane and 3 mL of saturated sodium chloride, mix vigorously and extract, and let stand until completely separated; 9) Take 1 mL of the upper organic phase and filter it into a sample vial; 10) Perform GC-MS detection: Analyze the upper organic phase containing fatty acid methyl esters in the sample vial using gas chromatography-mass spectrometry (GC-MS) to determine the fatty acid composition and the relative content of each component, especially to quantitatively detect the percentage of odd-numbered fatty acids (C15:0, C17:0) in the total lipids.

[0026] Example 1: Preparation of Schizochytrium mutant strain A1-11 1. Atmospheric pressure room temperature plasma (ARTP) mutagenesis treatment ARTP (Artificial Mutagenesis Propagation) induces gene mutations in target microorganisms by uniformly and efficiently damaging their DNA and other biomolecules through oxidizing ions and free radicals at room temperature and pressure. Compared with traditional mutagens such as ultraviolet light and X-rays, this mutagenesis technology has a wider radiation range and its mutagenesis efficiency can be dozens of times higher.

[0027] 1) Preparation of bacterial suspension: ... Aurantiochytrium sp. sdu080 was inoculated into seed culture medium, incubated overnight at 25°C, centrifuged, washed twice with physiological saline, and resuspended in physiological saline to allow its OD to adjust. 600 Between 0.6 and 0.8; 2) ARTP mutagenesis treatment: The bacterial suspension was divided into three groups and placed on the sample stage of the ARTP mutagenesis instrument. Each group was independently treated with plasma irradiation for 20 s, 40 s, and 60 s, respectively. After mutagenesis, each group of bacterial suspension was diluted 10... 4 The solution is spread evenly on the surface of a solid culture medium and incubated at a constant temperature of 25°C until a single colony is formed.

[0028] 2. Preliminary screening of mutant strains 1) Select individual single colonies from the mutagenized colonies, inoculate them into new solid culture medium and continue to isolate them to obtain single colonies with high purity. 2) Add 1 mL of seed liquid culture medium to the deep well plate, pick a single colony and inoculate it into the culture medium, and culture at 25°C and 600 rpm for 2 days with shaking; then aspirate the seed liquid, add fermentation liquid culture medium to the cell precipitate of the same deep well plate, and culture at 25°C with shaking for 4 days; 3) Extract the oil, methylate it, and then analyze and detect it by GC-MS.

[0029] Results: Analysis of selected single colonies (see Table 1) showed that, compared with the starting strain sdu080, the proportion of odd fatty acids in most colonies was slightly increased, while only the proportion of odd fatty acids in strain A1-11 was significantly increased, with the proportion of C15:0 increasing to 20.35%, and the total proportion of odd fatty acids (C15:0 and C17:0) reaching 29.12%.

[0030] Table 1 Preliminary screening of mutant strains

[0031] 3. Secondary screening of mutant strains Based on the results of the initial screening, strain A1-11, which has a high proportion of odd fatty acids (C15:0, C17:0), was selected for amplification culture.

[0032] 1) Select the superior mutant strain and inoculate it into a 100 mL shake flask containing 30 mL of seed liquid culture medium, and incubate overnight at 25°C with shaking. 2) Transfer the seed culture to a 250 mL shake flask containing 50 mL of fermentation liquid culture medium at an inoculation rate of 2%, and incubate at 25°C with shaking for 4 days; 3) Extract the oil, methylate it, and then analyze and detect it by GC-MS.

[0033] Results: Compared with the primary screening system, the secondary screening system had a higher dissolved oxygen level, which was more conducive to the metabolism of fatty acid synthesis pathway FAS, thereby promoting the synthesis of odd-number fatty acids. Therefore, after secondary screening, the proportion of odd-number fatty acids C15:0 and C17:0 in the A1-11 mutant strain reached 33.13%, of which C15:0 accounted for 23.04%. Figure 1 (As shown).

[0034] Example 2: Fermentation optimization of A1-11 mutant strain Propionyl-CoA is a key precursor for the accumulation of odd-number fatty acids. The synthesis of odd-number fatty acids can be promoted by adding propionate or propionyl-CoA precursors such as valine. To investigate the effects of different substrates on the accumulation of odd-number fatty acids in the mutant strain A1-11, sodium propionate, potassium propionate, calcium propionate, and L-valine were added to the fermentation liquid medium, respectively.

[0035] 1) Inoculate the A1-11 mutant strain into seed liquid culture medium and culture overnight at 25°C with shaking; 2) At an inoculation rate of 2%, the seed culture was inoculated into four independent fermentation liquid media: the first group was supplemented with 10 mM sodium propionate as the only exogenous precursor, the second group was supplemented with 10 mM potassium propionate as the only exogenous precursor, the third group was supplemented with 10 mM calcium propionate as the only exogenous precursor, and the fourth group was supplemented with 20 mM L-valine as the only exogenous precursor. 3) Incubate at 25℃ with shaking for 4 days, collect the bacterial cells, and extract the oil; 4) After methylation, GC-MS detection was performed; Result: As Figure 2As shown, the addition of potassium propionate, sodium propionate, and L-valine promoted the accumulation of C15:0. Specifically, the C15:0 proportion increased to 32.03% with the addition of potassium propionate; to 36.39% with the addition of sodium propionate; and to 44.38% with the addition of L-valine, which is the highest proportion reported. Therefore, potassium propionate, sodium propionate, and L-valine are beneficial for the accumulation of odd-number fatty acids in the A1-11 mutant strain.

[0036] Example 3: Scale-up test of 5 L fermenter for A1-11 1) Inoculate the A1-11 mutant strain into seed liquid culture medium and culture overnight at 25°C with shaking; 2) Inoculate 5-20% of the culture medium into a 5L fermenter (BIOTECH-5BG, Shanghai Baoxing) containing 3.0 L of fermentation broth pre-added with 20 mM L-valine, and incubate at 25-28℃ for 72-168 h. Maintain dissolved oxygen in the fermentation broth at approximately 15% by adjusting the stirring speed (300-700 rpm) and aeration ratio (1.0-2.0 vvm). Automatically add 2% malic acid and 10% ammonia to adjust the pH to maintain it between 6 and 8. During fermentation, maintain the residual sugar concentration between 1 and 30 g / L. If necessary, automatically add silicone defoamer (0.1% (v / v) PDMS emulsion) for defoaming.

[0037] 3) After fermentation, 50 mL of fermentation broth was taken to determine the cell dry weight and total lipid content; the remaining fermentation broth was centrifuged to collect the cells, and resuspended in 50 mM PBS (pH 7.0) at a ratio of 1:3 (w / v). 2% (w / w) alkaline protease was added, and the cells were enzymatically hydrolyzed at 55℃ for 2 h. Subsequently, the lipids were extracted and the data of various indicators were detected.

[0038] Measurements showed that the biomass of A1-11 cultured in a 5L fermenter reached 154.35 g / L, with an oil yield of 94.91 g / L and an oil content of 60.24% (biomass). The oil contained 48.80% C15:0 total lipids, 7.32% C17:0 total lipids, and 30.04% DHA total lipids. Odd-number fatty acids (C15:0 and C17:0) accounted for a combined 56.12%, with a total yield of 53.26 g / L.

[0039] Table 2. A1-11 cultured in a 5L fermenter

[0040] In summary, strain A1-11 exhibited highly efficient lipid accumulation capacity in a 5L fermenter (total yield 94.91 g / L). The significant advantage of its odd-chain fatty acids (C15:0+C17:0) (56.12% content, yield 53.26 g / L) combined with the high proportion of DHA (30.04%) indicates that this strain has dual application potential in the development of functional lipids, encompassing both odd-chain fatty acids and ω-3 polyunsaturated fatty acids.

[0041] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A Schizochytrium mutant strain A1-11, characterized in that, The preservation name of the Schizochytrium mutant strain A1-11 is: Aurantiochytrium sp. sdu080 A1-11 was deposited at the China Center for Type Culture Collection on August 22, 2025, with accession number CCTCC NO: M 20251884.

2. The Schizochytrium mutant strain A1-11 according to claim 1, characterized in that, The Schizochytrium mutant strain A1-11 was derived from the starting strain Aurantiochytrium sp. sdu080 was obtained by atmospheric pressure and room temperature plasma mutagenesis.

3. The Schizochytrium mutant strain A1-11 according to claim 2, characterized in that, The specific conditions for the atmospheric pressure room temperature plasma mutagenesis are as follows: the starting strain... Aurantiochytrium After preparing a bacterial suspension of sp. sdu080 with an OD600 of 0.6-0.8, it was placed on the sample stage of an ARTP mutagen and subjected to mutagenesis treatment with plasma irradiation for 20-60 s.

4. A method for producing odd-numbered carbon chain fatty acids, characterized in that, The method includes the following steps: 1) Seed culture: The Schizochytrium mutant strain A1-11 according to any one of claims 1 to 3 is inoculated into seed liquid culture medium and cultured overnight at 25°C with shaking at 600 rpm to obtain seed liquid; The seed liquid culture medium consists of: 10 g / L glucose, 20 g / L yeast extract, and 15 g / L sea salt. 2) Fermentation culture: At an inoculum rate of 2%, the seed culture obtained in step 1) was transferred to the fermentation liquid culture medium and cultured at 25°C and 600 rpm for 4 days with shaking. The fermentation broth containing the cells was then collected. The fermentation liquid culture medium consists of: 60 g / L glucose, 20 g / L yeast extract, and 15 g / L sea salt. 3) Oil extraction and detection: Extract the microbial oil from the fermentation broth in step 2) to obtain oil containing odd-numbered carbon chain fatty acids.

5. The method for producing odd-numbered carbon chain fatty acids according to claim 4, characterized in that, The odd-numbered carbon chain fatty acids include pentadecanoic acid and heptadecanoic acid.

6. The method for producing odd-numbered carbon chain fatty acids according to claim 4, characterized in that, The fermentation liquid culture medium contains precursors that promote the synthesis of odd-chain fatty acids.

7. A method for producing odd-numbered carbon chain fatty acids according to claim 6, characterized in that, The precursor is propionate or L-valine, and the concentration of the precursor in the fermentation liquid culture medium is: propionate 5-20 mM, or L-valine 10-30 mM.

8. The method for producing odd-numbered carbon chain fatty acids according to claim 7, characterized in that, The propionate is selected from one or more of sodium propionate, potassium propionate, and calcium propionate.

9. The method for producing odd-numbered carbon chain fatty acids according to claim 4, characterized in that, The specific steps for extracting bacterial cell oil and performing methyl esterification of the oil include: a) Take 2 mL of fermentation broth containing bacteria into a pre-weighed 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, and discard the supernatant; centrifuge at 500 rpm for 5 s to allow the liquid droplets on the tube wall to settle, open the centrifuge tube cap, and pre-freeze at -80℃ for at least 30 min; b) Transfer the pre-frozen sample to a freeze dryer and freeze-dry overnight under vacuum ≤10 Pa and temperature ≤-50℃. Determine the drying quality of the freeze-dried cells. c) Weigh 40-50 mg of lyophilized bacterial cells, add 600 μL of 1 M HCl solution, vortex at 2000 rpm for 30 s, and let stand at room temperature for 30 min; then perform boiling water bath for 5 min and freezing at -80℃ for 5 min in sequence, and repeat this temperature cycle once. d) Once the sample has recovered to 25±2℃, add 900 μL of chloroform-methanol mixture, vortex at 2000 rpm for 30 s, centrifuge at 12000 rpm for 3 min, and transfer the lower organic phase; add 600-750 μL of saturated sodium chloride solution, vortex, and centrifuge at 12000 rpm for 3 min. e) Transfer the lower oil phase to a pre-weighed 10 mL glass bottle and vacuum dry at 55 °C for 2-2.5 h to obtain bacterial oil.

10. A method for producing odd-numbered carbon chain fatty acids according to claim 9, characterized in that, The procedure also includes the methyl esterification and detection of the bacterial cell oil obtained in step f): 1 mL of chloroform and 2.5 mL of sulfuric acid-methanol mixture are added to the dried bacterial cell oil, and the mixture is capped and methylated at 85°C for 1-2.5 h; after returning to room temperature, the cap is opened, 3 mL of n-hexane and 3 mL of saturated sodium chloride are added, and the mixture is vigorously mixed and extracted until the layers are completely separated; 1 mL of the upper organic phase is taken and filtered, and the content of odd-chain fatty acids is detected by GC-MS analysis.

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