Method for producing squalene by inducing schizochytrium limacinum through salt stress strategy and application

Abstract

The invention belongs to the technical field of biochemical engineering, and discloses a method for producing squalene by inducing schizochytrium limacinum through a salt stress strategy and application, the method comprises the following steps: fermenting a schizochytrium limacinum strain for fermentation for 0-72 hours, adding 3g / L of NaCl into a fermentation medium, after 72 hours, adding NaCl into the fermentation liquid to 5g / L, continuing to ferment for 120 hours, measuring the sugar consumption every 12 hours in the fermentation process, and supplementing glucose to 80g / L. According to the method, the squalene yield of schizochytrium limacinum is remarkably increased through a salt stress strategy, an expensive inducer is prevented from being used, and the production cost is effectively reduced. 3 g / L of low-concentration salt is added in 0-72 h of the early stage of fermentation to serve as an adaptation period, so that schizochytrium limacinum grows rapidly and is induced to start an osmotic regulation mechanism, the concentration of NaCl is increased to 5 g / L in 72 h, at the moment, the schizochytrium limacinum enters a lipid accumulation period, and high-concentration NaCl is utilized to stress the schizochytrium limacinum to efficiently accumulate squalene.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bio-chemical industry, and particularly relates to a method for inducing Schizochytrium to produce squalene by a salt stress strategy and application. BACKGROUND

[0002] Squalene is a naturally occurring triterpenoid with strong antioxidant, anti-inflammatory and moisturizing properties. It participates in cholesterol metabolism in the human body and serves as a precursor of sterol substances, and exhibits significant functions in cardiovascular health, anti-tumor, hypoxia tolerance, skin repair, etc. At present, squalene is widely used in the fields of cosmetics, health care, medicine and chemical industry. Squalene was originally extracted from the livers of deep-sea sharks, but the ecological problems caused by overfishing limit its production and use. The extraction and purification process of plant-derived squalene is complicated, and it is also difficult to meet the huge market demand. The use of microbial fermentation to produce squalene can not only reduce production costs but also be environmentally friendly and sustainable, so it has become a research hotspot.

[0003] Schizochytrium is a kind of marine fungi, which has fast growth rate, short fermentation period, and oil content in the body of more than 50% of the dry weight of cells, and has a complete mevalonate (MVA) pathway, so it is an ideal host for producing squalene. However, the synthesis of squalene in Schizochytrium is limited by the inherent low efficiency of the MVA pathway, and the competitive pathways such as sterol synthesis in its metabolic network will divert carbon sources, resulting in a long-term low level of squalene production.

[0004] In view of the above problems, current researches are mostly focused on improving the squalene production of Schizochytrium by genetically engineering the metabolic pathway, or promoting the accumulation of squalene by adding exogenous chemicals. These methods have improved the yield of squalene to some extent, but still face the bottleneck of low gene editing efficiency, poor strain adaptability and difficulty in industrialization scaling up. At present, there is no report on inducing Schizochytrium to produce squalene by a salt stress strategy. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies in the prior art, and to provide a method for inducing Schizochytrium to produce squalene by a salt stress strategy and application.

[0006] The technical scheme adopted by the present application to solve its technical problems is as follows:

[0007] A method for inducing Schizochytrium to produce squalene by a salt stress strategy, wherein 3g / L of NaCl is added to the fermentation medium at 0-72h of fermentation of the Schizochytrium strain, and 5g / L of NaCl is added to the fermentation broth after 72h, and the fermentation is continued to 120h, and the sugar consumption is measured every 12h during the fermentation and the glucose is supplemented to 80g / L.

[0008] Further, the specific steps are as follows:

[0009] Schizochytrium sp. HX-308 is taken out from -80℃, inoculated into seed culture medium, and cultured at 28℃ and 180r / min for 24h to obtain a first generation seed; the first generation seed culture liquid is inoculated into seed culture medium at an inoculation amount of 0.8-1%, and cultured at 28℃ and 180r / min for 24h to obtain a second generation seed; the second generation seed culture liquid is inoculated into seed culture medium at an inoculation amount of 0.8-1%, and cultured at 28℃ and 180r / min for 24h to obtain a third generation seed, which is used as a fermentation Schizochytrium sp. strain;

[0010] The third generation seed is inoculated into fermentation culture medium at an inoculation amount of 10%, 3g / L of NaCl is added to the fermentation culture medium during 0-72h of fermentation, and the fermentation is carried out at 28-30℃ and 170-200rpm; 72h later, NaCl is added to the fermentation liquid to 5g / L, and the fermentation is continued at 28-30℃ and 170-200rpm until 120h; the sugar consumption is measured every 12h during the fermentation, and glucose is supplemented to 80g / L.

[0011] Further, the pH value of the seed culture medium is 6.0-6.5, and the seed culture medium comprises: 50g / L of glucose, 5g / L of yeast extract powder, 5g / L of sodium sulfate, 2g / L of magnesium sulfate, 6g / L of ammonium sulfate, 1g / L of potassium chloride, 0.1g / L of calcium chloride, 0.6g / L of potassium sulfate, 1g / L of potassium dihydrogen phosphate, 10g / L of sodium glutamate, 0.1% of trace minerals, 5mg / L of vitamin B6, and 0.5mg / L of vitamin B1, and the solvent is water; the percentages above are mass percentages; wherein the formula of the trace minerals is: 8g / L of MnCl2, 8g / L of ZnSO4, 0.1g / L of CoCl2, 0.1g / L of N-methyl morpholine oxide NaMo, 6g / L of CuSO4, 6g / L of NiSO4, 20g / L of FeSO4, and 10mL / L of concentrated H2SO4, and the solvent is water;

[0012] The pH value of the fermentation medium is 6.0-7.5, and the fermentation medium comprises: 80 g / L of glucose, 10 g / L of yeast extract powder, 10 g / L of sodium sulfate, 2 g / L of magnesium sulfate, 6 g / L of ammonium sulfate, 1 g / L of potassium chloride, 0.1 g / L of calcium chloride, 0.6 g / L of potassium sulfate, 1 g / L of potassium dihydrogen phosphate, 20 g / L of sodium glutamate, 0.1% of trace minerals, 5 mg / L of vitamin B6 and 0.5 mg / L of vitamin B1, and the solvent is water; the percentages are mass percentages; wherein the formula of the trace minerals is: 8 g / L of MnCl2, 8 g / L of ZnSO4, 0.1 g / L of CoCl2, 0.1 g / L of N-methyl morpholine oxide NaMo, 6 g / L of CuSO4, 6 g / L of NiSO4, 20 g / L of FeSO4 and 10 mL / L of concentrated H2SO4, and the solvent is water.

[0013] Further, in the method, NaCl can significantly improve the transcription level of key enzymes HMGS, HMGR, MVK, PMK and MDD of the MVA pathway.

[0014] Further, the determination method of the content of the squalene is as follows:

[0015] The fermentation liquid after fermentation for 120 h is treated to obtain squalene-containing oil, and the content of squalene is analyzed by gas chromatography, and the specific method is as follows:

[0016] (1) after adding NaOH solution to adjust the pH value to 10-13 in the fermentation liquid, 0.01-0.5% of cell wall breaking enzyme is added to the fermentation liquid, and the mixture is oscillated at 40-60 DEG C and 100-200 r / min for 5-15 h;

[0017] (2) cooling to room temperature, adding anhydrous ethanol to inactivate the cell wall breaking enzyme;

[0018] (3) adding n-hexane for extraction, and collecting the upper organic phase;

[0019] (4) repeating step 3) for several times, combining the organic phases, and evaporating the solvent to obtain the lipid;

[0020] (5) methyl esterification of the lipid: 10 μL of the above oil is taken in a 2 mL centrifuge tube, 600 μL of 1M sodium hydroxide-methanol solution is added, and the mixture is oscillated for 3-5 h, then 50 μL of concentrated sulfuric acid is added to terminate the reaction, 1 mL of n-hexane is added and the mixture is oscillated for 30 min, the upper n-hexane phase is taken and passed through a microporous filter membrane to remove impurities, and then the upper gas chromatography is obtained.

[0021] The method as described above is applied to the production of squalene.

[0022] The application has the following advantages and positive effects:

[0023] 1. This invention significantly improves the squalene yield of Schizochytrium through a salt stress strategy, avoiding the use of expensive inducers and effectively reducing production costs. A low concentration of salt (3 g / L) is added during the early fermentation stage (0–72 h) as an adaptation period, allowing Schizochytrium to grow rapidly and inducing it to activate its osmotic regulation mechanism. At 72 h, the NaCl concentration is increased to 5 g / L, at which point Schizochytrium enters the lipid accumulation phase. The high NaCl stress then forces Schizochytrium to efficiently accumulate squalene.

[0024] 2. Through transcriptome analysis, this invention found that NaCl induction can significantly increase the gene expression of key enzymes (HMGS, HMGR, MVK, PMK, MDD) in the MVA pathway, promote the synthesis of squalene precursors, and reveal the mechanism by which NaCl regulates the production of squalene by Schizochytrium.

[0025] 3. This invention realizes a method for dynamically gradient-regulated high squalene production by Schizochytrium fungi, which overcomes the limitations of traditional single stress strategies by using staged salt concentration shocks.

[0026] 4. This method is not only applicable to squalene production, but can also be extended to the biosynthesis research of other high-value-added terpenoid compounds, showing good application prospects. Attached Figure Description

[0027] Figure 1 The graph shows the biomass and squalene content of Schizochytrium under different NaCl concentrations in this invention.

[0028] Figure 2 This is a transcriptional level diagram of key enzymes in the MVA pathway when 5 g / L NaCl is added in this invention;

[0029] Figure 3 This is a comparison chart of squalene content between the control group and the stress group in this invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0031] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0032] A method for inducing squalene production by Schizochytrium using a salt stress strategy involves adding 3 g / L NaCl to the fermentation medium during the fermentation process from 0 to 72 h, then adding NaCl to the fermentation broth to a concentration of 5 g / L after 72 h, and continuing fermentation for 120 h. During the fermentation process, the sugar consumption is measured every 12 h and glucose is added to a concentration of 80 g / L.

[0033] Preferably, the specific steps are as follows:

[0034] The *Schizochytrium* HX-308 strain was removed from -80℃ and inoculated into seed culture medium. After culturing at 28℃ and 180 rpm for 24 hours, it was obtained as the first-generation seed. The first-generation seed culture was then inoculated into seed culture medium at an inoculation rate of 0.8-1%, and cultured at 28℃ and 180 rpm for 24 hours to obtain the second-generation seed. The second-generation seed culture was then inoculated into seed culture medium at an inoculation rate of 0.8-1%, and cultured at 28℃ and 180 rpm for 24 hours to obtain the third-generation seed, which was used as the *Schizochytrium* strain for fermentation.

[0035] The third-generation seeds were inoculated into the fermentation medium at an inoculation rate of 10%. During the fermentation period of 0-72h, 3g / L NaCl was added to the fermentation medium and cultured at 28-30℃ and 170-200rpm. After 72h, NaCl was added to the fermentation broth to bring the total concentration to 5g / L and fermentation continued at 28-30℃ and 170-200rpm for 120h. During the fermentation process, the sugar consumption was measured every 12h and glucose was added to bring the total concentration to 80g / L.

[0036] Preferably, the seed culture medium has a pH of 6.0–6.5 and comprises: 50 g / L glucose, 5 g / L yeast extract, 5 g / L sodium sulfate, 2 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L monosodium glutamate, 0.1% trace minerals, 5 mg / L vitamin B6, and 0.5 mg / L vitamin B12, with water as the solvent; the percentages mentioned above are mass percentages; wherein the formula for the trace minerals is: 8 g / L MnCl2, 8 g / L ZnSO4, 0.1 g / L CoCl2, 0.1 g / L N-methylmorpholine oxide NaMo, 6 g / L CuSO4, 6 g / L NiSO4, 20 g / L FeSO4, and 10 mL / L concentrated H2SO4, with water as the solvent;

[0037] The fermentation medium has a pH of 6.0–7.5 and includes: 80 g / L glucose, 10 g / L yeast extract, 10 g / L sodium sulfate, 2 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 20 g / L monosodium glutamate, 0.1% trace minerals, 5 mg / L vitamin B6, and 0.5 mg / L vitamin B12, all in water. All percentages are by mass. The trace minerals consist of: 8 g / L MnCl2, 8 g / L ZnSO4, 0.1 g / L CoCl2, 0.1 g / L N-methylmorpholine oxide (NaMo), 6 g / L CuSO4, 6 g / L NiSO4, 20 g / L FeSO4, and 10 mL / L concentrated H2SO4, all in water.

[0038] Preferably, in the method, NaCl can significantly increase the transcriptional levels of key enzymes in the MVA pathway, namely HMGS, HMGR, MVK, PMK, and MDD.

[0039] Preferably, the method for determining the squalene content is as follows:

[0040] The fermentation broth after 120 hours of fermentation was processed to obtain squalene-containing oil. The squalene content was analyzed by gas chromatography. The specific method is as follows:

[0041] (1) After adjusting the pH to 10-13 by adding NaOH solution to the fermentation broth, add 0.01-0.5% of cell wall breaking enzyme by weight of fermentation broth, shake at 100-200 r / min for 5-15 h at 40-60℃;

[0042] (2) Cool to room temperature and add an equal volume of anhydrous ethanol to the fermentation broth to inactivate the cell-wall breaking enzyme;

[0043] (3) Extract with n-hexane and collect the upper organic phase;

[0044] (4) Repeat step 3) several times, combine the organic phases, evaporate the solvent, and obtain lipids;

[0045] (5) Methyl esterification of the lipids: Take 10 μL of the above oil into a 2 mL centrifuge tube, add 600 μL of 1M sodium hydroxide-methanol solution, shake thoroughly for 3-5 h, add 50 μL of concentrated sulfuric acid to terminate the reaction, add 1 mL of n-hexane and shake for 30 min, take the upper n-hexane phase and filter it through a microporous membrane to remove impurities, and then perform gas chromatography.

[0046] The application of the method described above in the production of squalene.

[0047] Specifically, the relevant preparation and testing methods are as follows:

[0048] The Schizochytrium sp. HX-308 strain is a well-known strain in the prior art, as disclosed in patent publication CN118185850A, and has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M209059.

[0049] The culture media used in the following examples are as follows:

[0050] The seed culture medium has a pH of 6.0–6.5 and includes: 50 g / L glucose, 5 g / L yeast extract, 5 g / L sodium sulfate, 2 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L monosodium glutamate, 0.1% trace minerals, 5 mg / L vitamin B6, and 0.5 mg / L vitamin B12, all in water. All percentages are by mass. The trace minerals consist of: 8 g / L MnCl2, 8 g / L ZnSO4, 0.1 g / L CoCl2, 0.1 g / L N-methylmorpholine oxide (NaMo), 6 g / L CuSO4, 6 g / L NiSO4, 20 g / L FeSO4, and 10 mL / L concentrated H2SO4, all in water.

[0051] The fermentation medium has a pH of 6.0–7.5 and includes: 80 g / L glucose, 10 g / L yeast extract, 10 g / L sodium sulfate, 2 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 20 g / L monosodium glutamate, 0.1% trace minerals, 5 mg / L vitamin B6, and 0.5 mg / L vitamin B12, all in water. All percentages are by mass. The trace minerals consist of: 8 g / L MnCl2, 8 g / L ZnSO4, 0.1 g / L CoCl2, 0.1 g / L N-methylmorpholine oxide (NaMo), 6 g / L CuSO4, 6 g / L NiSO4, 20 g / L FeSO4, and 10 mL / L concentrated H2SO4, all in water.

[0052] Example 1. Determining the optimal NaCl concentration

[0053] Schizochytrium sp. HX-308 was removed from -80℃ and inoculated into a 250mL Erlenmeyer flask (containing 50mL of seed culture medium). After incubation at 28℃ and 180rpm for 24 hours, it became the first-generation seed culture. 1mL of the first-generation seed culture was inoculated into a 250mL Erlenmeyer flask (containing 50mL of seed culture medium) and incubated at 28℃ and 180rpm for 24 hours to obtain the second-generation seed culture. 1mL of the second-generation seed culture was inoculated into a 250mL Erlenmeyer flask (containing 50mL of seed culture medium) and incubated at 28℃ and 180rpm for 24 hours to obtain the third-generation seed culture, which was used as the inoculum for fermentation.

[0054] Three generations of seeds were inoculated into fermentation medium containing different concentrations of NaCl (0, 3 g / L, 5 g / L, 8 g / L, 10 g / L, and 15 g / L) at an inoculation rate of 10%. The cultures were incubated at 28-30℃ and 170-200 rpm for 120 h. Sugar consumption was monitored every 12 h, and glucose was replenished to a level of 80 g / L. Biomass and squalene content were measured at the end of fermentation.

[0055] Specifically, the method for determining the squalene content is as follows: (1) Add NaOH solution to the fermentation broth to adjust the pH to 10-13, then add 0.01-0.5% of the fermentation broth mass of cell wall breaking enzyme, shake at 100-200 r / min for 5-15 h at 40-60℃; (2) Cool to room temperature, add an equal volume of anhydrous ethanol to the fermentation broth to inactivate the cell wall breaking enzyme; (3) Add n-hexane for extraction, and collect the upper organic phase; (4) Repeat step 3) several times, combine the organic phases, evaporate the solvent, and obtain the lipid; (5) Perform methyl esterification on the lipid: Take 10 μL of the above oil into a 2 mL centrifuge tube, add 600 μL of 1M sodium hydroxide-methanol solution, shake thoroughly for 3-5 h, add 50 μL of concentrated sulfuric acid to terminate the reaction, add 1 mL of n-hexane and continue shaking for 30 min, take the upper n-hexane phase and filter it through a microporous membrane to remove impurities, and then perform gas chromatography.

[0056] The specific procedure for gas chromatography analysis is as follows: A GC-2010 (Shimadzu, Japan) gas chromatography system was used, equipped with a DB-23 capillary column (60m*0.22mm) and a flame ionization detector (FID). Nitrogen was used as the carrier gas. The injection volume was 1μL, and the injection temperature was 250℃. The column temperature was increased from 100℃ to 200℃ at a rate of 25℃ / min, then increased to 230℃ at a rate of 4℃ / min and held for 9 min. The FID detector temperature was 280℃. The squalene content could be determined by comparing with relevant external standards (Sigma, USA).

[0057] The results are as follows Figure 1As shown, the biomass of Schizochytrium slightly increased in the range of 0–3 g / L NaCl, and began to decrease at 5 g / L. The squalene content was highest at 5 g / L NaCl, reaching 110.73 mg / L. When the NaCl concentration was further increased to 15 g / L, the squalene content decreased slightly. Therefore, 5 g / L NaCl is optimal.

[0058] Example 2. Transcriptome analysis of salt stress-induced Schizochytrium fungi

[0059] Schizochytrium was fermented under the fermentation conditions of Example 1. Transcriptome analysis was performed on cells fermented for 48 hours with NaCl-5 g / L and NaCl-0 g / L, respectively. The transcriptional levels of key enzymes in the MVA pathway (HMGS, HMGR, MVK, PMK, MDD) were analyzed. The results are as follows: Figure 2 As shown, the transcriptional levels of key enzymes in the MVA pathway were significantly increased by more than 2-fold.

[0060] Example 3. Gradient regulation of NaCl stress fermentation in Schizochytrium

[0061] Three generations of seeds were obtained according to the method described in Example 1, and three groups of fermentation were carried out according to different NaCl addition methods. The rest were the same as in Example 1.

[0062] One group was given a single addition of NaCl to 5 g / L at 0 h of fermentation. During the 120 h fermentation period, sugar consumption was measured every 12 h and glucose was replenished to 80 g / L. Biomass and squalene content were measured at the end of fermentation.

[0063] One group fermented without adding NaCl to the fermentation medium from 0 to 72 hours. After 72 hours, NaCl was added to the fermentation broth to a concentration of 5 g / L, and fermentation continued for 120 hours. During fermentation, sugar consumption was measured every 12 hours, and glucose was replenished to a concentration of 80 g / L. Biomass and squalene content were measured at the end of fermentation.

[0064] In one group, 3 g / L NaCl was added to the fermentation medium from 0 to 72 h to achieve a high biomass. After 72 h, NaCl was added to the fermentation broth to bring the concentration to 5 g / L, and fermentation continued for 120 h. During fermentation, sugar consumption was measured every 12 h, and glucose was replenished to 80 g / L. At the end of fermentation, biomass and squalene content were measured.

[0065] The control group did not have NaCl added.

[0066] The fermentation results under different stress methods are shown in Table 1. Compared with the single addition of NaCl, the biomass of *Schizochytrium* with gradient NaCl addition increased by 1.97 times and the squalene content increased by 1.34 times. Therefore, gradient NaCl addition is more conducive to the efficient accumulation of squalene in *Schizochytrium*. The results of fermenting *Schizochytrium* using this method are as follows...Figure 3 As shown, compared with the control group, the squalene content of *Schizochytrium* in the stress group increased by 2.33 times. This invention achieves efficient production of squalene from *Schizochytrium* through a salt stress strategy.

[0067] Meanwhile, it can also be seen that the two steps in the method of the present invention, namely adding 3 g / L NaCl to the fermentation medium during fermentation from 0 to 72 h and adding NaCl to the fermentation broth to 5 g / L after 72 h, have a synergistic effect, which can synergistically improve the biomass of Schizochytrium and the content of squalene.

[0068] Table 1 Comparison of fermentation results under different salt stress methods

[0069]

[0070] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for inducing squalene production in Schizochytrium using a salt stress strategy, characterized in that: For the fermentation of Schizochytrium strain, 3 g / L NaCl was added to the fermentation medium during the first 0-72 hours of fermentation. After 72 hours, NaCl was added to the fermentation broth to bring the total concentration to 5 g / L. Fermentation continued for 120 hours. During the fermentation process, the sugar consumption was measured every 12 hours and glucose was added to bring the total concentration to 80 g / L.

2. The method according to claim 1, characterized in that: The specific steps are as follows: The *Schizochytrium* HX-308 strain was removed from -80℃ and inoculated into seed culture medium. After culturing at 28℃ and 180 rpm for 24 hours, it was obtained as the first-generation seed. The first-generation seed culture was then inoculated into seed culture medium at an inoculation rate of 0.8-1%, and cultured at 28℃ and 180 rpm for 24 hours to obtain the second-generation seed. The second-generation seed culture was then inoculated into seed culture medium at an inoculation rate of 0.8-1%, and cultured at 28℃ and 180 rpm for 24 hours to obtain the third-generation seed, which was used as the *Schizochytrium* strain for fermentation. The third-generation seeds were inoculated into the fermentation medium at an inoculation rate of 10%. During the fermentation period of 0-72h, 3g / L NaCl was added to the fermentation medium and cultured at 28-30℃ and 170-200rpm. After 72h, NaCl was added to the fermentation broth to bring the total concentration to 5g / L and fermentation continued at 28-30℃ and 170-200rpm for 120h. During the fermentation process, the sugar consumption was measured every 12h and glucose was added to bring the total concentration to 80g / L.

3. The method according to claim 2, characterized in that: The seed culture medium has a pH of 6.0–6.5 and comprises: 50 g / L glucose, 5 g / L yeast extract, 5 g / L sodium sulfate, 2 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L monosodium glutamate, 0.1% trace minerals, 5 mg / L vitamin B6, and 0.5 mg / L vitamin B12, with water as the solvent; the percentages mentioned above are mass percentages; wherein, the formula for the trace minerals is: 8 g / L MnCl2, 8 g / L ZnSO4, 0.1 g / L CoCl2, 0.1 g / L N-methylmorpholine oxide NaMo, 6 g / L CuSO4, 6 g / L NiSO4, 20 g / L FeSO4, and 10 mL / L concentrated H2SO4, with water as the solvent; The fermentation medium has a pH of 6.0–7.5 and includes: 80 g / L glucose, 10 g / L yeast extract, 10 g / L sodium sulfate, 2 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 20 g / L monosodium glutamate, 0.1% trace minerals, 5 mg / L vitamin B6, and 0.5 mg / L vitamin B12, all in water. All percentages are by mass. The trace minerals consist of: 8 g / L MnCl2, 8 g / L ZnSO4, 0.1 g / L CoCl2, 0.1 g / L N-methylmorpholine oxide (NaMo), 6 g / L CuSO4, 6 g / L NiSO4, 20 g / L FeSO4, and 10 mL / L concentrated H2SO4, all in water.

4. The method according to claim 1, characterized in that: In this method, NaCl can significantly increase the transcriptional levels of key enzymes in the MVA pathway: HMGS, HMGR, MVK, PMK, and MDD.

5. The method according to any one of claims 1 to 4, characterized in that: The method for determining the squalene content is as follows: The fermentation broth after 120 hours of fermentation was processed to obtain squalene-containing oil. The squalene content was analyzed by gas chromatography. The specific method is as follows: (1) After adjusting the pH to 10-13 by adding NaOH solution to the fermentation broth, add 0.01-0.5% of cell wall breaking enzyme by weight of fermentation broth, shake at 100-200 r / min for 5-15 h at 40-60℃; (2) Cool to room temperature and add an equal volume of anhydrous ethanol to the fermentation broth to inactivate the cell-wall breaking enzyme; (3) Extract with n-hexane and collect the upper organic phase; (4) Repeat step 3) several times, combine the organic phases, evaporate the solvent, and obtain lipids; (5) Methyl esterification of the lipids: Take 10 μL of the above oil into a 2 mL centrifuge tube, add 600 μL of 1M sodium hydroxide-methanol solution, shake thoroughly for 3-5 h, add 50 μL of concentrated sulfuric acid to terminate the reaction, add 1 mL of n-hexane and shake for 30 min, take the upper n-hexane phase and filter it through a microporous membrane to remove impurities, and then perform gas chromatography.

6. The use of the method according to any one of claims 1 to 5 in the production of squalene.

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