Method for preparing squalane by using biosynthesis method and chemical synthesis method

Abstract

The invention discloses a method for preparing squalane by using a biosynthesis method and a chemical synthesis method. The method comprises the following steps: (1) biosynthesis: 1) cloning an activated squalene synthetase gene ERG9 and a Ura auxotrophic gene leu2; co-transforming the genes into a strain Sc027 to obtain a recombinant bacterium; 2) preparing a recombinant strain seed solution; (3) carrying out protein induced expression on the seed solution; respectively adding absolute ethyl alcohol with the final concentration of 10g / L after culturing for 30 hours and 54 hours; culturing for 96 hours, centrifuging to obtain thalli, breaking walls, extracting, and removing an organic solvent to obtain squalene; (2) chemical synthesis: adding ethanol into a reaction container, adding a palladium catalyst, adding squalene in a hydrogen atmosphere, and reacting to obtain squalane; and the product yield is more than 95.5%. The method provided by the invention has the advantages of mild conditions, lower pollution, simple operation, high yield, simple product separation and purification, and good product purity (97.4% or above).

Description

Technical Field

[0001] The invention belongs to the field of combining biosynthesis and chemical synthesis, and specifically relates to a method for preparing squalane by utilizing biosynthesis and chemical synthesis. Background Art

[0002] Squalane is a saturated hydrocarbon with a molecular formula of C 30 H 62 , which is obtained by fully hydrogenating highly unsaturated squalene, is a colorless, transparent, viscous, oily liquid. Due to its high moisturizing and humectant properties, easy absorption by the skin, high chemical stability, and multiple functions such as improving immunity, anti-oxidation, promoting brain development, regulating blood lipids and improving vision, it has great potential in the nutrition, health care and pharmaceutical industries. There are four main sources of squalane: extraction from shark liver, artificial synthesis, plant sugarcane fermentation and olive extraction. Shark liver accounts for about 25% of a shark's body weight and contains about 50% oil, 80% of which is squalene. Generally speaking, it takes about 3,000 shark livers to produce one ton of squalene. However, due to environmental protection and sustainability considerations, plant squalene is now more used as a raw material to obtain squalane by hydrogenation. The current preparation methods have various defects. The chemical synthesis method has many challenges such as harsh and complex conditions, high temperature and high pressure, large pollution, and low conversion rate; and extraction from plants also has problems such as damage to the ecological environment and long cultivation time. Therefore, it is extremely important to develop a method for preparing squalane with mild process conditions, simple operation, environmental protection, low cost, high yield, simple product separation and purification, and good product purity. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing squalane by biosynthesis and chemical synthesis, which has the characteristics of short path, high efficiency, easy availability of raw materials, high safety, high yield, simple product separation and purification, good product purity, and environmental friendliness.

[0004] The technical solution of the present invention is summarized as follows:

[0005] The method for preparing squalane by biosynthesis and chemical synthesis comprises the following steps:

[0006] (1) Biosynthesis:

[0007] 1) cloning the activated squalene synthase gene ERG9 and the Ura auxotrophic gene leu2; transforming the gene ERG9 and the gene leu2 into competent cells of the Saccharomyces cerevisiae engineering strain Sc027 to obtain recombinant bacteria;

[0008] 2) Inoculate a single colony of the recombinant bacteria in SD-URA liquid medium and culture for 12-24 hours to obtain a seed solution;

[0009] 3) The seed solution obtained in step 2) was fermented in SD-URA liquid medium at 10-50° C. and 110-330 rpm for shake flask fermentation; galactose with a final concentration of 10 g / L was added at 24 h of culture to induce protein expression; anhydrous ethanol with a final concentration of 10 g / L was added at 30 h and 54 h of culture; after 96 hours of culture, the cells were obtained by centrifugation, cell wall breaking treatment was performed, extraction was performed with hexane, and the organic solvent was removed by rotary evaporation to obtain squalene;

[0010] (2) Chemical synthesis:

[0011] Add ethanol to a reaction container, add a palladium catalyst, replace the air in the reaction container with hydrogen, add the squalene to dissolve the squalene, react at a reaction temperature of 30-50° C. to obtain squalane; the molar amount of the palladium catalyst accounts for 5% of the molar amount of the squalene.

[0012] The nucleic acid sequence of the gene ERG9 is shown in SEQ ID NO.1; the nucleic acid sequence of the gene leu2 is shown in SEQ ID NO.2.

[0013] The step (2) of step (1) is: inoculating a single colony of the recombinant bacteria into SD-URA liquid culture medium, culturing in a shaker at 30° C. and 220 rpm for 24 hours to obtain a seed solution.

[0014] In step (2), the reaction temperature is 50°C.

[0015] The palladium catalyst is 10% palladium carbon, 5% palladium carbon, 3% palladium carbon, 2% palladium carbon, 1% palladium carbon, palladium chloride, palladium acetate or palladium oxide.

[0016] Beneficial effects of the present invention

[0017] The present invention obtains squalene by microbial fermentation, avoiding the shortcomings of the prior art in preparing squalene. The yield of the squalane product prepared by the method of the present invention is above 95.5%. Compared with the existing chemical synthesis method of squalane, the method of the present invention has milder conditions, lower pollution, simple operation, high yield, simple product separation and purification, and good product purity. DETAILED DESCRIPTION

[0018] The squalene synthase gene ERG9 was derived from Saccharomyces cerevisiae CEN.PK2-1C (commercially available), and the gene leu2 was derived from pESC-Ura plasmid (commercially available).

[0019] The engineered Saccharomyces cerevisiae strain Sc027 is derived from Chinese patent CN202311671548.4 (farnesene synthase mutant and encoding gene).

[0020] Construction of the engineered yeast strain Sc027: sc027 was constructed from Saccharomyces cerevisiae CEN.PK2-1C (commercially available, genotype: MAT a; ura3-52, trp1-289, leu2-3,112, his311, MAL2-8C, SUC2).

[0021] Based on homologous recombination, CYB5, ERG19, ERG8, ERG12, ERG10, tHMG1 (SEQ ID NO.3), IDI1, ERG13, ERG20, ALDH1, and CPR1 genes were inserted into the CEN.PK2-1C genome, wherein IDI1 and tHMG1 were inserted into the ADE1 site, ERG20 and tHMG1 were inserted into the TRP site, ERG13 and tHMG1 were inserted into the URA3 site, ALDH1, ERG10, and ERG12 were inserted into the HIS3 site, CYB5, ERG19, and ERG8 were inserted into the LEU2 site, and CPR1 was inserted into the GAL1 site, and the genes encoding HIS3, ADE1, and TRP1 were supplemented to finally obtain sc027. (Gene accession numbers are shown in Table 1)

[0022] (Article source: Metabolic Engineering of Saccharomyces cerevisiae forEnhanced Dihydroartemisinic Acid Production)

[0023] Table 1 Gene accession number (NCBI)

[0024]

[0025] The SD-URA liquid culture medium involved in this experiment included 1.7 g / L of amino-free yeast nitrogen source YNB, 20 g / L of glucose, 5 g / L of ammonium sulfate, and 2 g / L of uracil-deficient amino acid mixture. The solid culture medium required the addition of 2% agar powder.

[0026] The ingredients of the uracil-deficient amino acid mixture include: 2.0g alanine, 2.0g arginine, 2.0g asparagine, 2.0g aspartic acid, 2.0g cysteine, 2.0g glutamine, 2.0g glutamic acid, 2.0g glycine, 2.0g inositol, 2.0g isoleucine, 2.0g lysine, 0.2g p-toluic acid, 2.0g phenylalanine, 2.0g proline, 2.0g serine, 2.0g threonine, 2.0g tryptophan, 2.0g tyrosine, and 2.0g valine.

[0027] The present invention will be further described below with reference to specific embodiments.

[0028] Embodiment 1:

[0029] Cloning of the activated squalene synthase gene ERG9 and the Ura auxotrophic gene leu2;

[0030] Using Saccharomyces cerevisiae CEN.PK2-1C as a template and primer 1 (SEQ ID NO.4) and primer 2 (SEQ ID NO.5) as upstream and downstream primers, PCR was performed to obtain the squalene synthase gene ERG9 (SEQ ID NO.1);

[0031] Using pESC-Ura plasmid as template, primer 3 (SEQ ID NO.6) and primer 4 (SEQ ID NO.7) as upstream and downstream primers, PCR was performed to obtain the Ura auxotrophic gene leu2 (SEQ ID NO.2)

[0032] The above biological elements were assembled into a complete expression cassette using fusion PCR. The total size of the expression cassette was 5302 bp after electrophoresis detection.

[0033] Example 2

[0034] The expression cassette was expressed in Saccharomyces cerevisiae as follows:

[0035] The competent cells of the engineering strain Sc027 of Saccharomyces cerevisiae were mixed with 600ng of the expression cassette obtained in Example 1, and 240μL of sterilized PEG3350, 36μL of sterilized 1.0M LiAc, 10μL of salmon sperm DNA cooled after boiling were added, and sterile water was added to 360μL and mixed. After 30min in a 42℃ water bath, the supernatant was discarded by centrifugation, 1mL of YPD medium was added to the centrifuge tube containing the bacteria, and the centrifuge tube was placed in a 30℃ shaker for 2h. The supernatant was discarded by centrifugation again, and the bacteria were washed twice with sterile water, and a small amount of bacteria in the precipitate was picked and applied to SD-URA solid medium, and cultured at 30℃ for 96 hours to obtain recombinant bacteria.

[0036] Example 3

[0037] The synthesis method of squalene is as follows:

[0038] A single colony of the recombinant bacteria was inoculated into 20 mL of SD-URA liquid culture medium and cultured at 220 rpm in a shaker at 30° C. for 24 h (or any value between 12 and 24 h, such as 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 23 h) to obtain a seed solution.

[0039] The seed solution was transferred to 1000 mL SD-URA liquid culture medium, the temperature was 30°C (any value in the range of 10-50°C can also be selected, such as 10°C, 15°C, 20°C, 25°C, 35°C, 40°C, 50°C), the rotation speed was 220 rpm (any value in the range of 110-330 rpm can also be selected, such as 110 rpm, 150 rpm, 210 rpm, 300 rpm, 330 rpm) for shake flask fermentation culture, galactose with a final concentration of 10 g / L was added at 24 hours of culture to induce protein expression, and anhydrous ethanol with a final concentration of 10 g / L was added at 30 hours and 54 hours of culture to supplement the carbon source required for microbial growth; after 96 hours of culture, the bacteria were obtained by centrifugation, the cell wall was broken, extracted with hexane, and the organic solvent was removed by rotary evaporation to obtain squalene; the yield was 12 g / L.

[0040] Example 4

[0041] The chemical synthesis method of squalane is as follows:

[0042] Add 60 ml of ethanol to a 200 ml Shrek bottle, add 10% palladium carbon as a catalyst, the molar amount of 10% palladium carbon accounts for 5% of the molar amount of squalene, replace the air in the bottle with hydrogen, the pressure is 1 atm, and then slowly add 8.4 g of squalene to dissolve the squalene, raise the temperature to 50°C and stir for about 6 hours, filter after the reaction is completed, remove the ethanol with a rotary evaporator to obtain 8.13 g of product squalane, with a purity of 99.6% and a yield of 96.8%.

[0043] In addition, a reuse test was conducted on the 10% palladium carbon catalyst used in the reaction process, and the results showed that the catalyst 10% palladium carbon could be reused 26 times.

[0044] The product squalane can be obtained by replacing the 10% palladium carbon in this embodiment with 5% palladium carbon, 3% palladium carbon, 2% palladium carbon, 1% palladium carbon, palladium chloride, palladium acetate or palladium oxide as the catalyst.

[0045] Embodiment 5:

[0046] The chemical synthesis method of squalane is as follows:

[0047] In a 200 ml Shrek bottle, add 60 ml of ethanol and 10% palladium carbon as a catalyst, the molar amount of 10% palladium carbon accounts for 5% of the molar amount of squalene, replace the air in the bottle with hydrogen, the pressure is 1 atm, and then slowly add 8.4 g of squalene to dissolve the squalene, heat to 30°C and stir for about 8 hours, filter after the reaction is completed, remove the ethanol with a rotary evaporator to obtain 8.02 g of product squalane, with a purity of 97.4% and a yield of 95.5%.

[0048] In addition, the catalyst palladium carbon used in the reaction process was tested for reuse, and the results showed that the catalyst palladium carbon could be reused 30 times.

[0049] The product squalane was tested by GC-MS, and the gas chromatography conditions were as follows: chromatographic column: a capillary column with a stationary phase of 5% phenyl-95% dimethylpolysiloxane and a size of 30m×0.25mm×0.5μm; column temperature: 150°C for 4min, heated to 280°C at a rate of 10°C / min, and maintained for 3min; column flow rate: 1.0mL / min; injection port temperature: 250°C; detector temperature: 250°C; carrier gas: helium; injection mode: split injection, split ratio of 5:1; injection volume: 1μL, mass spectrometry conditions: EI ion source temperature: 230°C; quadrupole temperature: 150°C; solvent delay 3min; scanning mode: SIM.

Claims

1. A method for preparing squalane by biosynthesis and chemical synthesis, Its characteristics are The steps include: (1) Biosynthesis: 1) cloning the activated squalene synthase gene ERG9 and the Ura auxotrophic gene leu2; transforming the gene ERG9 and the gene leu2 into competent cells of the Saccharomyces cerevisiae engineering strain Sc027 to obtain recombinant bacteria; 2) Inoculate a single colony of the recombinant bacteria in SD-URA liquid medium and culture for 12-24 hours to obtain a seed solution; 3) The seed solution obtained in step 2) was fermented in SD-URA liquid medium at 10-50° C. and 110-330 rpm for shake flask fermentation; galactose with a final concentration of 10 g / L was added at 24 h of culture to induce protein expression; anhydrous ethanol with a final concentration of 10 g / L was added at 30 h and 54 h of culture; after 96 hours of culture, the cells were obtained by centrifugation, cell wall breaking treatment was performed, extraction was performed with hexane, and the organic solvent was removed by rotary evaporation to obtain squalene; (2) Chemical synthesis: Add ethanol to a reaction container, add a palladium catalyst, replace the air in the reaction container with hydrogen, add the squalene to dissolve the squalene, and react at a reaction temperature of 30-50° C. to obtain squalane.

2. The method according to claim 1, Features The nucleic acid sequence of the gene ERG9 is shown in SEQ ID NO.1; the nucleic acid sequence of the gene leu2 is shown in SEQ ID NO.

2.

3. The method according to claim 1, Features The step (2) of step (1) is: inoculating a single colony of the recombinant bacteria into SD-URA liquid culture medium, culturing in a shaker at 30° C. and 220 rpm for 24 hours to obtain a seed solution.

4. The method according to claim 1, Features In step (2), the reaction temperature is 50°C.

5. The method according to claim 1 or 5, Features The palladium catalyst is 10% palladium carbon, 5% palladium carbon, 3% palladium carbon, 2% palladium carbon, 1% palladium carbon, palladium chloride, palladium acetate or palladium oxide.

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