CN110656055A - A kind of eukaryotic engineering strain and its preparation method and application - Google Patents

Abstract

The invention discloses a eukaryotic engineering strain and a preparation method and application thereof, belonging to the field of biotechnology. The protein expressed by the eukaryotic engineering strain is diacylglycerol acetyltransferase, which is shown in SEQ ID NO: 1 in the amino acid sequence table of the diacylglycerol acetyltransferase. The invention provides a eukaryotic engineering strain and a preparation method and application thereof. The eukaryotic engineering strain can synthesize acetylated triglycerides in large quantities, and the eukaryotic engineering strain can realize large-scale production of acetylated triglycerides. The preparation method provided in the embodiment of the invention can obtain a large number of eukaryotic engineering strains, which provides a basis for large-scale production of acetylated triglycerides. The application includes using the eukaryotic engineering strains to synthesize acetylated triglycerides.

Description

A kind of eukaryotic engineering strain and its preparation method and application

technical field

The invention relates to the field of biotechnology, in particular to a eukaryotic engineering strain and a preparation method and application thereof.

Background technique

Acetylated triglycerides are specialized triglycerides that contain an acetyl group at the sn-3 terminus. Compared with conventional long-chain triglycerides, acetylated triglycerides have the advantages of low viscosity and good activity at low temperature, so acetylated triglycerides can be directly used in diesel fuel as biofuels. In addition, the acetylated triglyceride derived from the acyl group at the sn-3 end can be used as a semi-synthetic product of 1,2-diacetic acid triglyceride, which can be used in food emulsifiers, lubricating oils, and PVC additives. Plasticizers and other plastic products.

Tam et al. in the United States tried to use the acetylated triglyceride synthase gene derived from Euonymus plants to transgenic Camelina sativa, soybean, Arabidopsis thaliana and Saccharomyces cerevisiae Through engineering transformation, a recombinant Saccharomyces cerevisiae is obtained, and the engineering strain is used to synthesize acetylated triglyceride.

In the process of realizing the present invention, the inventor found that the prior art has at least the following problems:

In addition to synthesizing acetylated triglycerides, the recombinant Saccharomyces cerevisiae also synthesized conventional triglycerides. The amount of acetylated triglycerides in each gram of the sample was 2.4 mg, and the synthesized acetylated triglycerides were only 2.4 mg per gram. About 0.24% of the dry weight of recombinant Saccharomyces cerevisiae, it can be seen that the yield of acetylated triglycerides synthesized by recombinant Saccharomyces cerevisiae is low, which is not conducive to the large-scale production of acetylated triglycerides.

SUMMARY OF THE INVENTION

In order to solve the problems of the prior art, the embodiments of the present invention provide a eukaryotic engineering strain and a preparation method and application thereof. The technical solution is as follows:

On the one hand, the embodiment of the present invention provides a kind of eukaryotic engineering strain, the protein expressed by the eukaryotic engineering strain is diacylglycerol acetyltransferase, and SEQ ID in the amino acid sequence table of the diacylglycerol acetyltransferase NO:1 shown.

On the other hand, the embodiment of the present invention provides a preparation method of the above-mentioned eukaryotic engineering strain, and the preparation method comprises:

connecting the codon-optimized diglyceride acetyltransferase encoding gene EfDAcT with the carrier to obtain a ligated product;

transferring the ligation product into competent cells to obtain the first transformation product;

The first transformation product is incubated at 37°C in a shaker for 0.6 to 1 hour to obtain a first resuscitation solution;

Coating the first resuscitation solution on a solid medium containing ampicillin resistance, and culturing at 37°C for 8-12 hours to obtain transformants;

The plasmid of the transformant is extracted, and the plasmid is digested to obtain the digested product;

Transferring the enzyme cleavage product into Yarrowia lipolytica competent cells to obtain a second transformation product;

The second transformation product is cultured in a yeast extract peptone glucose liquid medium at 28° C. in a shaker for 0.6-1.2 hours to obtain a second resuscitation solution;

The second resuscitation solution is coated on the yeast complete supplemented mixed medium, and cultured at 37° C. for 8-12 hours to obtain the eukaryotic engineering strain.

Specifically, the preparation method of the EfDAcT gene comprises: using the synthesized EfDAcT gene sequence as a cDNA template, using an upstream primer and a downstream primer to amplify to obtain the EfDAcT gene, and the sequence of the upstream primer is as SEQ ID in the sequence table. As shown in NO: 2, the sequence of the downstream primer is shown in SEQ ID NO: 3 in the sequence listing.

Specifically, the ligation product is transferred into the competent cells by heat shock method.

Further, the heat shock method includes: after mixing the ligation product and the competent cells, placing it on ice for 30-40 minutes to obtain a mixed solution, and placing the mixed solution in a 42°C water bath to heat shock After 45 s, cooling on ice for 2 min gave the conversion product.

Specifically, the vector is pYLXP'.

Specifically, the transformants are verified by PCR amplification, and the verified transformants are transferred into the Yarrowia lipolytica competent cells.

In another aspect, the embodiment of the present invention provides an application of the above-mentioned eukaryotic engineering strain, the application comprising: transferring the eukaryotic engineering strain into a yeast extract peptone glucose medium for subculture, After three subcultures, a third-generation eukaryotic strain is obtained, and the third-generation eukaryotic strain is transferred to a yeast-completely supplemented mixed medium, and the third-generation eukaryotic strain is transferred to a yeast-completely complemented mixed medium. On the mixed medium, an engineered strain is obtained, and the engineered strain is fermented and cultured for synthesizing acetylated triglycerides.

Specifically, the temperature of the fermentation culture is 28°C, and the time of the fermentation culture is 48h.

Specifically, the eukaryotic engineering strain is fermented and cultured at a rotational speed of 200-250 rpm.

The beneficial effects brought by the technical solutions provided in the embodiments of the present invention are as follows: the embodiments of the present invention provide a eukaryotic engineering strain and a preparation method and application thereof. The eukaryotic engineering strain can synthesize a large amount of acetylated triglycerides, and can pass The eukaryotic engineering strain realizes large-scale production of acetylated triglycerides, and the preparation method provided in the embodiment of the present invention can obtain a large number of eukaryotic engineering strains, which provides a basis for the large-scale production of acetylated triglycerides. This eukaryotic engineering strain is used to synthesize acetylated triglycerides.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative effort.

Fig. 1 is the agarose gel electrophoresis figure of the amplification product provided in the embodiment of the present invention;

Fig. 2 is the agarose gel electrophoresis figure of the verification amplification product provided in the embodiment of the present invention;

Fig. 3 is the agarose gel electrophoresis figure of the enzyme cleavage product provided in the embodiment of the present invention;

FIG. 4 is a graph of lipid distribution on a silica gel plate provided in an embodiment of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

Example

The embodiment of the present invention provides a eukaryotic engineering strain, the protein expressed by the eukaryotic engineering strain is diacylglycerol acetyltransferase (Diacylglycerol Acetyltransferase, DAcT), and SEQ ID in the amino acid sequence table of diacylglycerol acetyltransferase NO: 1 shows that the diacylglycerol acetyltransferase is derived from Fu Fang.

The embodiment of the present invention provides a preparation method of a eukaryotic engineering strain, and the preparation method includes:

The gene EfDAcT encoding a plant-derived diglyceride acetyltransferase was optimized according to the codon preference of Yarrowia lipolytica.

The codon-optimized plasmid of the diacylglycerol acetyltransferase encoding gene EfDAcT was ligated with the vector to obtain the ligation product pYLXP'::EfDAcT. Wherein, the nucleotide sequence of the plasmid of the codon-optimized diglyceride acetyltransferase encoding gene EfDAcT is shown in SEQ ID NO: 4 in the sequence listing.

At the time of implementation, the codon-optimized diglyceride acetyltransferase encoding gene EfDAcT was provided by the Chinese Academy of Agricultural Sciences. The specific preparation method of the codon-optimized diglyceride acetyltransferase encoding gene EfDAcT includes: using the chemically synthesized EfDAcT gene sequence as a cDNA template, using forward primer EfDAcT-F and reverse primer EfDAcT-R to carry out PCR amplification , the amplification product is the codon-optimized diacylglycerol acetyltransferase encoding gene EfDAcT. The sequence of the forward primer is shown in SEQ ID NO: 2 in the sequence listing, and the sequence of the reverse primer is shown in SEQ ID NO: 3 in the sequence listing. The PCR amplification system is 25 μL in total, and the PCR amplification system is shown in Table 1.

Table 1 shows the PCR amplification system

The PCR amplification procedure is as follows:

The amplification product was detected by agarose gel electrophoresis with a concentration of 1%. The electrophoresis result is shown in Figure 1. It can be seen from Figure 1 that the electrophoresis detection result is consistent with the actual situation, that is, the amplification product is correct, and the codon is passed through. The optimized diglyceride acetyltransferase encoding gene EfDAcT. The target gene fragment (amplification product) was recovered by using a gel recovery kit (purchased from Beijing Qingke Xinye Biotechnology Co., Ltd.).

The target gene fragment is ligated with the vector to obtain the ligated product. In this embodiment, the vector can be pYLXP', and T4-DNA ligase (purchased from New England Biolabs) can be used for ligation.

The ligation product is transformed into competent cells to obtain the first transformation product. The competent cells can be Escherichia coli DH5α competent cells (purchased from Nanjing Novozymes). When realized, the ligation product was transferred into E. coli DH5α competent cells by heat shock method. Specifically, on the ultra-clean workbench, take the prepared bacterial solution of Escherichia coli DH5α competent cells, add the ligation product, shake gently, and place on ice for about 30 minutes. Heat shock for 45s in the prepared 42°C constant temperature water bath, and quickly cool on ice for 2min to obtain the first transformation product.

The first transformation product was fermented and cultured at 37°C for 0.6 to 1 hour to obtain the first recovery solution; specifically, the first transformation product was cultured in 1 mL of LB liquid resistance-free medium, and recovered in a shaker at 37°C for about 1 hour. . Among them, the preparation method of LB liquid resistance-free medium includes: taking 5g of imported yeast extract, 10g of imported peptone, 10g of anhydrous sodium chloride and 1L of sterile water, mixing, and sterilizing at 121°C for 20min before use.

Coat 200 μL of the first resuscitation solution on the ampicillin-resistant LB solid medium, and place the coated LB solid plate medium face up for 20 minutes. After the LB solid plate medium was absorbed, it was inverted and cultured in a constant temperature incubator at 37°C for 8 to 12 hours to obtain transformants. The preparation method of the 1 L ampicillin-resistant LB solid plate medium comprises: weighing 5 g of imported ampicillin powder (purchased from BioSharp Company) and dissolving it in 100 mL of deionized water, so that the final concentration is 50 mg/mL. Sterile suction filtration was performed on a 0.22 μm suction filter, and the suction filtration product was divided into sterilized EP tubes and stored at -20°C.

The obtained transformants were verified by PCR amplification. The PCR amplification verification system is 25 μL in total, and the PCR amplification verification system is shown in Table 2.

Table 2 is the PCR amplification verification system

The PCR amplification procedure is as follows:

The verification amplification product is detected by agarose gel electrophoresis with a concentration of 1%. The electrophoresis result is shown in FIG. 2 . It can be seen from FIG. 2 that the electrophoresis detection result is consistent with the actual situation.

The Escherichia coli DH5α strain containing the ligation product was activated, and the plasmid was extracted, and the plasmid was digested to recover the digested product. The enzyme digestion system is 30 μL in total, and the enzyme digestion system is shown in Table 3.

Table 3 is the enzyme digestion system

The enzyme cleavage product was detected by agarose gel electrophoresis with a concentration of 1%, and the electrophoresis result was shown in Fig. 3. It can be seen from Fig. 3 that the electrophoresis detection result was consistent with the actual situation. The target gene fragment (enzyme digestion product) was recovered using a gel recovery kit (purchased from Beijing Qingke Xinye Biotechnology Co., Ltd.).

The enzyme cleavage product is transferred into the Yarrowia lipolytica competent cell to obtain the second transformation product; wherein, the preparation method of the Yarrowia lipolytica competent cell is as follows:

1. Culture of host Yarrowia lipolytica Po1g

In this example, competent cells were prepared from Yarrowia lipolytica Po1g, which was preserved and provided by the Laboratory of Applied Microbiology, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences. Pick a single colony of newly activated Yarrowia lipolytica Po1g, inoculate it with a toothpick in 2 mL of yeast extract powder peptone glucose liquid medium (YPD liquid medium), and cultivate at 28 °C for 12 h on a shaker at a constant temperature of 200 rpm. The strain of Yarrowia lipolytica Po1g was grown to the late logarithmic growth phase to obtain a bacterial suspension. 200 μL of bacterial suspension was inoculated into yeast extract powder peptone glucose solid medium (YPD solid medium), and cultured at 28° C. for 22 h to obtain the recovered Yarrowia lipolytica Po1g. Among them, the preparation method of YPD liquid culture medium is as follows: take 10 g of imported yeast extract, 20 g of imported peptone, 20 g of glucose and 1 L of sterile water, mix well, and sterilize at 121°C for 20 minutes before use.

2. Preparation of Yarrowia lipolytica Po1g competent cells by lithium acetate method

Transfer the recovered Yarrowia lipolytica Po1g into a 1.5mL sterile EP tube, add 5 μL of salmon essence treated at 100°C, 90 μL of PEG6000 with a concentration of 50% and 5 μL of lithium acetate with a concentration of 2 mol/L and mix well. , to obtain Yarrowia lipolytica Po1g competent cells, and the obtained bacterial liquid of Yarrowia lipolytica Po1g competent cells can be directly used as host bacteria for transformation experiments.

The enzyme-digested product was transformed into Yarrowia lipolytica Po1g competent cells by heat shock method to obtain the second transformation product. Specifically, on the ultra-clean workbench, take the prepared bacterial solution of Yarrowia lipolytica Po1g competent cells, add the enzyme digestion product, and shake gently. Heat shock for 30min in the prepared 39°C constant temperature water bath, and mix evenly every 10min to obtain the second conversion product.

The second transformation product was cultured in 1 mL of YPD liquid medium, and recovered in a shaker at 28° C. for about 1 hour to obtain a recovery solution.

All the resuscitation liquid was coated on the yeast complete supplemented mixed medium (CSM solid medium), and cultured at 37°C for 12 hours to obtain a strain containing the target gene. The preparation method of the CSM solid medium was as follows: take 0.69 g of CSM -Leu, 1.7g YNB, 20g glucose, 5g ammonium sulfate, 10g agar powder and 1L sterile water were mixed, sterilized at 121℃ for 20min before use.

Single colonies of transformants grown on CSM solid medium were used as eukaryotic engineering strains.

When realized, the eukaryotic engineering strain can be transferred to YPD solid medium for three subcultures to obtain the third-generation eukaryotic strain, and the third-generation eukaryotic strain can be transferred to the CSM medium to obtain the engineered strain (Yarrowialipolytica/pYLXP'::EfDAcT), the engineered strain was used to synthesize acetylated triglycerides.

The engineered strains were fermented and cultured to synthesize acetylated triglycerides, which were fermented and cultured in a shaker at a rotational speed of 220 rpm. The fermentation medium was YPD medium, the temperature of fermentation was 28°C, and the time of fermentation was 48h.

Detection of acetylated triglycerides synthesized by engineered strains.

After the fermentation, the bacterial liquid was transferred to a 50 mL centrifuge tube, centrifuged at 6000 rpm for 8 min, and the sediment (cell) was collected, and the cell was placed in an ultra-low temperature refrigerator at -80 °C for 12 h. The pre-cooled cells were dried in a vacuum freeze dryer for 24 h and weighed. Grind the bacterial cells into powder with a mortar to obtain bacterial powder, weigh 2 mg of bacterial powder, and transfer it to a 4 mL threaded glass bottle. Add 1 mL of methanol, 1 mL of chloroform, and 10 μL to the threaded glass bottle in sequence to a concentration of 0.5 mg/mL. The standard pentadecanoic acid triglyceride and 20 μL of the standard pentadecanoic acid with a concentration of 1 mg/mL were ultrasonically shaken for 15 min in the screw glass bottle, so that the bacteria powder and the reagent were fully dissolved in each other. Then centrifuge at 3500rpm for 10min in a low-speed centrifuge, and then centrifuge at 4000rpm for 5min to obtain the supernatant (sample to be tested). save.

Thin-layer chromatography (TLC) was used to separate and analyze different types of oils and fats. The specific methods are as follows:

1. Prepare TLC chromatography agent: prepare TLC chromatography agent in a fume hood. TLC chromatography agent includes chromatographic grade n-hexane with a volume ratio of 70:30:1, analytically pure ether and analytically pure acetic acid, n-hexane, ether and acetic acid, and shake the prepared TLC chromatography agent.

2. Spotting: Draw a horizontal straight line with a pencil at a distance of 1.5 cm from the edge on one side of the silica gel plate (Silica gel 60, 20×20 cm; EMD Chemicals, Germany), and mark the samples to be tested on the straight line at certain intervals. . After drying, the sample to be tested was reconstituted with 100 μL of chromatographic grade chloroform, and the sample was spotted at the corresponding mark on the silica gel plate.

3. About 10 minutes before the end of sampling, pour the configured TLC chromatography agent into the chromatography cylinder. After spotting the sample, place the silica gel plate in the layer cylinder for development. When the TLC chromatography agent runs to about 1 cm from the edge of the silica gel plate, take out the silica gel plate and dry it in a fume hood.

4. Color rendering. Mix acetone and water at a volume ratio of 4:1 to obtain a mixed solution. Weigh 10 mg of primuline and dissolve it in 20 mL of mixed solution to prepare a color developing solution. The color developing solution is uniformly sprayed on the surface of the silica gel plate for color development. The lipid distribution on the silica gel plate was observed under UV light, and the results were photographed. The results are shown in Figure 4. From Figure 4, acetylated triglyceride (AcTAG) bands, Free fatty acid (FFA), diglyceride (DAG) and monoglyceride (MAG) bands. The acetylated triglyceride strip is scraped off for methyl esterification to obtain methyl esterified acetylated triglyceride.

Gas-phase quantitative analysis of acetylated triglycerides:

The methylated acetylated triglyceride was transferred to a 4mL thread transparent glass bottle, 0.75mL sulfuric acid methanol (the volume ratio of sulfuric acid and methanol in sulfuric acid methanol was 5:95) was added to the thread transparent glass bottle, and the thread The cap of the transparent glass bottle was sealed and mixed by shaking. The threaded transparent glass bottle was placed in a water bath at 92°C for a water bath, and the water bath time was 1.5h. After the threaded transparent glass bottle was cooled to room temperature, 500 μL of 0.9% NaCl solution and 300 μL of chromatographic grade n-hexane were added to the threaded transparent glass bottle, vortexed for 1 min, and centrifuged at 3500 rpm for 20 min to obtain the upper layer solution (organic phase) , transfer the upper layer solution to a 1.5mL EP tube, centrifuge at 12000rpm for 10min to obtain a supernatant, transfer the supernatant to a GC bottle, and use gas chromatography to detect the fatty acid composition in the product.

The present embodiment adopts Agilent 7890A gas chromatograph to detect fatty acid methylation samples, and the chromatographic conditions are as follows:

The chromatographic column used for the separation of fatty acid methyl esters is HP-FFAP (30m×250μm×0.25μm), and a flame ionization detector (FID); the carrier gas is nitrogen with a purity of 99.999%; the temperature is programmed according to the initial start Temperature, 150°C, 10°C/min increase the temperature to 210°C, hold for 7min; continue to increase the temperature at 20°C/min to 230°C, hold for 6min; the split ratio is 30:1; the injection port temperature is 260°C.

After testing, the amount of acetylated triglycerides in each gram of engineering strain samples was 30 mg, the proportion of acetylated triglycerides in the dry weight of each gram of engineering strains was 3%, and the amount of acetylated triglycerides accounted for the total glycerol. The proportion of triesters was 18%. It can be seen that the acetylated triglyceride obtained by this engineering strain has a higher content.

The embodiment of the present invention provides a eukaryotic engineering strain and a preparation method and application thereof, the eukaryotic engineering strain can synthesize acetylated triglycerides in large quantities, and the large-scale production of acetylated triglycerides can be realized by the eukaryotic engineering strain , the preparation method provided in the embodiment of the present invention can obtain a large number of eukaryotic engineering strains, which provides a basis for the large-scale production of acetylated triglycerides. The application includes using the eukaryotic engineering strains to synthesize acetylated triglycerides.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

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Claims (10)

1. a eukaryotic engineering strain, is characterized in that, the protein expressed by described eukaryotic engineering strain is diglyceride acetyltransferase, and SEQ ID NO:1 in the amino acid sequence table of described diglyceride acetyltransferase shown. 2. a preparation method of eukaryotic engineering strain as claimed in claim 1, is characterized in that, described preparation method comprises: connecting the codon-optimized diglyceride acetyltransferase encoding gene EfDAcT with the carrier to obtain a ligated product; transferring the ligation product into competent cells to obtain the first transformation product; The first transformation product is incubated at 37°C in a shaker for 0.6 to 1 hour to obtain a first resuscitation solution; Coating the first resuscitation solution on a solid medium containing ampicillin resistance, and culturing at 37°C for 8-12 hours to obtain transformants; The plasmid of the transformant is extracted, and the plasmid is digested to obtain the digested product; Transferring the enzyme cleavage product into Yarrowia lipolytica competent cells to obtain a second transformation product; The second transformation product is cultured in a yeast extract peptone glucose liquid medium at 28° C. in a shaker for 0.6-1.2 hours to obtain a second resuscitation solution; The second resuscitation solution is coated on the yeast complete supplemented mixed medium, and cultured at 37° C. for 8-12 hours to obtain the eukaryotic engineering strain. 3. preparation method according to claim 2, is characterized in that, the preparation method of described EfDAcT gene comprises: using synthetic EfDAcT gene sequence as cDNA template, utilize upstream primer and downstream primer to carry out amplification, obtain described EfDAcT gene , the sequence of the upstream primer is shown in SEQ ID NO: 2 in the sequence listing, and the sequence of the downstream primer is shown in SEQ ID NO: 3 in the sequence listing. 4 . The preparation method according to claim 2 , wherein the ligation product is transferred into the competent cells by a heat shock method. 5 . 5 . The preparation method according to claim 4 , wherein the thermal shock method comprises: after mixing the ligation product and the competent cells, placing it on ice for 30-40 min to obtain a mixed solution, The mixture was placed in a water bath at 42° C. for 45 s and then cooled on ice for 2 min to obtain the conversion product. 6. The preparation method according to claim 2, wherein the carrier is pYLXP'. 7 . The preparation method according to claim 2 , wherein the transformant is subjected to PCR amplification verification, and the verified transformant is transferred into the Yarrowia lipolytica competent cell. 8 . 8. the application of a eukaryotic engineering strain as claimed in claim 1, is characterized in that, described application comprises: described eukaryotic engineering strain is transferred in yeast leaching peptone glucose medium to carry out subculture, through Subculture for three times to obtain a third-generation eukaryotic strain, transfer the third-generation eukaryotic strain to a yeast complete supplemented mixed medium, and transfer the third-generation eukaryotic strain to a yeast complete complementary mixed medium On the medium, an engineered strain is obtained, and the engineered strain is fermented and cultured for synthesizing acetylated triglycerides. 9 . The application according to claim 8 , wherein the temperature of the fermentation culture is 28° C., and the time of the fermentation culture is 48 hours. 10 . 10 . The application according to claim 8 , wherein the engineered strain is fermented and cultured at a rotational speed of 200-250 rpm. 11 .

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