CN110656076A - Prokaryotic engineering strain and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a prokaryotic engineering strain and a preparation method and application thereof, belonging to the technical field of biology. The protein expressed by the prokaryotic engineering strain is diglyceride acetyltransferase, and the amino acid sequence table of the diglyceride acetyltransferase is shown as SEQ ID NO 1. The preparation method comprises the following steps: connecting the DAcT gene with a vector to obtain a connection product; transferring the ligation product into a competent cell to obtain a transformation product; carrying out shake cultivation on the transformation product at 37 ℃ and 200rpm for 0.6-1 h to obtain a resuscitation solution; centrifuging the resuscitation solution to obtain a concentrated solution; the concentrated solution is coated on a solid medium containing kanamycin resistance, and cultured for 8-12 h at 37 ℃. The prokaryotic engineering strain is cultured for 24-48h at 25 ℃, high-purity acetylated triglyceride is synthesized in cells, and the large-scale production of the high-purity acetylated triglyceride can be realized through the prokaryotic engineering strain. The preparation method can obtain prokaryotic engineering strains and provide a foundation for large-scale production of acetylated triglyceride.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a prokaryotic engineering strain and a preparation method and application thereof.
Background
Acetylated triglycerides are a special kind of triglycerides that contain an acetyl group at the sn-3 end. Compared with the conventional long-chain triglyceride, the acetylated triglyceride has the advantages of low viscosity and good activity in a low-temperature environment, so that the acetylated triglyceride can be directly applied to diesel oil as biofuel. In addition, the acyl group derived from the sn-3 terminal, acetylated triglyceride can be used as a semisynthetic product of 1, 2-diacetic triglyceride, and the lipid can be applied to an emulsifier for food, a lubricating oil, a plasticizer for PVC and other plastic products.
Tam et al in the U.S. attempted to genetically engineer Camelina sativa (Camelina sativa), soybean, Arabidopsis thaliana (Arabidopsis thaliana), and Saccharomyces cerevisiae (Saccharomyces cerevisiae) with acetylated triglyceride synthase genes derived from plants of genus Euonymus (Euonymus), to obtain recombinant Saccharomyces cerevisiae, and synthesized acetylated triglyceride using the engineered strains.
In the process of implementing the invention, the inventor finds that the prior art has at least the following problems:
the recombinant saccharomyces cerevisiae can synthesize conventional triglyceride besides acetylated triglyceride, and the synthesized acetylated triglyceride is only about 0.24% of the dry weight of the recombinant saccharomyces cerevisiae per gram and accounts for 31% of the total triglyceride weight. If the encoding gene of the conventional triglyceride synthesized by the recombinant saccharomyces cerevisiae is knocked out, only acetylated triglyceride can be synthesized by the recombinant saccharomyces cerevisiae, but the simultaneously generated acetylated triglyceride is only about 0.15 percent of the dry weight of each gram of the recombinant saccharomyces cerevisiae, and the cell biomass is greatly reduced. This greatly limits the extraction and purification of acetylated triglycerides and is not conducive to large scale production of acetylated triglycerides.
Disclosure of Invention
The embodiment of the invention provides a prokaryotic engineering strain, a preparation method and an application thereof, and the prokaryotic engineering strain can singly synthesize high-purity acetylated triglyceride. The technical scheme is as follows:
on one hand, the embodiment of the invention provides a prokaryotic engineering strain, wherein the protein expressed by the prokaryotic engineering strain is diacylglycerol acetyltransferase, and the amino acid sequence table of the diacylglycerol acetyltransferase is shown as SEQ ID NO 1.
In another aspect, an embodiment of the present invention provides a preparation method of the above prokaryotic engineering strain, where the preparation method includes:
connecting the EfDAcT gene with a vector to obtain a connection product;
transferring the ligation product into a competent cell to obtain a transformation product;
fermenting and culturing the transformation product at 37 ℃ for 0.6-1 h to obtain a recovery solution;
centrifuging the resuscitation solution to obtain a concentrated solution;
and (3) coating the concentrated solution on a solid culture medium containing spectinomycin resistance, and performing shake cultivation for 8-12 h at 37 ℃ to obtain the prokaryotic engineering strain.
Specifically, the ligation product is transferred into the competent cells by heat shock.
Specifically, the heat shock method includes: and mixing the connecting product and the competent cells, placing the mixture on ice for 30-40 min to obtain a mixed solution, placing the mixed solution in a 42 ℃ water bath kettle for heat shock for 45s, and cooling the mixed solution on ice for 2min to obtain the conversion product.
Specifically, the vector is pCDFDuet-1 or pRSFDuet-1.
Specifically, the competent cell is a competent cell of common Escherichia coli BL21(DE 3).
In another aspect, an embodiment of the present invention provides an application of the above prokaryotic engineered strain, where the application includes: and (3) fermenting and culturing the prokaryotic engineering strain to synthesize the acetylated triglyceride.
Specifically, the prokaryotic engineering strain is cultured in a fermentation medium containing 2-10% of glycerol.
Specifically, the prokaryotic engineering strain is subjected to shake cultivation in the fermentation medium at the rotating speed of 120-180 rpm.
Specifically, the culture temperature is 25 ℃, and the culture time is 36-48 h.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the embodiment of the invention provides a prokaryotic engineering strain, a preparation method and an application thereof, wherein the prokaryotic engineering strain can singly synthesize high-purity acetylated triglyceride, and large-scale production of the acetylated triglyceride can be realized through the prokaryotic engineering strain.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a diagram of the electrophoresis of the EfDAcT amplification product of the present invention on a 1% agarose gel;
FIG. 2 is a diagram of agarose gel electrophoresis at a concentration of 1% for verifying amplification products provided by the embodiments of the present invention;
FIG. 3 is a graph showing the distribution of lipids on a silica gel plate according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
Examples
The embodiment of the invention provides a prokaryotic engineering strain, wherein the protein expressed by the prokaryotic engineering strain is Diacylglycerol Acetyltransferase (DAcT), the amino acid sequence table of the Diacylglycerol Acetyltransferase is shown as SEQ ID NO:1, and the Diacylglycerol Acetyltransferase is derived from wintercreeper.
The embodiment of the invention provides a preparation method of a prokaryotic engineering strain, which comprises the following steps:
ligating a plasmid carrying the EfDAcT (Euonymus fortunei diacetyltransferase enzyme) gene to a vector to obtain a ligation product pCDF:: EfDAcT (2560);
when implemented, the EfDAcT gene is provided by the chinese academy of agricultural sciences. The specific preparation method of the EfDAcT gene comprises the following steps: and (3) performing PCR amplification by using a chemically synthesized EfDAcT gene sequence as a cDNA template and using a forward primer EfDAcT-F and a reverse primer EfDAcT-R, wherein an amplification product is the EfDAcT gene. Wherein, the sequence of the forward primer is shown as SEQ ID NO. 2 in the sequence table, and the sequence of the reverse primer is shown as SEQ ID NO. 3 in the sequence table. The total volume of the PCR amplification system was 25. mu.L, and the PCR amplification system is shown in Table 1.
Table 1 shows PCR amplification System
The PCR amplification procedure was as follows:
the amplification product was detected by electrophoresis using 1% agarose gel, and the results of the electrophoresis are shown in FIG. 1, and it is clear from FIG. 1 that the results of the electrophoresis were consistent with those of the actual products. The target gene fragment (amplification product) is recovered by using a gel recovery kit (purchased from Beijing Optimalaceae, New Biotechnology Co., Ltd.).
The target gene fragment is ligated to a vector, which in this example may be pRSFDuet-1, using T4-DNA ligase (available from New England Biolabs), to obtain a ligation product.
Transferring the ligation product into a competent cell to obtain a transformation product; in this embodiment, the method for preparing the competent cell comprises:
1. cultivation of host E.coli
The competent cell is common Escherichia coli BL21(DE3), and the Escherichia coli BL21(DE3) is provided by the laboratory preservation of microorganisms applied to the institute of oil crops of Chinese academy of agricultural sciences. A single colony of newly activated escherichia coli BL21(DE3) is picked up, inoculated in 2mL of LB liquid non-resistant medium by using a toothpick, subjected to shake cultivation at constant temperature of 37 ℃ and 200rpm for 12h until a strain of escherichia coli BL21(DE3) grows to the late stage of logarithmic growth, and a bacterial suspension is obtained. Inoculating 1mL of bacterial suspension into 100mL of LB fermentation medium, and performing shake cultivation at 37 ℃ for 3-4 h until OD is reached600And (5) 0.4-0.5, and obtaining the recovered escherichia coli liquid. The preparation method of the LB liquid non-resistant culture medium comprises the following steps: 5g of imported yeast extract, 10g of imported peptone, 10g of anhydrous sodium chloride and 1L of sterile water are uniformly mixed, and the mixture is sterilized at 121 ℃ for 20min for use.
2. Using CaCl2Method for preparing competent cells of Escherichia coli
The fermented E.coli solution was transferred to 1.5mL of a precooled and sterile EP tube, allowed to stand on ice for 10min, and then centrifuged at 4000rpm at 4 ℃ for 2 min.
The supernatant was aspirated off with a micropipette, the pellet was retained, the cells in the pellet were gently suspended with 100. mu.L of pre-cooled Solution A Solution and left on ice for about 2min, after which they were centrifuged at 4000rpm for 2min at 4 ℃.
And (3) sucking the supernatant by using a micropipette, reserving the precipitate, adding 50-100 mu L of precooled Solution B Solution into the precipitate, slightly suspending the cells in the precipitate, subpackaging the cells into 25-50 mu L of small parts in an aseptic operation table, and placing the small parts on ice for more than 2min to obtain the bacterial liquid of the escherichia coli competent cells, wherein the bacterial liquid can be directly used as host bacteria for carrying out transformation experiments.
The ligation products were transferred into E.coli competent cells by heat shock. Specifically, on a clean bench, the prepared bacterial liquid of the escherichia coli competent cells is taken, the ligation product is added, the mixture is gently shaken up and placed on ice for about 30 min. Heating in a prepared constant temperature water bath kettle at 42 deg.C for 45s, and rapidly cooling on ice for 2min to obtain the converted product.
Fermenting and culturing the conversion product at 37 ℃ for 0.6-1 h to obtain a recovery solution; specifically, the transformed product was cultured in 1mL of LB liquid non-resistant medium and then subjected to shake recovery at 37 ℃ for about 1 hour. The preparation method of the LB liquid non-resistant culture medium comprises the following steps: 5g of imported yeast extract, 10g of imported peptone, 10g of anhydrous sodium chloride and 1L of sterile water are uniformly mixed, and the mixture is sterilized at 121 ℃ for 20min for use.
Centrifuging the resuscitation solution to obtain a concentrated solution; specifically, the resuscitation solution was concentrated by centrifugation and shaken up.
And (3) coating the concentrated solution on a solid culture medium containing spectinomycin resistance, and culturing at 37 ℃ for 8-12 h to obtain the prokaryotic engineering strain. Specifically, 50. mu.L of the medium was spread on a spectacular-resistant LB solid plate medium, and the preparation method of 1L of the spectacular-resistant LB solid plate medium included: 5g of an imported spectinomycin powder (purchased from BioSharp) was weighed, dissolved in 100mL of deionized water to a final concentration of 50mg/mL, subjected to sterile suction filtration using a suction filter having a size of 0.22 μm, and the suction-filtered product was dispensed into sterilized EP tubes and stored at-20 ℃. And (3) placing the LB solid plate culture medium containing the spectacular resistance with the front side facing upwards for half an hour to one hour until the resuscitation solution is completely absorbed by the LB solid plate culture medium containing the spectacular resistance, inverting the culture medium, and culturing the culture medium in a constant temperature incubator at 37 ℃ for 8-12 hours.
And carrying out PCR amplification verification on the obtained transformant to obtain the prokaryotic engineering strain. The total volume of the PCR amplification verification system was 25. mu.L, and the PCR amplification verification system is shown in Table 2.
Table 2 shows the PCR amplification verification system
The PCR amplification procedure was as follows:
the amplification product was detected by electrophoresis on 1% agarose gel, and the results are shown in FIG. 2, which shows that the results are consistent with the actual results.
Fermenting and culturing by a shaking table at the rotating speed of 120rpm, wherein a fermentation culture medium is a ZYP culture medium containing 2-10% of glycerol, and the prokaryotic engineering strain is synthesized into acetylated triglyceride through fermentation culture at the temperature of 25 ℃ for 36-48 h. Detecting the acetylated triglyceride synthesized by the prokaryotic engineering strain. The preparation method of 1L of fermentation medium comprises the following steps: taking 10g of zymolytic casein, 5g of yeast extract, 17.9g of disodium hydrogen phosphate dodecahydrate, 6.845g of potassium dihydrogen phosphate, 3.3035g of ammonium sulfate and 20g of glycerol, uniformly mixing, sterilizing at 115 ℃ for 20min, and then adding 10mL of 100 multiplied magnesium sulfate mother liquor, 10mL of 100 multiplied glucose and lactose mixed liquor and 1mL of 1000 multiplied trace element mixed liquor. The preparation method of the 100 multiplied magnesium sulfate mother liquor comprises the following steps: 4.92g of magnesium sulfate was weighed, dissolved in 100mL of deionized water, and sterilized separately. The preparation method of the mixed solution of 100 Xglucose and lactose comprises the following steps: 5g of glucose and 20g of lactose are weighed and dissolved in 100mL of deionized water, sterile filtered through a 0.22 mu m filter and stored in an environment at 4 ℃ for standby. The preparation method of the 1000 multiplied trace element mixed solution comprises the following steps: weighing 13.51g of ferric chloride hexahydrate, 2.22g of calcium chloride, 2.03g of manganous chloride tetrahydrate, 2.88g of zinc sulfate heptahydrate, 0.48g of cobalt chloride hexahydrate, 0.34g of cuprous chloride dihydrate, 0.12g of nickel chloride, 0.48g of sodium molybdate tetrahydrate, 0.35g of sodium selenite and 0.12g of boric acid to obtain a mixture, dissolving 2mL of concentrated hydrochloric acid in 1L of deionized water to obtain a mixed solution, adding the mixture into the mixed solution, performing sterile suction filtration through a 0.22 mu m suction filter, subpackaging the mixture into a sterilized EP tube, and storing the sterilized EP tube at the temperature of-20 ℃ for later use.
Detection of acetylated triglycerides: after fermentation, transferring the bacterial liquid into a 50mL centrifuge tube, centrifuging at 6000rpm for 8min, collecting thalli (precipitates), and placing the thalli in an ultra-low temperature refrigerator at-80 ℃ for precooling for 1-2 h. And (4) drying the precooled thalli in a vacuum freeze dryer for 24 hours, and weighing. Grinding the thalli into powder by using a mortar, weighing 20mg of bacteria powder, transferring the bacteria powder into a 4mL threaded glass bottle, sequentially adding 1mL of methanol, 1mL of chloroform, 10 muL of a pentadecanoic acid triglyceride standard substance with the concentration of 0.5mg/mL and 20 muL of a pentadecanoic acid standard substance with the concentration of 1mg/mL into the threaded glass bottle, and ultrasonically oscillating the glass bottle for 15min to ensure that the bacteria powder and the reagent are fully dissolved mutually. The supernatant (sample) was then carefully transferred to a new 4mL screw glass vial and stored at-20 ℃ in a low speed centrifuge at 3500rpm for 10min and then 4000rpm for 5 min.
Separating different kinds of oil by Thin-layer chromatography (TLC) and analyzing, wherein the specific method comprises the following steps:
1. preparing a TLC chromatographic agent: preparing a TLC (thin layer chromatography) agent in a fume hood, wherein the TLC agent comprises chromatographic grade n-hexane, analytically pure ether and analytically pure acetic acid in a volume ratio of 70:30:1, and the prepared TLC agent is shaken up;
2. sample application: a horizontal straight line was drawn with a pencil on one side of a Silica gel plate (Silica gel 60, 20X 20 cm; EMD Chemicals, Germany) at a distance of 1.5cm from the edge, and samples of all points were marked on the straight line at regular intervals. Re-dissolving the dried sample by using 100 mu L of chromatographic grade chloroform, spotting the sample at a corresponding mark position of a silica gel plate, repeating the operation once, and ensuring the diameter of each point as small as possible during spotting;
3. about 10min before the end of spotting, the prepared TLC chromatography was poured into the chromatography cylinder. After the sample is spotted, the silica gel plate is placed in a spreading layer cylinder for spreading. When the TLC chromatography agent runs to the position about 1cm away from the upper edge of the silica gel plate, taking out the silica gel plate and airing the silica gel plate in a fume hood;
4. and (4) developing color. Mixing acetone and water according to the volume ratio of 4:1 to obtain a mixed solution, weighing 10mg of primuline, dissolving in 20mL of the mixed solution to prepare a color development solution, and uniformly spraying the color development solution on the surface of a silica gel plate for color development. Lipid distribution on the silica gel plate was observed under an ultraviolet lamp, and the retention results were photographed, and the results are shown in FIG. 3, in which an acetylated triglyceride (Acetyl-TAG) band, a Triglyceride (TAG) band, a Free Fatty Acid (FFA) band, a Diglyceride (DAG) and a Monoglyceride (MAG) band are seen in FIG. 3. Scraping off the acetylated triglyceride strip to carry out methyl esterification to obtain methyl esterified acetylated triglyceride.
The acetylated triglycerides were subjected to gas phase quantitative analysis:
transferring the acetylated triglyceride subjected to methyl esterification into a 4mL threaded transparent glass bottle, adding 0.75mL of methanol sulfate (the volume ratio of sulfuric acid to methanol in the methanol sulfate is 5:95) into the threaded transparent glass bottle, sealing the bottle cap of the threaded transparent glass bottle, and then shaking and mixing uniformly. And (3) putting the threaded transparent glass bottle into a water bath kettle at the temperature of 92 ℃ for water bath, wherein the water bath time is 1.5 h. After the threaded transparent glass bottle is cooled to room temperature, 500 mu L of NaCl solution with the concentration of 0.9% and 300 mu L of chromatographic grade n-hexane are added into the threaded transparent glass bottle, vortex oscillation is carried out for 1min, centrifugation is carried out at 3500rpm for 20min to obtain an upper layer solution (organic phase), the upper layer solution is taken and transferred into a 1.5mL EP tube, centrifugation is carried out at 12000rpm for 10min to obtain a supernatant, the supernatant is transferred into a GC bottle, and the fatty acid composition in a product is detected by adopting gas chromatography.
In this example, an agilent 7890A gas chromatograph is used to detect fatty acid methyl esterification samples, and the chromatographic conditions are as follows:
the chromatographic column used for separating fatty acid methyl ester is HP-FFAP (30m × 250 μm × 0.25 μm), Flame Ionization Detector (FID); the carrier gas is nitrogen with the purity of 99.999 percent; heating according to a program: the temperature is initially started, 150 ℃, increased to 210 ℃ at the speed of 10 ℃/min and kept for 7 min; continuously raising the temperature to 230 ℃ at the speed of 20 ℃/min, and keeping the temperature for 6 min; the split ratio is 30: 1; the injection port temperature was 260 ℃.
The proportion of acetylated triglycerides in the sample (BL21-pCDFDuet-1:: EfDAcT) was determined to be 100% of total triglycerides. It can be seen that the acetylated triglycerides obtained by the prokaryotic engineered strain have a very high purity.
The embodiment of the invention provides a prokaryotic engineering strain, a preparation method and an application thereof, wherein the prokaryotic engineering strain can singly synthesize high-purity acetylated triglyceride, and the large-scale production of the acetylated triglyceride can be realized through the prokaryotic engineering strain.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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Leu Val Ile Tyr Cys Cys His Val Tyr Val Leu Leu Asp Ile Ser Leu
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Claims (10)
1. A prokaryotic engineering strain is characterized in that a protein expressed by the prokaryotic engineering strain is diacylglycerol acetyltransferase, and an amino acid sequence table of the diacylglycerol acetyltransferase is shown as SEQ ID NO 1.
2. A method for the preparation of a prokaryotic engineered strain as claimed in claim 1, characterized in that it comprises:
connecting the EfDAcT gene with a vector to obtain a connection product;
transferring the ligation product into a competent cell to obtain a transformation product;
carrying out shake culture on the transformation product at 37 ℃ for 0.6-1 h to obtain a resuscitation solution;
centrifuging the resuscitation solution to obtain a concentrated solution;
and (3) coating the concentrated solution on a solid culture medium containing kanamycin resistance, and culturing at 37 ℃ for 8-12 h to obtain the prokaryotic engineering strain.
3. The method according to claim 2, wherein the ligation product is transferred into the competent cell by heat shock.
4. The method of claim 3, wherein the thermal shock method comprises: and mixing the connecting product and the competent cells, placing the mixture on ice for 30-40 min to obtain a mixed solution, placing the mixed solution in a 42 ℃ water bath kettle for heat shock for 45s, and cooling the mixed solution on ice for 2min to obtain the conversion product.
5. The method according to claim 2, wherein the vector is pCDFDuet-1 or pRSFDuet-1.
6. The method according to claim 2, wherein the competent cell is a competent cell of Escherichia coli BL21(DE 3).
7. Use of the prokaryotic engineered strain according to claim 1, characterised in that it comprises: and (3) fermenting and culturing the prokaryotic engineering strain to synthesize the acetylated triglyceride.
8. The use according to claim 7, wherein the prokaryotic engineered strain is cultured in a fermentation medium containing glycerol at a concentration of 2-10%.
9. The use of claim 8, wherein the prokaryotic engineered strain is shake-cultured in the fermentation medium at a rotation speed of 120-180 rpm.
10. The use according to claim 7, wherein the temperature of the culture is 25 ℃ and the time of the culture is 36-48 h.
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