CN117801968A - Genetically engineered bacterium for over-expressing phosphorylcholine cytidine transferase gene, construction method and application thereof - Google Patents

Genetically engineered bacterium for over-expressing phosphorylcholine cytidine transferase gene, construction method and application thereof Download PDF

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CN117801968A
CN117801968A CN202311560651.1A CN202311560651A CN117801968A CN 117801968 A CN117801968 A CN 117801968A CN 202311560651 A CN202311560651 A CN 202311560651A CN 117801968 A CN117801968 A CN 117801968A
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phosphorylcholine
cct
transferase gene
genetically engineered
expressing
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凌雪萍
崔潇文
卢英华
陈翠雪
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Xiamen University
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Xiamen University
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Abstract

The invention provides a genetic engineering bacterium for over-expressing a phosphorylcholine cytidine transferase gene, a construction method and application thereof, wherein the genetic engineering bacterium is obtained by over-expressing the phosphorylcholine cytidine transferase gene CCT in a starting bacterium; the starting strain is schizochytrium sp 21; the nucleotide sequence of the phosphorylcholine cytidine transferase gene is shown as SEQ ID NO: 1. The total grease and phospholipid content of the engineering bacteria are obviously increased, the accumulation of polyunsaturated fatty acids is increased, the grease content is increased by 20% and the PUFAs content is increased by 15% under the condition of keeping the dry weight of cells unchanged, and theoretical basis is provided for regulating and controlling schizochytrium limacinum to improve fatty acid synthesis.

Description

Genetically engineered bacterium for over-expressing phosphorylcholine cytidine transferase gene, construction method and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a genetically engineered bacterium for over-expressing a phosphorylcholine cytidine transferase gene, and a construction method and application thereof.
Background
Schizochytrium sp is a marine fungus capable of synthesizing various beneficial active substances, such as fatty acids, squalene, pigments (lutein, astaxanthin, carotenoids) and the like. The oil content of the microbial strain can reach more than 50% of that of dry fungus cells, and the proportion of polyunsaturated fatty acids (PUFAs) in the total lipid can reach 40% -70%.
PUFAs are mainly produced by the anaerobic pathway of polyketide synthases, released as free fatty acids and accumulated in polar phospholipids such as Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) or Phosphatidylinositol (PI), and eventually migrate to nonpolar lipids such as Diacylglycerol (DAG) or Triacylglycerol (TAG) for storage.
In the related art, the regulation and control of polyunsaturated fatty acids (PUFAs) produced by schizochytrium is mainly focused on the regulation and control by using a regulator in the fermentation process, and the regulation and control mode is limited.
At present, the research on phosphorylcholine cytidine transferase (CCT) is limited, mainly focused on animal organs and insects, and has not been reported in the field of genetic engineering of schizochytrium.
Disclosure of Invention
The invention aims to solve at least one of the technical problems in the technology to a certain extent, and provides a genetically engineered bacterium for over-expressing phosphorylcholine cytidine transferase gene, and a construction method and application thereof. The schizochytrium Schizochytrium limacinum SR is taken as an original strain, and the phospholipid metabolic pathway is modified to promote the synthesis of grease and the accumulation of polyunsaturated fatty acids.
Therefore, in an embodiment of the present invention, the present invention proposes, in a first aspect, a genetically engineered bacterium that overexpresses a phosphorylcholine cytidine transferase gene, wherein the genetically engineered bacterium is obtained by overexpressing a phosphorylcholine cytidine transferase gene CCT in a starting bacterium; the starting strain is schizochytrium sp 21; the nucleotide sequence of the phosphorylcholine cytidine transferase gene is shown as SEQ ID NO: 1.
According to the embodiment of the invention, the phospholipid content in the engineering bacteria is greatly improved. In schizochytrium, PUFAs bind to phospholipids and are then transferred to DAG and TAG by enzymes such as PDAT, PDCT and DGAT. Therefore, by over-expressing the phosphocholine cytidine transferase gene CCT to improve phospholipid metabolism, the synthesis of grease and the accumulation of PUFAs are obviously promoted, the content of grease is increased by 20 percent, and the content of PUFAs is increased by 15 percent under the condition of keeping the dry weight of cells unchanged.
The invention provides a construction method of the genetically engineered bacterium in a second aspect, which comprises the following steps:
(1) S.limacinum SR21 genomic DNA is used as a template, and SEQ ID NO:2 and SEQ ID NO:3, PCR amplifying the phosphorylcholine cytidine transferase gene CCT by using the sequence shown in the formula 3 as a primer;
(2) Inserting the phosphorylcholine cytidine transferase gene CCT into a homologous recombination region of a pBlue-zeo-18sUD overexpression vector, and constructing a CCT overexpression vector pBlue-zeo-18sUD-CCT with bleomycin as resistance;
(3) Linearizing the homologous recombination region of the overexpression vector pBlue-zeo-18sUD-CCT, electrically converting into the genome of S.limacinum SR21 for homologous recombination, and screening and identifying the resistance of bleomycin to obtain the genetically engineered bacterium SR21 over-expressing the CCT gene.
According to the embodiment of the invention, a key step of phospholipid pathway construction-phosphorylcholine cytidine transferase (CCT) gene overexpression vector is established in escherichia coli based on a genetic engineering means, then an expression frame with bleomycin resistance screening genes is inserted into an 18sDNA locus of an SR21 genome to construct schizochytrium engineering bacteria, the total grease and phospholipid content of the engineering bacteria are obviously increased, polyunsaturated fatty acid accumulation is increased, and a theoretical basis is provided for regulating and controlling schizochytrium to improve fatty acid synthesis.
In a second aspect of the invention, the use of the genetically engineered bacterium over-expressing a phosphorylcholine cytidine transferase gene in the production of fatty acids is provided.
In a third aspect of the present invention, there is provided a method for enhancing schizochytrium limacinum phospholipid metabolism to increase fatty acid production, comprising inoculating the genetically engineered bacterium overexpressing a phosphorylcholine cytidine transferase gene described above to a seed culture medium for activation to obtain a fermentation strain; inoculating the fermentation strain into a fermentation culture medium for fermentation culture, and collecting thalli for identification and analysis of oil components.
Optionally, streaking the genetically engineered bacteria over-expressing the phosphorylcholine cytidine transferase gene on a solid seed culture medium, and carrying out stationary culture at the temperature of 27-29 ℃; selecting a monoclonal on a flat plate to a liquid seed culture medium, and culturing at the temperature of 27-29 ℃ and the speed of 200-250 rpm to obtain first-stage seeds; inoculating the primary seeds into a new liquid seed culture medium according to the inoculum size of 4% -8%, and culturing at the temperature of 27-29 ℃ and the speed of 200-250 rpm to obtain secondary seeds; inoculating the secondary seeds into a fermentation culture medium in an inoculum size of 4% -8%, culturing for 120 hours at the temperature of 27-29 ℃ and the rpm of 200-250 rpm, and sampling every 24 hours in the culturing process for relevant analysis.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram of a gene overexpression vector pBlue-zeo-18sUD-CCT in the examples of the present invention;
FIG. 2 is an agarose gel electrophoresis diagram of the identification of the genetically engineered strain according to the embodiment of the invention;
FIG. 3 shows the results of CCT fermentation biomass and oil weight of the S.limacinum SR21 and the genetically engineered strain SR21 of the embodiment of the invention;
FIG. 4 shows the ratio of CCT polar oil to nonpolar oil of a wild strain of S.limacinum SR21 to a genetically engineered strain SR21;
FIG. 5 shows the fatty acid content change of S.limacinum SR21 wild-type strain and genetically engineered strain SR21 of the embodiment of the invention;
FIG. 6 shows the change of important phospholipid content of S.limacinum SR21 wild-type strain and genetically engineered strain SR21 of the embodiment of the invention, wherein the CCT is changed in 72 hours and 120 hours.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The following disclosure provides many different embodiments, or examples, for implementing different embodiments of the invention. In order to simplify the present disclosure, specific embodiments or examples are described below. Of course, they are merely examples and are not intended to limit the invention. In addition, one of ordinary skill in the art will recognize the applicability of other processes and/or the use of other materials, as examples of the various specific processes and materials provided by the present invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques in the fields of chemistry, molecular biology, etc., which are within the ability of a person skilled in the art. In addition, unless otherwise indicated, herein, nucleic acids are written in a5 'to 3' direction from left to right, and amino acid sequences are written in an amino-to carboxy-terminal direction from left to right.
In the following examples, the following examples were carried out:
the formula of the solid seed culture medium is as follows: 30-35 g/L glucose, 8-11 g/L yeast powder, 50-52 mL 20 Xcomponent A, 500 XCaCl 2 1.5-2.5 mL, 15-20 g/L of agar, and regulating the pH value to 6.4-6.6.
The formula of the liquid seed culture medium is as follows: 30-35 g/L glucose, 8-11 g/L yeast powder, 50-52 mL 20 Xcomponent A, 500 XCaCl 2 1.5 to 2.5mL, and the pH value is adjusted to 6.4 to 6.6.
The formula of the fermentation medium is as follows: glucose 89-92 g/L, corn steep liquor 4-6 g/L, tryptone 4-6 g/L, 20 Xcomponent A48-52 mL, 500 XCaCl 2 2mL, and the pH is adjusted to 6.4-6.6.
The formula of the 20X component A is as follows: naSO (Naso-doped sodium sulfonate) 4 238～242g/L，MgSO 4 38～42g/L，(NH 4 ) 2 SO 4 19～21g/L，KH 2 PO 4 19～21g/L，K 2 SO 4 12～14g/L，KCl 9～11g/L；
500×CaCl 2 The formula is as follows: caCl (CaCl) 2 ·2H 2 O 83～87g/L。
The whole period of fermentation culture is 120h, and sampling detection is carried out every 24 h.
Schizochytrium s.limacinum SR21 was purchased from the american type culture collection (ATCC, USA).
The invention is described below by way of illustrative specific examples, which are not intended to limit the scope of the invention in any way. Specifically described are: the reagents used in the present invention are commercially available unless otherwise specified.
EXAMPLE 1 construction of the overexpression vector pBlue-zeo-18sUD-CCT
1. PCR amplification of CCT genes
Designing an amplification primer CCT-F/CCT-R according to the sequence information of the CCT gene of schizochytrium limacinum SR21, and then obtaining the CCT gene by using PrimeStar high-fidelity polymerase and the primer and adopting a PCR mode by taking the S.limocinum SR21 wild strain gene as a template, wherein the nucleotide sequence of the CCT gene is shown as SEQ ID NO: 1.
CCT-F：TGCTCTAGAC CTTCAAAGCA CTTCCCAG(SEQ ID NO：2)；
CCT-R：CCGCTCGAGA TCGACAAAAA CAAAAGAATT GGA(SEQ ID NO：3)；
The PCR reaction system is shown in the following table:
PCR reaction procedure is shown in the following Table
2. Construction of the overexpression vector pBlue-zeo-18sUD-CCT
The vector pBlue-zeo-18sUD and the CCT gene obtained above were double digested with restriction enzymes at 37℃for 2h.
The double enzyme digestion system is shown in the following table
The CCT gene was ligated to the vector pBlue-zeo-18sUD fragment using T4 ligase and reacted at 16℃for 12 hours.
The connection system is shown in the following table
The ligation product was transformed into E.coli DH 5. Alpha. Competent cells to obtain the overexpression vector pBlue-zeo-18sUD-CCT (refer to FIG. 1). The transformation method is as follows:
the ligation product was precooled on ice.
Competent E.coli (100. Mu.L) stored in an ultra-low temperature refrigerator at-80℃was removed and placed on ice for 10min.
mu.L of the ligation product was pipetted into competent E.coli, mixed well and placed on ice for 30min.
The mixture of ligation product and E.coli competent cells was placed in a water bath at 42℃for 45 to 90 seconds. Immediately after the completion of the reaction, the mixture was placed on ice for 2 minutes to 3 minutes.
900. Mu.L of E.coli LB liquid medium was added to the above ice-bathed competent cells, and the mixture was subjected to shaking culture at 37℃and 200rpm for 1 hour.
100. Mu.L of the bacterial liquid was aspirated from the shake flask containing competent cells after 1 hour of culture, and the culture was performed on a plate (E.coli LB solid medium) containing an antibiotic (bleomycin) at 37℃for 12 hours to 16 hours.
Example 2 construction of genetically engineered Strain SR 21::: CCT
Extracting the overexpression vector pBlue-zeo-18sUD-CCT, linearizing and recovering with restriction endonuclease Not I, transferring electric shock into schizochytrium competence for homologous recombination, coating bleomycin (Zeocin) resistance plates, culturing at 28 ℃ for 3-5d, and screening transformants. After genomic extraction, transformants were verified by PCR (as shown in fig. 2). Thus obtaining schizochytrium engineering bacteria SR21 over-expressing CCT genes.
Preparation of schizochytrium competence:
(1) Selecting schizochytrium single colony to 10mL of schizochytrium basal liquid seed culture medium, culturing for 24 hours at 28 ℃ in a shaking table at 200rpm, and transferring 50mL of liquid seed culture medium according to 4% of inoculum size for culturing for 20 hours;
(2) Taking 10mL of bacterial liquid, and centrifuging at a rotating speed of 4000 Xg under the condition of room temperature to obtain bacterial cells;
(3) Then treated with 25mM DTT in PBS for 30min, and centrifuged to discard the supernatant at 4 ℃;
(4) Respectively cleaning thalli twice by using precooled sterile water and precooled sorbitol solution;
(5) The cells were resuspended in an appropriate amount of sorbitol solution and then sub-packaged in sterilized 1.5mL centrifuge tubes, 100. Mu.L per tube, and placed on ice for use.
Electric conversion of schizochytrium:
(1) Mixing 100 μl schizochytrium competent cells and about 10 to 15 μl of the ligation product (about 3 to 5 μg) uniformly, transferring to a pre-chilled electric shock cup, placing in ice bath for 30min, and then shocking for 6 ms at 2KV for one pulse;
(2) Sucking 1mL of schizochytrium basal liquid seed culture medium, adding the schizochytrium basal liquid seed culture medium into an electric rotating cup, fully and uniformly mixing the culture medium and the electric rotated schizochytrium, transferring the mixture to a sterile 1.5mL centrifuge tube, and culturing the mixture in a shaking table at 28 ℃ and 200rpm for 2 to 3 hours;
(3) After the bacterial liquid is centrifuged and the supernatant is discarded, the bacterial liquid is coated on schizochytrium basal solid seed culture medium containing antibiotics and is cultured for 3 to 5 days in a constant temperature incubator at 28 ℃.
Example 3 identification analysis of schizochytrium engineering bacteria SR 21::: CCT
1. Cultivation of schizochytrium:
(1) CCT and schizochytrium original bacteria (schizochytrium S.limocinum SR 21) seeds preserved at-80 ℃ are streaked on a solid seed culture medium, and are cultured for 36 hours at 28 ℃;
(2) First-stage seed: selecting high-quality monoclonal on the cultured solid seed culture medium, and culturing in 10mL seed culture medium at 28 ℃ and 200rpm for 24-36 h;
(3) Secondary seed: 2mL of the primary seed culture medium is sucked and transferred into 50mL of the seed culture medium, and the primary seed culture medium is cultured for 20 to 24 hours at the temperature of 28 ℃ and the rpm of 200 rpm;
(4) Shaking and fermenting: from the secondary seed medium, a certain amount of seed culture solution was transferred to 50mL of fermentation medium at an inoculum size of 8%, and cultured at 28℃and 200rpm for 144 hours.
2. Measurement of the biomass:
(1) 1mL of the fermentation broth was accurately aspirated, added to a 1.5mL centrifuge tube that had been weighed in advance, centrifuged in a centrifuge at 10000 Xg for 2 minutes at room temperature, and then the supernatant was decanted.
(2) The cells were washed by air-sucking and mixing with 1mL of physiological saline, centrifuged at 10000 Xg for 2 minutes at room temperature in a centrifuge, and then the supernatant was discarded.
(3) Repeating the operation step (2) once.
(4) The samples were placed in a vacuum freeze dryer and after 24 hours of freeze drying, the centrifuge tube was removed and weighed. Biomass is calculated.
As shown in FIG. 3, the biomass of S.limacinum SR21 was always higher than that of the mutant with the increase of the culture time, and the difference was maximum at 72 hours, which was about 24.3% higher. The turning points of the wild type and SR21, which are respectively 72h and 96h when CCT enters the later growth stage, but the biomass tends to be the same at 144h, about 37.7g/L.
3. Determination of total oil content:
(1) 5mL of fermentation broth is taken into a50 mL centrifuge tube, and 5mL of hydrochloric acid is added into the centrifuge tube and heated at the constant temperature of 65 ℃ for 1h until the thalli are completely digested.
(2) The centrifuge tube was removed and after cooling to room temperature, 15mL of n-hexane was added for three extractions, and the upper organic phase was taken into a weighed 50mL centrifuge tube.
(3) The centrifuge tube was purged with nitrogen to evaporate the hexane.
(4) And taking out the centrifuge tube, cooling to room temperature, and weighing, wherein the weight of the empty centrifuge tube is subtracted from the weight of the centrifuge tube to obtain the total grease yield.
As shown in FIG. 3, the total oil weight of the S.limacinum SR21 is higher than that of SR21:: CCT only for 96 hours, the total oil yield of the SR21:: CCT is increased by about 31.23%, the total oil yield of the SR21:: CCT is higher than that of the wild type at other times, the total oil reaches the maximum value for 120 hours, and the total oil yield is increased by about 17.80%.
4. Determination of fatty acid content
(1) To the oil obtained in step 3, 5mL of 0.5M KOH-CH was added 3 And (3) placing the centrifuge tube in a constant-temperature water bath kettle at 65 ℃ for heating for 10min until the grease is completely dissolved.
(2) After the centrifuge tube was taken out and cooled to room temperature, 5mL of 30% boron trifluoride diethyl etherate was added to the centrifuge tube, and the centrifuge tube was heated in a 65℃thermostat water bath for 30min.
(3) Taking out the centrifuge tube, cooling to room temperature, adding 5mL of normal hexane, shaking, mixing uniformly, adding 1mL of saturated sodium chloride solution to prevent emulsification, standing for 5min, and layering.
(4) The upper organic phase was pipetted into a 5mL centrifuge tube containing an appropriate amount of anhydrous sodium sulfate for dehydration.
(5) The solution in the 5mL centrifuge tube was filtered through a 0.22 μm organic filter and filled into a gas bottle for gas chromatography. The gas chromatography detection conditions were as follows:
instrument: agilentGC7890A gas chromatography; chromatographic column: supelco-2560 (100 m 0.25mm ID,0.20 μm film); sample injection setting: the sample injection amount is 1 mu L, the sample injection temperature is 260 ℃, and the split ratio is 50:1; carrier gas: nitrogen, 20cm/s; detector temperature: 260 ℃; column temperature control: the initial temperature is 140 ℃, and the temperature is maintained for 5min; then the temperature was raised to 260℃at a rate of 3℃per minute and maintained for 10 minutes.
The results are shown in FIG. 5, in which SFA content was significantly reduced and PUFA content was significantly increased after the overexpression of CCT. C14:0, C16:0 and C18:0 are the major components of SFA. The C14:0 and C18:0 in the CCT are slightly reduced, the C16:0 is greatly changed, the reduction amplitude is 32% -39%, and the tendency of rising and then falling is presented. PUFAs are the opposite trend, with significant changes in DPA and DHA. SR21 the proportion of DPA and DHA in CCT is lower than S.limacinum SR21 in early stage of cell growth, but is significantly higher than S.limacinum SR21 in middle and later stage of cell growth. Taking the last day (120 h) of fermentation as an example, the ratio of SR21 to C14:0, C16:0 and C18:0 in the total fatty acids of CCT is respectively reduced by 41.08%, 33.60% and 25.31%, and the ratio of DPA and DHA is respectively increased by 78.41% and 45.86%.
5. Isolation and identification of phospholipids
25mg of total lipid was dissolved in 1mL of chloroform/methanol solution (2:1, v/v), 20. Mu.L of PC (19:0/19:0) was added as an internal standard, and Solid Phase Extraction (SPE) with n-hexane added was performed. The nonpolar lipids were eluted with 4mL of two volume ratios (4:1 and 1:1 v/v) of n-hexane/ether solution, respectively. The polar lipids (principally phospholipids) were then filtered with 5mL of methanol and 6mL of a trifluoromethane/methanol/water solution (3:25:2, v/v/v). The obtained phospholipids were then used for UPLC-Q/TOF-MS analysis. Analysis was performed using an ACQUITY UPLC system (Waters Corporation, milford, USA) equipped with a Waters UPLC BEH C chromatographic column (150 mm. Times.2.1 mm. Times.1.7 μm).
The results are shown in FIGS. 4 and 6, wherein the non-polar lipid content of S.limacinum SR21 and SR21 is 81.07% and 77.31% at 48 hours and peaks at 95.41% and 92.72% at 96 hours, respectively. In the fermentation process, the content of the polar lipid of the SR21 is always higher than that of the S.limacinum SR21, and the content of the polar lipid is highest (22.69%) at 48 hours and is 3.9% higher than that of the S.limacinum SR21. Subsequent analysis of the phospholipid composition showed that the phospholipids remained predominantly C16:0, DPA and DHA, consistent with previous results from gas phase detection of various fatty acid components in the phospholipids. The PC mainly comprises PC-C16:0/C22:6, PC-C22:5/C22:6 and PC-C22:6/C22:6. In the wild type, these phospholipid contents decrease significantly over time, whereas in the mutant strain the opposite is true. LPC consists essentially of LPC-C22:5 and LPC-C22:6, which are present in a SR 21:CCT lower than the wild type at 72h, but higher than the wild type at 120 h. PI is mainly composed of PI-C16:0/C22:5 and PI-C16:0/C22:6, and the change is similar to that of PC-C16:0/C22:6. At 120h, the content of PI-C16:0/C22:5 in the SR 21:CCT is obviously changed, and the reduction is about 11%. PG is mainly PG-C16:0/C16:0, and the maximum amplitude reduction is about 7.7% at 72 h. Thus, the CCT strain has significantly increased phospholipid content containing DPA and DHA in the late fermentation stage, while the phospholipid content containing C16:0 is reduced or has no significant change.
In summary, according to the embodiment of the invention, schizochytrium Schizochytrium limacinum SR is adopted as an original strain, a key step of constructing a phospholipid pathway in escherichia coli based on a genetic engineering means, namely a phosphorylcholine cytidine transferase (CCT) gene overexpression vector, then an expression frame with a bleomycin resistance screening gene is inserted into an 18s rDNA locus of an SR21 genome to construct schizochytrium engineering bacteria, and phospholipid metabolic pathways of the schizochytrium engineering bacteria are modified, so that the total grease and the phospholipid content of the engineering bacteria are obviously increased, the accumulation of polyunsaturated fatty acids is increased, the phospholipid ratio is improved by nearly 20% compared with that of the original strain, and the phospholipid ratio is doubled at 120 h; in addition, as the phospholipid is used as an important intermediate in the accumulation process of polyunsaturated fatty acids, the content of PUFAs such as DHA and the like in important phospholipids such as PC/PI is obviously increased, and a theoretical basis is provided for regulating and controlling schizochytrium limacinum to improve fatty acid synthesis.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (5)

1. The genetically engineered bacterium for over-expressing the phosphorylcholine cytidine transferase gene is characterized in that the genetically engineered bacterium is obtained by over-expressing the phosphorylcholine cytidine transferase gene CCT in a starting bacterium; the starting strain is schizochytrium sp S.
limacinum SR21; the nucleotide sequence of the phosphorylcholine cytidine transferase gene is shown as SEQ ID NO: 1.
2. The method for constructing genetically engineered bacteria of claim 1, comprising the steps of:
(1) S.limacinum SR21 genomic DNA is used as a template, and SEQ ID NO:2 and SEQ ID NO:3, PCR amplifying the phosphorylcholine cytidine transferase gene CCT by using the sequence shown in the formula 3 as a primer;
(2) Inserting the phosphorylcholine cytidine transferase gene CCT into a homologous recombination region of a pBlue-zeo-18sUD overexpression vector, and constructing a CCT overexpression vector pBlue-zeo-18sUD-CCT with bleomycin as resistance;
(3) Linearizing the homologous recombination region of the overexpression vector pBlue-zeo-18sUD-CCT, electrically converting into the genome of S.limacinum SR21 for homologous recombination, and screening and identifying the resistance of bleomycin to obtain the genetically engineered bacterium SR21 over-expressing the CCT gene.
3. The use of the genetically engineered bacterium over-expressing a phosphorylcholine cytidine transferase gene as defined in claim 1 in the production of fatty acids.
4. A method for enhancing the metabolism of schizochytrium limacinum phospholipids and improving the production of fatty acids, which is characterized in that the genetically engineered bacterium which overexpresses the phosphorylcholine cytidine transferase gene according to claim 1 is inoculated in a seed culture medium for activation to obtain a strain for fermentation; inoculating the fermentation strain into a fermentation culture medium for fermentation culture, and collecting thalli for identification and analysis of oil components.
5. The method according to claim 4, wherein the genetically engineered bacterium over-expressing the phosphorylcholine cytidine transferase gene is streaked on a solid seed culture medium and subjected to stationary culture at a temperature of 27-29 ℃; selecting a monoclonal on a flat plate to a liquid seed culture medium, and culturing at the temperature of 27-29 ℃ and the speed of 200-250 rpm to obtain first-stage seeds; inoculating the primary seeds into a new liquid seed culture medium according to the inoculum size of 4% -8%, and culturing at the temperature of 27-29 ℃ and the speed of 200-250 rpm to obtain secondary seeds; inoculating the secondary seeds into a fermentation culture medium in an inoculum size of 4% -8%, culturing for 120 hours at the temperature of 27-29 ℃ and the rpm of 200-250 rpm, and sampling every 24 hours in the culturing process for relevant analysis.
CN202311560651.1A 2023-11-21 2023-11-21 Genetically engineered bacterium for over-expressing phosphorylcholine cytidine transferase gene, construction method and application thereof Pending CN117801968A (en)

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