CN115975823A - Schizochytrium limacinum gene engineering strain with phospholipase D gene knockout function and construction method and application thereof - Google Patents
Schizochytrium limacinum gene engineering strain with phospholipase D gene knockout function and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a gene engineering strain of schizochytrium limacinum for knocking out phospholipase D gene, a construction method and application thereof. The invention adopts Schizochytrium limacinum SR21 as an original strain, constructs a knockout carrier in escherichia coli by means of genetic engineering, takes upstream and downstream sequences of a PLD gene as homologous arms, and uses bleomycin gene to replace a PLD gene and as a screening resistance gene to obtain a genetic engineering strain with high DHA yield, thereby providing a new idea for regulating and controlling the high DHA yield of the Schizochytrium by genetic engineering.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a schizochytrium genetic engineering strain for knocking out a phospholipase D gene, and a construction method and application thereof.
Background
Schizochytrium (Schizochytrium) is a representative strain for industrially producing PUFAs and DHA as a high-quality producer of polyunsaturated fatty acids (PUFAs) due to high growth speed, high fat content and high DHA proportion. Scientists at home and abroad have conducted extensive studies on the synthesis mechanism of PUFAs in Schizochytrium, and found that the synthesis pathways of PUFAs include the Fatty Acid Synthase (FAS) pathway and the polyketide synthase (PKS) pathway, wherein the synthesis of DHA in Schizochytrium is considered to be mainly related to the PKS pathway. Recent studies have found that the production of DHA in schizochytrium depends not only on the DHA synthesis process, but also on the assembled form of DHA migration, accumulation and storage after its synthesis. The storage form of DHA in Schizochytrium is mainly triglyceride, and is also in the form of phospholipid and sterol ester, and the process of mutual conversion of glyceride, phospholipid and sterol ester is also accompanied by the migration of fatty acid, and the process is closely related to phospholipid metabolism. Therefore, the optimization of the phospholipid metabolic process of the schizochytrium has important significance for the synthesis of DHA in the schizochytrium.
Phospholipase D specifically hydrolyzes phosphodiester bonds in phospholipids, mainly catalyzing two types of reactions: (1) hydrolysis reaction; and (2) transphosphatidylation reaction. Phospholipase D can regulate the partitioning of TAG and phospholipids by hydrolysis and also can convert between different types of phospholipids by transphosphatidylation. The regulation and control of the expression of phospholipase D can influence the phospholipid metabolism of the schizochytrium limacinum, and further can influence the synthesis of grease and DHA of the schizochytrium limacinum. However, no report that the regulation and control of phospholipase D in the schizochytrium influence the lipid synthesis and lipid storage form of the schizochytrium has been reported at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art to a certain extent, and provides a schizochytrium genetic engineering strain for knocking out a phospholipase D gene and a construction method and application thereof. The method takes Schizochytrium limacinum SR21 as an original strain, and regulates the storage form of Schizochytrium limacinum lipid by knocking out a PLD gene, so as to enhance the synthesis of DHA.
One of the technical schemes adopted by the invention for solving the technical problems is as follows:
a gene engineering strain of Schizochytrium limacinum for knocking out a phospholipase D (PLD) gene is constructed by taking Schizochytrium limacinum SR21 as an original strain, and the PLD gene in the genome of the gene engineering strain is knocked out, so that the PLD expression level in the gene engineering strain is reduced.
The second technical scheme adopted by the invention for solving the technical problems is as follows:
a construction method of a PLD gene-knocked-out Schizochytrium limacinum gene engineering strain comprises the following steps:
(1) Cloning an upstream and a downstream homologous arms of a PLD gene in a genome of a wild strain Schizochytrium limacinum SR21, inserting the upstream and the downstream homologous arms into a homologous recombination region of a pBlue-zeo plasmid, and constructing a PLD gene knockout vector pBlue-zeo-PLD taking bleomycin as resistance;
(2) After linearization is carried out on the homologous recombination region of the PLD gene knockout vector pBlue-zeo-PLD, electrotransformation is conducted into Schizochytrium limacinum SR21 competent cells, and the Schizochytrium genetic engineering strain with the phospholipase D gene PLD knocked out is obtained.
Further, in the step (1), the method for constructing the PLD gene knockout vector pBlue-zeo-PLD includes: according to the sequence information of the PLD gene of Schizochytrium limacinum SR21, primers shown in SEQ ID No. 3-SE 1 ID No.6 are designed, and the upstream homology arm of the PLD gene and the downstream homology arm of the PLD gene are obtained through PCR amplification; and sequentially carrying out enzyme digestion and connection on the obtained upstream homology arm of the PLD gene and the downstream homology arm of the PLD gene with the plasmid pBlue-zeo, and then transforming the upstream homology arm and the downstream homology arm of the PLD gene into an escherichia coli DH5 alpha competent cell to obtain the knockout vector pBlue-zeo-PLD.
Further, in the step (2), the construction method of the gene engineering strain of schizochytrium for knocking out the phospholipase D gene PLD comprises the following steps: extracting pBlue-zeo-PLD knockout vector plasmid, after double enzyme digestion linearization, electric shock transformation is conducted and introduced into schizochytrium receptive state for homologous recombination, positive transformants are obtained by bleomycin resistance plate screening, PCR verification is utilized, and schizochytrium genetic engineering strains with PLD gene knockout are finally obtained.
Wherein:
SEQ ID No.1: upstream homology arm sequence of PLD gene in SR21 genome
acgcacgtgatgaattccaagcctagctatgtgaacgttccaatctgggaagactgaccttacaaaccaaatctcacggtgaaatgaagatatattgcatcagaacggtttttatcttggctagaaataagatttgagcgctcggccaatctggaggctctgcgtttgggttgctaagttttcattactgttgtgaggaagggcggagaaagttaaagtggctcacttcaatggttttctcaactccttcatttcaactacaagaatgatgtttaaggcttgtaattgtgaattttgtttattcatctttgagattgagatcaagtatacttcgttctgcaagcctcaatcatggtattctaaaactctattctcaaggctcttacatattttatgagatataaatgcatgaaccctttctgcagaacaacatagggttgcggcagcgtatggtgccaacaaaatagacacaattgtgggagaactcaaggcaatgctcaacagcaactaatataaaataagagtatgtggccattcagacaggcaggggtatagcaacctttgaatgtgctgcgatggataatgagtggaagcagaaca
SEQ ID No.2: downstream homology arm sequence of PLD gene in SR21 genome
ttgcgtctcccatgctagcatagctttgcatgtattcatatctctgattccatatgtttacgatattgaaatcagagaacgttgttaatttcattcataaaaatcgagtatttctgcgaagtctgaagtagccagtgttttgtggtagttgatgtcgttcagcttcccgttactttgactcaacagcaaagagaatatatagtttattgtttgttcgttttcaaacaaaacacacacgcccgaaaaacattaaagaaacatgtagttctagtaacaaatttaaatgtcttacagagcctaatcttatgttatgtaaaggcccaagtatgttgagcccagtaaattgtaaatgaaatatgcctcgcataccacatgccctcttcagcgttttggccttagtcaactcttctattcaaggaagaaaatctaacaaagtaaatctatagctacacaacaaatatagaagttacgctcaatccaaataccttacatgttattcagttgtacacggagcgaatcacgttcttcgatgaactcgtaaggggttccatagtacatcggatcaaggttttcgtttccttcgtaagcctcggcagcagc
The third technical scheme adopted by the invention for solving the technical problems is as follows:
an application of the schizochytrium genetic engineering strain with the phospholipase D gene knockout function in DHA synthesis.
The fourth technical scheme adopted by the invention for solving the technical problems is as follows:
a method for producing DHA by using the constructed PLD gene-knocked-out Schizochytrium limacinum genetic engineering strain is characterized in that the PLD gene-knocked-out Schizochytrium limacinum genetic engineering strain is inoculated to a seed culture medium for activation to obtain a strain for fermentation; inoculating the fermentation strain into a fermentation culture medium for fermentation culture; collecting thalli to carry out grease component identification analysis to obtain DHA.
Further, the method for obtaining the strain for fermentation is as follows: firstly, inoculating a PLD gene-knocked-out Schizochytrium limacinum gene engineering strain on a solid seed culture medium, standing and culturing at the temperature of 27-29 ℃, and activating; then, selecting a single colony with plump shape, inoculating the single colony to a liquid seed culture medium, and culturing at the temperature of 27-29 ℃ and the rpm of 150-250 to obtain a first-class seed; finally, inoculating the primary seeds to a new liquid seed culture medium according to the inoculation amount of 2-10%, and culturing at the temperature of 27-29 ℃ and the speed of 150-250 rpm to obtain secondary seeds serving as the strains for fermentation; inoculating the second-level seeds to a fermentation culture medium by the inoculation amount of 2-10%, culturing at the temperature of 27-29 ℃ under the condition of 150-250 rpm, and sampling in the culture process to perform grease component identification analysis to obtain DHA.
Further, the formula of the solid seed culture medium is as follows: 29-31 g/L of glucose, 9-11 g/L of yeast powder, 20 multiplied by 48-52 mL of inorganic salt component A and 500 multiplied by CaCl 2 1.5-2.5 mL of agar and 15-20 g/L of agar, and the pH is adjusted to 6.4-6.6.
Further, the formula of the liquid seed culture medium is as follows: 29 to 31g/L of glucose, 9 to 11g/L of yeast powder, 20 times of inorganic salt component A, 48 to 52mL of inorganic salt component A, and 500 times of CaCl 2 1.5-2.5mL, and adjusting the pH value to 6.4-6.6.
Further, the formula of the fermentation medium is as follows: 88 to 92g/L of glucose, 4 to 6g/L of corn starch, 4 to 6g/L of tryptone, 48 to 52mL of 20 multiplied by inorganic salt component A and 500 multiplied by CaCl 2 1.5 to 2.5mL, and adjusting the pH value to 6.4 to 6.6.
Wherein the 20 x inorganic salt component a comprises: naSO 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 12-14 g/L, KCl 9-11 g/L and deionized water as a solvent.
Wherein, the 500 XCaCl 2 The method comprises the following steps: caCl 2 ·2H 2 O83-87 g/L or anhydrous CaCl 2 63-67 g/L, and deionized water as solvent.
The equipment, reagents, processes, parameters and the like related to the invention are conventional equipment, reagents, processes, parameters and the like except for special description, and no embodiment is needed.
All ranges recited herein include all point values within the range.
In the present invention, the "room temperature", i.e., the normal ambient temperature, may be 10 to 30 ℃.
Compared with the background technology, the technical scheme has the following advantages:
the invention takes Schizochytrium limacinum SR21 as a wild strain, constructs a PLD gene knockout vector pBlue-zeo-PLD in escherichia coli, takes sequences at two ends of a PLD gene as homologous arms and bleomycin as a screening marker, and introduces a functional fragment into a Schizochytrium genome through electric transformation for homologous recombination to obtain a Schizochytrium engineering strain with the PLD gene knocked out, thereby improving the yield of DHA and providing a new idea for regulating and controlling the high yield of DHA in Schizochytrium through genetic engineering.
Drawings
FIG. 1 is a schematic diagram of the knockout vector pBlue-zeo.
FIG. 2 is a schematic diagram of PLD gene knock-out vector pBlue-zeo-PLD.
FIG. 3 is agarose gel electrophoresis picture of genetic engineering strain identification. Wherein PC is a positive control, NC is a negative control, M is marker, and PT is a knockout strain.
FIG. 4 is a diagram showing the result analysis of the PLD gene RT-qPCR of the wild type strain and the genetically engineered strain of Schizochytrium limacinum SR 21.
FIG. 5 is a graph comparing the DHA production of Schizochytrium limacinum SR21 wild strain and genetically engineered strain.
Detailed Description
The invention is further illustrated by the following figures and examples.
The culture medium adopted in the embodiment of the invention is as follows:
solid seed culture medium: 29 to 31g/L of glucose, 9 to 11g/L of yeast powder, 20 multiplied by inorganic salt component A48 to 52mL,500 multiplied by CaCl 2 1.5-2.5 mL, 15-20 g/L agar, and adjusting the pH value of the culture medium to 6.5.
Liquid seed culture medium: 29 to 31g/L of glucose, 9 to 11g/L of yeast powder, 20 multiplied by inorganic salt component A48 to 52mL,500 multiplied by CaCl 2 1.5～2.5mL, medium pH was adjusted to 6.5.
Fermentation medium: 88 to 92g/L of glucose, 4 to 6g/L of tryptone, 4 to 6g/L of corn steep liquor powder, 20 times of inorganic salt component A48 to 52mL,500 times of CaCl 2 1.5 to 2.5mL, and the pH was adjusted to 6.5.
Wherein: 20 x inorganic salt component a: mgSO (MgSO) 4 38～42g/L，(NH4) 2 SO 4 19～21g/L，Na 2 SO 4 238～242g/L，KH 2 PO 4 19～21g/L，KCl 9～11g/L，K 2 SO 4 12-14 g/L, and the solvent is deionized water.
500×CaCl 2 ：CaCl 2 ·2H 2 O83-87 g/L or anhydrous CaCl 2 63-67 g/L, and the solvent is deionized water.
The whole period of fermentation culture is 144h, and sampling detection is carried out every 24h.
TABLE 1 summary of primer sequences used in the examples of the invention
Example 1 construction of PLD Gene knock-out vector pBlue-zeo-PLD
1. Amplification of upstream and downstream homology arms of PLD gene
Designing an upstream homologous arm amplification primer PLD-U (shown as SEQ ID No.3 and SEQ ID No. 4) of the PLD gene and a downstream homologous arm amplification primer PLD-D (shown as SEQ ID No.5 and SEQ ID No. 6) of the PLD gene according to sequence information of the PLD gene of the Schizochytrium, inserting enzyme cutting sites Xho I and HindIII before and after the upstream homologous arm sequence through primer design, inserting enzyme cutting sites BamH I and Xba I before and after the downstream homologous arm sequence, and then obtaining the upstream homologous arm of the PLD gene and the downstream homologous arm of the PLD gene through a PCR mode by using Primer Star high-fidelity polymerase and the primers by taking a genome of a wild strain as a template.
The PCR procedure was: 5min at 94 ℃ (1min at 94 ℃, 1min at 60 ℃, 1min at 72 ℃) multiplied by 35 cycles, 10min at 72 ℃ and forever at 4 ℃.
2. Construction of PLD Gene knockout vector pBlue-zeo-PLD
(1) The PCR-purified product fragments from the knock-out vector pBlue-zeo (FIG. 1) and upstream homology arms of the PLD gene were digested simultaneously with restriction enzymes Xho I and Hind III for 2 hours at 37 ℃. The double enzyme cutting system is as follows: mu.g DNA template, 5. Mu.L 10 XQuickCut Buffer, 1.5. Mu.L each of the two fast-cutting enzymes, and adding a proper amount of pre-cooled sterile water to make up the system to 50. Mu.L.
(2) The upstream homology arm fragment of the PLD gene and the vector pBlue-zeo fragment were ligated by T4 ligase, and reacted at 16 ℃ for 12 hours. A connection system: 10 XT 4 DNA Ligase Buffer 1μL，T 4 1 mu L of DNA Ligase, 1.5 mu L of vector fragment and 6.5 mu L of target gene fragment.
(3) The ligation product was transformed into E.coli DH 5. Alpha. Competent cells by the following method:
i. 100 mu L of Escherichia coli DH5 alpha competent cells were placed on ice to melt for 10min, 10. Mu.L of the pre-cooled ligation product was added, and the mixture was allowed to stand on ice for 30min.
The competent cells containing the ligation product were placed in a water bath at 42 ℃ for 45s and then immediately placed on ice for 2-3 min.
Adding 900 μ L LB medium into the competent cells containing the ligation product, and culturing at 37 ℃ and 200rpm for 1h by shaking.
And iv, taking 150 mu L of bacterial liquid from the cultured liquid culture medium, coating the bacterial liquid on an LB plate containing antibiotics, and culturing for 12-16 h at 37 ℃.
And (3) selecting positive transformants, extracting plasmids, after PCR verification connection is successful, repeating the operation on the pBlue-zeo vector inserted with the upstream homology arm of the PLD gene, inserting the downstream homology arm of the PLD gene, and finally obtaining the PLD gene knockout vector pBlue-zeo-PLD after PCR verification and sequencing verification are successful (figure 2).
Example 2 construction of Gene engineering Strain of Schizochytrium limacinum with PLD Gene knockout
Extracting pBlue-zeo-PLD knockout carrier plasmid, after double digestion linearization with restriction enzymes Apa I and Not I, electric shock transformation is conducted and introduced into schizochytrium receptive condition for homologous recombination, positive transformants are obtained by screening bleomycin resistant plates, PCR verification is carried out (figure 3), and finally the schizochytrium genetic engineering strain with the PLD gene knocked out is obtained. The specific process is as follows:
1. preparation of schizochytrium competent cells:
(1) And picking the activated schizochytrium single colony to 10mL of liquid seed culture medium, and culturing for 24h in a shaker at the temperature of 28 ℃ and the speed of 200 rpm.
(2) Transferring 2mL of the first-order seed solution to 50mL of liquid seed culture medium, and culturing for 6-10 h at 28 ℃ in a shaking table at 200rpm until the seed solution reaches OD 600 About 1.5 to about 3.
(3) 10mL of the cultured bacterial solution was transferred to a sterilized 50mL centrifuge tube, centrifuged at 4,500rpm for 2min at room temperature, and the supernatant was discarded.
(4) 20mL of pre-cooled sterile water was added to wash the cells, and the cells were centrifuged at 4 ℃ and 500rpm for 2min, and the supernatant was discarded.
(5) 25mL of DTT-PBS buffer was added to resuspend the cells, and the cells were cultured at 28 ℃ for 30min on a shaker at 200 rpm. Followed by centrifugation at 4,500rpm for 2min at room temperature and discarding the supernatant.
(6) 20mL of pre-cooled sterile water was added to resuspend the cells, and the cells were centrifuged at 4 ℃ and 500rpm for 2min, and the supernatant was discarded.
(7) Add 20mL of sterile pre-cooled sorbitol solution to resuspend the cells, centrifuge at 4 ℃ for 4,500rpm for 4min, and discard the supernatant. And this operation is repeated once.
(8) Adding a small amount of sterile precooled sorbitol solution into a centrifuge tube, blowing and beating the mixture evenly, then subpackaging the mixture into 1.5mL sterile centrifuge tubes according to the amount of 100 mu L per tube, and placing the sterile centrifuge tubes on ice for later use.
2. Electro-transformation of schizochytrium:
(1) Adding 10-20 mu L of functional gene segment (about 2-3 mu g) into schizochytrium infected cells, blowing, uniformly mixing, transferring into an electric shock cup, and carrying out ice bath for 30min.
(2) After the electric shock cup is wiped, the electric shock cup is arranged at the corresponding position of the electric rotating instrument. The electric shock program is as follows: 2kv,6ms, one pulse.
(3) 1mL of precooled liquid seed culture medium (containing 1M sorbitol) is taken to be put into an electric rotating cup, the electric rotating cup is blown and beaten evenly, then the electric rotating cup is transferred to a 1.5mL sterile centrifuge tube, and the mixture is cultured for 2 to 3 hours in a shaking table at the temperature of 28 ℃ and the rpm of 200.
(4) And after slight centrifugation, removing part of supernatant, taking 150 mu L of bacterial liquid rich in bacteria, coating the bacterial liquid on a solid seed culture medium containing antibiotics, and culturing in a constant temperature incubator at 28 ℃ until colonies with full shapes appear on a flat plate.
3. Identification of PLD gene-knocked-out Schizochytrium limacinum genetic engineering strain
(1) The plate colony is selected and inoculated into a liquid seed culture medium containing 30-50 mg/L bleomycin, and cultured for 24 hours at 28 ℃ and 200 rpm.
(2) And carrying out shake flask subculture for 5-7 times according to the operation, so as to ensure the stable inheritance of the knockout vector.
(3) Extracting stably inherited positive strain genome, designing primers (SEQ ID No.7 and SEQ ID No. 8) specifically combined with the bleomycin resistance gene for PCR verification, and identifying the genetic engineering strain by agarose gel electrophoresis as shown in figure 3, wherein the result shows that the PLD gene has been successfully knocked out.
(4) The successfully constructed schizochytrium genetic engineering strain with the PLD gene knocked out is preserved at-80 ℃.
Example 3 measurement of transcription level of PLD Gene in Positive transformant
Designing primers shown as SEQ ID No.9 to SEQ ID No.12 according to the PLD gene sequence and the internal reference Actin sequence, and then measuring the transcription level by RT-q-PCR, wherein the specific experimental steps are as follows:
1. sample pretreatment:
1mL of the mixed fermentation broth was put into a 1.5mL centrifuge tube and centrifuged at 10,000rpm for 2min at room temperature to collect the cells. The cells were washed with physiological saline, centrifuged in the same manner, and the procedure was repeated twice. After washing, quenching the mixture by liquid nitrogen, and storing the mixture in a refrigerator at the temperature of 80 ℃ below zero for later use.
2. RNA extraction
(1) And (3) placing the pretreated thalli in a mortar, grinding for 5-6 times by using liquid nitrogen, adding 600 mu L of Buffer RL solution when the thalli are powdered, repeatedly blowing and beating the powder into the mortar without obvious precipitation, and transferring the powder into a 1.5mL sterilized centrifuge tube.
(2) The lysate was centrifuged at 12,000rpm for 5min at 4 ℃ and the supernatant carefully pipetted into a fresh 1.5mL RNase Free Tube.
(3) The supernatant was transferred to a gDNA Eraser Spin Column,12,000rpm, which had been mounted in the Collection Tube and centrifuged for 1min.
(4) Abandoning gDNA Eraser Spin Column, adding 1/2 volume of anhydrous ethanol into the Collection Tube, blowing, beating and mixing uniformly, and immediately transferring the mixed solution into RNA Spin Column (containing the Collection Tube). (if the mixture is more than 600. Mu.L, add in portions.) centrifuge at 12,000rpm for 1min, and discard the filtrate.
(5) mu.L of Buffer RWA was added to the RNA Spin Column, centrifuged at 12,000rpm for 30s, and the filtrate was discarded.
(6) mu.L of Buffer RWB was added to the RNA Spin Column along the periphery of the tube wall, centrifuged at 12,000rpm for 30s, and the filtrate was discarded. The operation was repeated once.
(7) Centrifuge at 12000rpm for 2 minutes at idle.
(8) The RNA Spin Column was mounted on 1.5mL of RNase Free Collection Tube, and 50. Mu.L of RNase Free dH was added to the center of the membrane 2 O, standing at room temperature for 5min, and then centrifuging at 12,000rpm for 2min to elute RNA.
3. Reverse transcription of RNA
(1) The various reagents required for the reaction were added to the PCR tube according to Table 2.
(2) After mixing, incubation was carried out at 50 ℃ for 5 minutes.
(3) Heating at 85 deg.C for 5 seconds to
Uni RT/RI and gDNA Removal were inactivated.
TABLE 2 RNA reverse transcription System
4. Real-time fluorescent quantitative PCR
(1) The reagents were added as per Table 3. The reaction was carried out by a PCR machine, and the reaction procedure is shown in Table 4.
TABLE 3 RT-qPCR reaction System
TABLE 4 RT-qPCR reaction procedure
The results show that the PLD gene-knocked-out Schizochytrium limacinum gene engineering strain obtained by the method of the embodiment of the invention shows lower transcription levels in the middle and later fermentation periods than the PLD gene of the wild strain.
Example 4 determination of DHA content in Gene engineering Strain of Schizochytrium limacinum with PLD Gene knocked out
1. Cultivation of Schizochytrium limacinum
(1) Activating strains: and taking the schizochytrium limacinum seeds preserved at the temperature of minus 80 ℃ to perform streak culture on a solid seed culture medium, and culturing for 36h at the temperature of 28 ℃.
(2) First-stage seed: and selecting single strains with plump shapes on the cultured solid seed culture medium, placing the single strains in 50mL of seed culture medium, and culturing for 24h under the conditions of 28 ℃ and 200 rpm.
(3) Secondary seeds: from the primary seed medium, a certain amount of the seed culture solution was transferred to 50mL of the seed medium at an inoculum size of 4%, and cultured at 28 ℃ for 24 hours at 200 rpm.
(4) And (3) shaking flask fermentation: from the secondary seed medium, a certain amount of the seed culture broth was transferred to 50mL of the fermentation medium at an inoculum size of 4%, and cultured at 28 ℃ for 144 hours at 200 rpm.
2. Determination of the Total oil content
(1) Taking 5mL of fermentation liquid to a 50mL centrifuge tube, adding 5mL of concentrated hydrochloric acid to the centrifuge tube, adding a magnetic rotor, and heating the mixture in a magnetic stirrer at the constant temperature of 65 ℃ for 50min until the thalli are completely digested.
(2) And taking out the centrifuge tube, cooling to room temperature, adding 5mL of n-hexane for extraction, reversing, uniformly mixing, standing for 5min, centrifuging for 1min at 6,000rpm, taking the upper organic phase into a weighed 50mL centrifuge tube, and repeating the operation twice until the upper organic phase is colorless.
(3) And (3) blowing the n-hexane in the centrifugal tube by using nitrogen, and drying the n-hexane in an oven at 60 ℃ for 2 hours to completely volatilize the n-hexane.
(4) And taking out the centrifuge tube, cooling to room temperature, weighing, and subtracting the weight of the centrifuge tube from the obtained weight to obtain the total grease yield.
3. Determination of fatty acid content
(1) 5mL of 0.5M KOH-CH was added to a 50mL centrifuge tube containing the oil 3 And (4) putting the centrifugal tube into a constant-temperature water bath kettle at 65 ℃ to heat the OH solution for 10min until the grease is completely dissolved.
(2) Taking out the centrifuge tube, cooling to room temperature, adding 5mL of 30% boron trifluoride diethyl etherate into the centrifuge tube, and placing the centrifuge tube in a constant-temperature water bath kettle at 65 ℃ to heat for 30min.
(3) And taking out the centrifuge tube, cooling to room temperature, adding 5mL of n-hexane and 50 mu L of 40g/L of methyl eicosanoate, shaking, uniformly mixing, adding 1mL of saturated sodium chloride solution to prevent emulsification, and standing for 5min for 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:
the instrument comprises: agilentGC7890A gas chromatography; a chromatographic column: supelco-2560 (100 m × 0.25mm ID,0.20 μmfilm); sample introduction setting: the sample introduction amount is 1 mu L, the sample introduction temperature is 260 ℃, and the split ratio is 50; carrier gas: nitrogen gas, 20cm/s; detector temperature: 260 ℃; controlling the column temperature: the initial temperature is 140 ℃, and the temperature is maintained for 5min; then the temperature is raised to 260 ℃ at the speed of 3 ℃/min and maintained for 10min.
The DHA yield of the modified strain and the wild strain is shown in FIG. 5, and the result shows that the yield of DHA of the PLD gene-knocked schizochytrium genetic engineering strain is obviously improved compared with that of the wild strain, and is improved by 12.3% at 120h (P is less than 0.01), which is mainly attributed to the influence of the PLD knock-out on the DHA proportion (Table 5), and the DHA proportion is improved by 13.3% at 120h (P is less than 0.01). The result shows that the DHA yield of the schizochytrium can be obviously improved by knocking out the PLD gene.
TABLE 5 fatty acid composition of wild type and genetically engineered strains at late stage of fermentation
Note: the significant differences were compared by one-way anova, and were not significant when P > 0.05, significant when P <0.01 < 0.05, and very significant when P <0.01, and indicated by.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, and all equivalent variations and modifications made within the scope of the present invention and the content of the description should be included in the scope of the present invention.
Claims (10)
1. A gene engineering strain of schizochytrium for knocking out phospholipase D gene is characterized in that: the genetic engineering strain is constructed by selecting Schizochytrium limacinum SR21 as an original strain, and a phospholipase D gene in a genome of the genetic engineering strain is knocked out.
2. The method for constructing a gene engineering strain of Schizochytrium limacinum with a phospholipase D gene knockout function according to claim 1, comprising the following steps: the method comprises the following steps:
1) Designing primers shown as SEQ ID No. 3-SEQ ID No.6 by taking Schizochytrium limacinum SR21 genome as a template;
2) Cloning upstream and downstream sequences of a PLD gene from a Schizochytrium limacinum SR21 genome by using the primers in the step 1) as homology arms, inserting the upstream homology arm of the obtained PLD gene and the downstream homology arm of the PLD gene into a homologous recombination region of a pBlue-zeo knockout vector, and constructing the pBlue-zeo-PLD of the PLD gene knockout vector;
3) Linearizing the homologous recombination region of the constructed PLD gene knockout vector pBlue-zeo-PLD, and electrically transforming the linearized PLD gene into the genome of Schizochytrium limacinum SR21 for homologous recombination to obtain the PLD gene knockout Schizochytrium genetic engineering strain.
3. The use of the Schizochytrium limacinum gene engineering strain with the phospholipase D gene knockout function in the synthesis of DHA according to claim 1.
4. A method for producing DHA by using the Schizochytrium limacinum genetically engineered strain with the phospholipase D gene knockout function according to claim 1, wherein the method comprises the following steps: inoculating the schizochytrium genetic engineering strain with the phospholipase D gene knocked out to a seed culture medium for activation to obtain a strain for fermentation; inoculating the fermentation strain to a fermentation culture medium for culturing; collecting thallus to obtain DHA as oil component.
5. The method of claim 4, wherein: inoculating the PLD gene-knocked-out Schizochytrium limacinum gene engineering strain on a solid seed culture medium, standing and culturing at the temperature of 27-29 ℃, and activating; inoculating the activated seeds to a liquid seed culture medium, and culturing at the temperature of 27-29 ℃ and the rpm of 150-250 to obtain first-grade seeds; inoculating the primary seeds to a liquid seed culture medium according to the inoculation amount of 2-10%, and culturing at the temperature of 27-29 ℃ and the rpm of 150-250 to obtain secondary seeds; inoculating the secondary seed serving as the fermentation strain to a fermentation culture medium in an inoculation amount of 2-10%, and culturing at 27-29 ℃ under 150-250 rpm to obtain the DHA oil component.
6. The method of claim 5, wherein: the formula of the solid seed culture medium is as follows: 29 to 31g/L of glucose, 9 to 11g/L of yeast powder, 20 times of inorganic salt component A, 48 to 52mL of inorganic salt component A, and 500 times of CaCl 2 1.5-2.5 mL of agar and 15-20 g/L of agar, and the pH is adjusted to 6.4-6.6.
7. The method of claim 5, wherein: the formula of the liquid seed culture medium is as follows: 29-31 g/L of glucose, 9-11 g/L of yeast powder, 20 multiplied by 48-52 mL of inorganic salt component A and 500 multiplied by CaCl 2 1.5 to 2.5mL, and adjusting the pH value to 6.4 to 6.6.
8. The method of claim 4, wherein: the formula of the fermentation medium is as follows: 88 to 92g/L of glucose, 4 to 6g/L of corn steep liquor powder, 4 to 6g/L of tryptone, 48 to 52mL of 20 times of inorganic salt component A and 500 times of CaCl 2 1.5 to 2.5mL, and adjusting the pH value to 6.4 to 6.6.
9. The method according to any one of claims 6 to 8, wherein: wherein, the formula of the 20X inorganic salt component A is as follows: naSO 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。
10. The method according to any one of claims 6 to 8, wherein: the 500 XCaCl 2 The formula is as follows: caCl 2 ·2H 2 O83-87 g/L or anhydrous CaCl 2 63～67g/L。
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