CN114574373B - Recombinant schizochytrium for producing tocopherol, construction method and application thereof - Google Patents

Recombinant schizochytrium for producing tocopherol, construction method and application thereof Download PDF

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CN114574373B
CN114574373B CN202210320776.6A CN202210320776A CN114574373B CN 114574373 B CN114574373 B CN 114574373B CN 202210320776 A CN202210320776 A CN 202210320776A CN 114574373 B CN114574373 B CN 114574373B
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苟元元
杨璐
郭建琦
牛永洁
孟永宏
邢武
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Shaanxi Healthful Biological Engineering Co ltd
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Abstract

The invention provides a recombinant schizochytrium limacinum for producing tocopherol, a construction method and application thereof. The recombinant schizochytrium comprises a geranylgeranyl diphosphate reductase gene GGH derived from Arabidopsis thaliana, a homogentisate phytyl transferase gene HPT derived from Chlorella vulgaris, and a gamma-tocopherol methyltransferase gene gamma-TMT derived from Fusarium sp. The recombinant schizochytrium limacinum is adopted to carry out fermentation culture to synthesize the tocopherol, the yield of the tocopherol can reach 126.5 mu g/g DCW, wherein the content of gamma-tocopherol reaches 82.3 mu g/g DCW, the content of alpha-tocopherol reaches 44.2 mu g/g DCW, a tocopherol heterologous synthesis way is successfully constructed in the schizochytrium limacinum, and high yield is realized.

Description

Recombinant schizochytrium for producing tocopherol, construction method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant schizochytrium limacinum for producing tocopherol, a construction method of the recombinant strain, and application of the recombinant strain in producing the tocopherol.
Background
Tocopherols (tocopherols) belong to a class of Vitamin E (vitamine E) and are mainly derived from the oil of plant seeds, tocopherols and tocotrienols having the ability to resist oxidation of lipids in the daily diet, which is generally defined as "Vitamin E activity". The vitamin E has wide application field, can be added into cosmetics and skin care products, can also be used as a medical health care product for preventing various diseases, has irreplaceable significance in animal and plant life, and has huge demand on the vitamin E by human beings. However, some current methods for chemical synthesis and biological extraction have the problems of low yield, low purity, large waste, easy environmental pollution, expensive equipment, complex operation, poor quality and the like. Therefore, the expression of some key enzyme genes is regulated and controlled by a biotechnology means, and the method becomes a new way for synthesizing natural tocopherol.
Schizochytrium (Schizochytrium) belongs to the family of thraustochytriaceae, is a marine heterotrophic microorganism, is propagated in a fission mode, and a large amount of active substances beneficial to a human body, such as grease, pigments (carotenoid, lutein, astaxanthin and the like), cantharene and the like, can be accumulated in a single cell of the Schizochytrium, and a common precursor used for synthesizing the substances, namely acetyl coenzyme A, has a high content in the Schizochytrium, which is also a reason for the large amount accumulation of the active substances. One precursor for tocopherol synthesis is Phytyl Diphosphate (PDP), which is synthesized from geranylgeranyl diphosphate (GGPP) under the action of 4-hydroxyphenylpyruvate dioxygenase (HPPD), and geranylgeranyl diphosphate is also a precursor for carotenoid synthesis, so that the construction of a tocopherol synthesis pathway on the basis of a carotenoid synthesis pathway is attempted, and a new pathway for natural tocopherol synthesis is sought.
According to the method, a key enzyme gene in a tocopherol synthesis way is excavated, and a tocopherol heterologous synthesis way is constructed in the schizochytrium to obtain a schizochytrium recombinant strain capable of synthesizing tocopherol.
Disclosure of Invention
The invention aims to construct a tocopherol heterologous synthesis way in schizochytrium and excavate key genes in the tocopherol synthesis way, and provides a recombinant engineering strain for producing tocopherol, a construction method and application of the recombinant strain.
The idea of the invention is to obtain a recombinant strain of schizochytrium limacinum capable of synthesizing tocopherol by screening a tocopherol synthesis gene capable of being successfully expressed in the schizochytrium limacinum and constructing a tocopherol heterologous synthesis way in the schizochytrium limacinum.
Based on the above, the invention provides a recombinant schizochytrium, which contains geranylgeranyl diphosphate reductase gene derived from arabidopsis thaliana, homogentisate phytotransferase gene HPT derived from chlorella, and gamma-tocopherol methyltransferase gene gamma-TMT derived from fusarium.
In the invention, the nucleic acid sequence of the geranylgeranyl diphosphate reductase gene derived from arabidopsis thaliana is shown as SEQ ID No.1, the nucleic acid sequence of the gene HPT is shown as SEQ ID No.2, and the nucleic acid sequence of the gene gamma-TMT is shown as SEQ ID No. 3.
The invention also provides a construction method of the recombinant schizochytrium limacinum, which comprises the following steps:
(1) Construction of recombinant plasmid containing geranylgeranyl diphosphate reductase Gene, HPT Gene and Gamma-TMT Gene derived from Arabidopsis thaliana
Obtaining sequences of geranylgeranyl diphosphate reductase gene from arabidopsis thaliana, homogentisate phytotransferase gene HPT from chlorella and gamma-tocopherol methyltransferase gene gamma-TMT from fusarium, synthesizing and adding enzyme cutting sites, and connecting EGFP gene at the tail end of the sequences; the synthetic fragment and the plasmid pBluZeo-18S are respectively subjected to enzyme digestion and connection to construct recombinant plasmids pBluZeo-18S-AtGGPR, pBluZeo-GaHPT-18S and pBluZeo-Ft gamma-TMT-18S, the obtained recombinant plasmids are subjected to heat shock to transform escherichia coli competent cells, positive transformants are screened, and sequencing is carried out to verify correctness;
(2) Schizochytrium limacinum transformed by recombinant plasmid
After the above recombinant plasmid is subjected to enzyme digestion linearization treatment, taking 10 mul, adding 100 mul of schizochytrium limacinum ATCC20888 competent cells, uniformly mixing, transferring to a precooled electric rotating cup, standing on ice for 30min, then carrying out electric shock, immediately adding 1mL of precooled seed culture medium containing 1M sorbitol after the electric shock is finished, gently mixing uniformly, transferring to 4mL of precooled seed culture medium containing 1M sorbitol, and culturing at 28 ℃ and 200rpm for 2h; then uniformly coating 300 mu L of bacterial liquid on a solid plate containing 50 mu g/mL bleomycin, placing the solid plate in an incubator at 28 ℃ for culturing for 24h, and performing PCR verification and correct verification on the obtained single colony extracted genome;
(3) Construction of recombinant Schizochytrium ATCC-20889
And (3) carrying out enzyme digestion connection on the AtGGPR, gaHPT and Ft gamma-TMT fragments in the recombinant plasmids pBluZeo-AtGGPR-18S, pBluZeo-GaHPT-18S and pBluZeo-Ft gamma-TMT-18S which are successfully verified in the step (2), then connecting the fragments to the plasmid pBluZeo-18S to obtain the recombinant plasmid pBluZeo-AtGGPR-GaHPT-Ft gamma-TMT-18S, and after enzyme digestion linearization, transferring the fragments to schizochytrium by an electric shock method to obtain a recombinant strain ATCC-20889 (ATCC-pBl-AtGGPR-GaHPT-Ft gamma-TMT).
According to a preferred embodiment, the Schizochytrium sp of step (3) above, i.e. the starting strain of the invention, is Schizochytrium sp ATCC-20889.
The invention also provides application of the recombinant schizochytrium limacinum in producing tocopherol.
Preferably, the obtained recombinant Schizochytrium limacinum is cultured in a seed culture medium for 48-72h in a constant-temperature shaking table at 180-220rpm and 27-29 ℃, then is inoculated into a fermentation culture medium by 3-6% of inoculum size, and is cultured in the constant-temperature shaking table at 180-220rpm and 27-29 ℃ for 3-5d.
According to a preferred embodiment, the seed culture medium contains: 40g/L glucose, 2g/L yeast extract, 10g/L sodium glutamate and KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、CaCl2·2H2O 1g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L；
The fermentation medium contains: 40g/L glucose, 2g/L yeast extract, 10g/L sodium glutamate and KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、(NH4)2SO4 6g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L。
The geranylgeranyl diphosphate reductase is selected from rape, sunflower, peanut and arabidopsis thaliana, and verification confirms that only AtGGPR gene from arabidopsis thaliana can realize effective expression.
The homogentisate phytotransferase is selected from pyrrophytic all-green-filament-derived bacteria, chlamydomonas reinhardtii, chlorella and lemongrass, and verification proves that only GaHPT genes derived from the chlorella can realize effective expression.
The gamma-tocopherol methyltransferase is selected from Fusarium, acorus gramineus, anthrax and rape, and the effective expression of Ft gamma-TMT gene only from Fusarium is confirmed through verification.
According to the invention, a tocopherol synthetic gene capable of being successfully expressed in the schizochytrium is screened, a tocopherol heterogenous synthetic approach is constructed in the schizochytrium, and the recombinant schizochytrium with high tocopherol yield is screened through gene mining.
Although key genes such as 4-hydroxyphenylpyruvate dioxygenase (HPPD), 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT), tocopherol Cyclase (TC) gene, geranylgeranyl diphosphate reductase, homogentisate Phytotransferase (HPT) and gamma-tocopherol methyltransferase (gamma-TMT) are all related to high-yield tocopherol, experiments show that the expression levels of the three genes of geranylgeranyl diphosphate reductase, homogentisate Phytotransferase (HPT) and gamma-tocopherol methyltransferase (gamma-TMT) in the recombinant schizochytrium are very low, and cannot play a role in improving the tocopherol yield of the recombinant schizochytrium.
The schizochytrium limacinum recombinant strain ATCC 20889 (ATCC-pBl-AtGGPR-GaHPT-Ft gamma-TMT) is adopted to carry out fermentation culture to synthesize tocopherol, the yield of the tocopherol can reach 126.5 mu g/g DCW, wherein the content of the gamma-tocopherol reaches 82.3 mu g/g DCW, and the content of the alpha-tocopherol reaches 44.2 mu g/g DCW.
The invention successfully expresses the tocopherol synthetic gene in the schizochytrium through gene excavation, constructs a tocopherol heterologous synthetic approach in the schizochytrium and realizes higher yield.
[ description of the drawings ]
FIG. 1 is a pathway diagram of the tocopherol synthesis pathway;
FIG. 2 shows the real-time fluorescent quantitative PCR results of 6 key genes in the original strain.
[ detailed description ] A
The present invention will be described in further detail with reference to the drawings and examples, but the scope of the present invention is not limited to these examples.
In the present invention, "%" used for specifying the concentration is mass percent, ": "is a mass ratio.
The present invention relates to the following media:
the seed culture medium contains:40g/L glucose, 2g/L yeast extract, 10g/L sodium glutamate and KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、CaCl2·2H2O 1g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L；
The fermentation medium contains: 40g/L glucose, 2g/L yeast extract, 10g/L sodium glutamate and KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、(NH4)2SO4 6g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L。
Solid seed culture medium: adding 1.5-2% agar based on seed culture medium, sterilizing, and pouring into sterilized plate in ultra-clean bench.
And B, bleomycin seed screening culture medium: after the 1.5% solid seed culture medium is sterilized, when the temperature of the culture medium is reduced to 50 ℃, bleomycin (Z-cin) with the final concentration of 100 mu g/ml is added, the medium is immediately poured into a flat plate, and the medium is stored in a refrigerator at 4 ℃ in a dark place.
The present invention relates to the following gene sequences:
TABLE 1 sources of Key genes and reference genes in the tocopherol Synthesis pathway and primer information
Figure GDA0003857685550000051
Figure GDA0003857685550000061
TABLE 2 sources of geranylgeranyl diphosphate reductase genes
Figure GDA0003857685550000062
TABLE 3 homogentisate phytotransferase HPT Gene sources
Figure GDA0003857685550000063
TABLE 4 sources of gamma-tocopherol methyltransferase gamma-TMT genes
Figure GDA0003857685550000064
The invention relates to the following primers:
TABLE 5 primer information
Figure GDA0003857685550000071
Figure GDA0003857685550000081
Example 1 verification of Tocopherol Synthesis Key Gene qPCR
Firstly extracting original strain Schizochytrium ATCC20888 genome, simultaneously extracting RNA of peanut sample capable of synthesizing tocopherol, reverse transcribing to obtain cDNA as control sample, and then according to the formula
Figure GDA0003857685550000082
qPCR
Figure GDA0003857685550000083
Green Master Mix (Without ROX) kit instructions, primers were designed using Primer 5.0 software, and qRT-PCR analysis was performed using GAPDH as an internal reference gene (see Table 1 for sequences of gene primers). Real-time fluorescent quantitative PCR (qRT-PCR) was performed on a LineGene 9600Plus fluorescent quantitative PCR instrument (Boy, hangzhou), and the quantitative reverse transcription step was performed according to PrimeScript RT regent kit with gDNA Eraser instructions; fluorescent quantitative PCR procedure
Figure GDA0003857685550000084
qPCR
Figure GDA0003857685550000085
Green Master Mix(Without ROX) kit. Three biological replicates and three technical replicates were performed per sample. By using 2-ΔΔCtThe method of (3) calculates the relative quantitative qRT-PCR expression of the gene. The relative expression quantity of 6 key genes in the original strain ATCC20888 is verified, and whether the original strain contains the key genes for generating tocopherol is preliminarily determined.
The results are shown in fig. 2, the expression levels of 6 genes in the original strain are different, wherein 4-hydroxyphenylpyruvate dioxygenase (HPPD), 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT) and Tocopherol Cyclase (TC) genes are expressed and are equivalent, while the expression levels of geranylgeranyl diphosphate reductase, homogentisate Phytotransferase (HPT) and gamma-tocopherol methyltransferase (gamma-TMT) genes are very low, and it is preliminarily determined that the original strain ATCC20888 may not have the three genes, which is also the reason that the strain cannot synthesize tocopherol.
Example 2 mining of the Geranylgeranyl diphosphate reductase Gene, the homogentisate Phytase HPT Gene and the Gamma-tocopherol methyltransferase Gamma-TMT Gene
1. Construction of geranylgeranyl diphosphate reductase gene, HPT and gamma-TMT gene recombinant expression plasmid
Obtaining a geranylgeranyl diphosphate reductase gene sequence from NCBI (NCBI) derived from rape (BnGGPR), sunflower (HaGGPR), peanut (AhGGPR) and arabidopsis thaliana (AtGGPR), an HPT gene sequence from thermoluminescent whole green filament bacteria (CaHPT), chlamydomonas reinhardtii (CrHPT), chlorella (GaHPT) and lemon grass (LiHPT), and a gamma-TMT gene sequence from fusarium (Ft gamma-TMT), acorus gramineus (Cg gamma-TMT), anthracnose bacteria (Ca gamma-TMT) and rape (Bn gamma-TMT), carrying out biological synthesis, adding enzyme cutting sites, and connecting EGFP genes at the tail ends of the sequences. The synthesized fragment and plasmid pBluZeo-18S (plasmid source: purchased from Shanghai biological engineering (Shanghai) Co., ltd.) are respectively subjected to enzyme digestion and connection to obtain recombinant plasmids pBluZeo-BnGGPR-18S, pBluZeo-HaGGPR-18S, pBluZeo-AhGGPR-18S, pBluZeo-AtGGPR-18S; pBlueZeo-CaHPT-18S, pBlueZeo-CrHPT-18S, pBlueZeo-GaHPT-18S, pBlueZeo-LiHPT-18S; pBluZeo-Ft gamma-TMT-18S, pBluZeo-Cg gamma-TMT-18S, pBluZeo-Ca gamma-TMT-18S, pBluZeo-Bn gamma-TMT-18S. And (3) carrying out heat shock transformation on the obtained recombinant plasmid into an escherichia coli competent cell, culturing and screening positive transformants on an LB plate containing 100 mu g/ml ampicillin resistance, and extracting the plasmid for sequencing verification.
2. Preparation of schizochytrium competent cells
Selecting activated Schizochytrium sp.ATCC20888 Schizochytrium single colony on a plate to 50ml of seed culture medium, culturing at 28 ℃ and 200r/min for 24h; transferring the strain to 50ml of seed culture medium again according to the inoculation amount of 4%, culturing at 200r/min for 24h, taking 20ml of bacterial liquid, centrifuging at 4000rpm for 2min, removing supernatant, collecting the bacterial body, re-suspending the bacterial body by using 25ml of pretreatment agent (20mM PH6.5 phosphoric acid buffer solution containing 25ml of DTT), and shaking at 150rpm for 30min to loosen cell walls. Then respectively washing the thalli twice by using 20ml of precooled sterile water and 1M of precooled sterile sorbitol solution, centrifuging for 2min at 4 ℃ at 4000rpm, finally re-suspending the thalli by using 200 mul of 1M of precooled sterile sorbitol solution, and subpackaging in 1.5ml of sterile centrifuge tubes.
3. Transformation and fluorescence comparison of recombinant expression plasmids
After the above-identified correct recombinant plasmid enzyme digestion linearization treatment, 10 μ l of the competent cells of schizochytrium limacinum ATCC20888 was added, and after mixing, the mixture was transferred to a precooled electric rotor, and allowed to stand on ice for 30min, and then the electric rotor was wiped dry and placed in an electric rotating machine with a voltage set at 2kV and an electric shock time of one pulse. Immediately after the electric shock is finished, 1mL of pre-cooled seed culture medium containing 1M sorbitol is added, the mixture is gently mixed uniformly and transferred to 4mL of pre-cooled seed culture medium containing 1M sorbitol (filled in a 25mL conical flask), and the mixture is cultured for 2h at 28 ℃ and 200 rpm. 300 mu L of the bacterial liquid is evenly smeared on a solid plate containing 50 mu g/mL bleomycin and is placed in an incubator at 28 ℃ for 24h.
Inoculating a single colony growing on the plate to a seed culture medium, culturing at 28 ℃, and extracting a genome for PCR verification after culturing for 24 hours at 200 rpm. The results show that the resistance selection marker has been recombined to the genome of the schizochytrium, the homologous recombination of the exogenous gene is realized, and the recombinant strains 1 (ATCC-pBl-BnGGPR), 2 (ATCC-pBl-HaGGPR), 3 (ATCC-pBl-AhGGPR), 4 (ATCC-pBl-AtGGPR) are obtained; 5 (ATCC-pBl-CaHPT), 6 (ATCC-pBl-CrHPT), 7 (ATCC-pBl-GaHPT), 8 (ATCC-pBl-LiHPT), 9 (ATCC-pBl-Ft. Gamma. -TMT), 10 (ATCC-pBl-Cg. Gamma. -TMT), 11 (ATCC-pBl-Ca. Gamma. -TMT), 12 (ATCC-pBl-Bn. Gamma. -TMT).
Then qualitatively observing whether the recombinant strain has fluorescence through a fluorescence microscope to preliminarily judge whether the gene can be expressed in the host or not, wherein the method comprises the following steps:
(1) Tabletting: a proper amount of single colony was picked up from the plate cultured for 3 days, mixed well in 200ml of distilled water, spotted on a glass slide at 10. Mu.l, and covered with a cover glass.
(2) Microscopic examination: and (3) placing the prepared sheet on a fluorescence microscope for observation, firstly finding out a clear schizochytrium thallus visual field in a bright visual field, then turning on blue light exciting light, turning off an illuminating lamp, and observing the existence of fluorescence in a dark visual field.
The results showed that only recombinant strains 4 (ATCC-pBl-AtGGPR), 7 (ATCC-pBl-GaHPT) and 9 (ATCC-pBl-Ft. Gamma. -TMT) were fluorescent, and none of the other recombinant strains was fluorescent, indicating that only the geranylgeranyl diphosphate reductase gene derived from Arabidopsis thaliana (Arabidopsis thaliana), and the GaHPT gene derived from Chlorella vulgaris and the Ft. Gamma. -TMT gene derived from Fusarium were expressed in the host, while none of the GGPR gene, the HPT gene and the. Gamma. -TMT gene derived from other sources were expressed in the host.
3. Construction of recombinant Strain ATCC-20889
The 3 recombinant plasmids pBluZeo-AtGGPR-18S, pBluZeo-GaHPT-18S, pBluZeo-Ft gamma-TMT-18S in AtGGPR, gaHPT and Ft gamma-TMT fragments were connected by digestion, and then connected to pBluZeo-18S to obtain recombinant plasmid pBluZeo-AtGGPR-GaHPT-Ft gamma-TMT-18S, after digestion linearization in the same manner, the recombinant plasmid was transferred to Schizochytrium ATCC20888 by the above method to obtain recombinant strain ATCC-20889 (ATCC-pBl-AtGGPR-GaHPT-Ft gamma-TMT), and the recombinant strain obtained was cultured in fermentation medium at constant temperature of 220rpm and 28 ℃ for 96h, then 5ml of fermentation broth was centrifuged and the strain was collected, after grinding the cells, extracted twice with 2m1 acetone, the supernatant was filtered, and then subjected to HPLC analysis.
Whether the strain can produce the tocopherol or not is verified by detecting the tocopherol content in the recombinant strain ATCC-20889.
Preparation of a detection sample: taking 1m1SD fermentation liquid in a 1.5ml centrifuge tube, centrifuging at 12000rpm for 5min, taking supernatant in a new centrifuge tube, adding 40ml glacial acetic acid, and filtering with a 0.22 μm water system pinhole filter head.
HPLC detection conditions: the mobile phase is 0.01M KH2PO4Solution (a) and methanol (B); ratio 90% A/10% B; the flow rate is 0.8ml/min; the detection wavelength is 290nm; column YMC-Pack ODS-AQ (4.6X 250 mm).
The standard curve was prepared as follows:
the gamma-tocopherol standard curve is prepared by weighing 10mg of gamma-tocopherol by a ten-thousandth balance, dissolving the 10mg of gamma-tocopherol in 10ml of pure methanol solution, sequentially diluting the mother solution with the pure methanol solution in a gradient way to obtain gamma-tocopherol solutions with the concentrations of 250mg/L,100mg/L,50mg/L,20mg/L and 5mg/L, diluting the gamma-tocopherol solutions by the same method to obtain two batches of gamma-tocopherol solutions with the same concentration, filtering all samples by a 0.22 mu m organic pinhole filter head, and analyzing by HPLC.
The preparation method of the standard curve of the alpha-tocopherol is the same as that of the standard curve of the gamma-tocopherol.
As a result, as shown in Table 6, the tocopherol contents of the recombinant strain ATCC-20889 (ATCC-pBl-AtGGPR-GaHPT-Ft. Gamma. -TMT) were measured, and it was found that the total tocopherol content of the recombinant strain ATCC-20889 (ATCC-pBl-AtGGPR-GaHPT-Ft. Gamma. -TMT) reached 126.5. Mu.g/g DCW, in which the gamma-tocopherol content reached 82.3. Mu.g/g DCW and the alpha-tocopherol content reached 44.2. Mu.g/g DCW. Therefore, the initial construction of the heterologous synthesis way of the tocopherol in the schizochytrium is successful. The geranylgeranyl diphosphate reductase gene derived from Arabidopsis thaliana, the GaHPT gene derived from Chlorella vulgaris, and the Ft gamma-TMT gene derived from Fusarium are respectively selected.
TABLE 6 content of various tocopherols in the fermentation broth of various recombinant strains
Figure GDA0003857685550000121
Sequence listing
<110> Shaanxi Hai Sff bioengineering, inc
<120> recombinant schizochytrium for producing phenol, construction method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1404
<212> DNA
<213> Geranylgeranyl diphosphate reductase gene GGH (Arabidopsis thaliana) derived from Arabidopsis thaliana
<400> 1
atggcgacga cggttacact caaatccttc accggacttc gtcaatcatc aacggagcaa 60
acaaacttcg tctctcatgt accgtcatca ctttctctcc ctcaacgacg gacctctctc 120
cgagtaaccg cagccagagc cactcccaaa ctctccaacc gtaaactccg tgtcgccgtc 180
atcggtggtg gaccagcagg cggggcagct gcagagactc tagcacaagg aggaatcgag 240
acgattctca tcgagcgtaa gatggacaat tgcaagcctt gcggtggcgc gattcctctc 300
tgtatggtcg gagaattcaa cttgccgttg gatattattg atcggagagt gacgaagatg 360
aagatgattt cgccgtcgaa cattgctgtt gatattggtc gtacgcttaa ggagcatgag 420
tatataggta tggtgagaag agaagttctt gatgcttatc tgagagagag agctgagaag 480
agtggagcca ctgtgattaa cggtctcttc cttaagatgg atcatccgga gaattgggac 540
tcgccgtaca ctttgcatta cactgagtac gatggtaaaa ctggagctac agggacgaag 600
aaaacaatgg aggttgatgc tgtcattgga gctgatggag ctaactctag ggttgctaaa 660
tctattgatg ctggtgatta cgactacgca attgcatttc aggagaggat taggattcct 720
gatgagaaaa tgacttacta tgaggattta gctgagatgt atgttggaga tgatgtgtcg 780
ccggatttct atggttgggt gttccctaag tgcgaccatg tagctgttgg aacaggtact 840
gtgactcaca aaggtgacat caagaagttc cagctcgcga ccagaaacag agctaaggac 900
aagattcttg gagggaagat catccgtgtg gaggctcatc cgattcctga acatccgaga 960
ccacgtaggc tctcgaaacg tgtggctctt gtaggtgatg ctgcagggta tgtgactaaa 1020
tgctctggtg aagggatcta ctttgctgct aagagtggaa gaatgtgtgc tgaagccatt 1080
gtcgaaggtt cacagaatgg taagaagatg attgacgaag gggacttgag gaagtacttg 1140
gagaaatggg ataagacata cttgcctacc tacagggtac ttgatgtgtt gcagaaagtg 1200
ttttacagat caaatccggc tagagaagcg tttgtggaga tgtgtaatga tgagtatgtt 1260
cagaagatga cattcgatag ctatctgtac aagcgggttg cgccgggtag tcctttggag 1320
gatatcaagt tggctgtgaa caccattgga agtttggtta gggctaatgc tctaaggaga 1380
gagattgaga agcttagtgt ttaa 1404
<210> 2
<211> 927
<212> DNA
<213> homogentisate phytotransferase gene HPT (Gomphosphaeria aponina) derived from Chlorella vulgaris
<400> 2
atgagtcaaa gttccccttc tcgccccaaa aacttattgc aagaaccttt ttcctggctg 60
tacagtttct ggaaattttc ccgcccccat actattattg gtacaagtct cagtatcttt 120
gcattatatc tgataacttt tgacagtagg atcacaccag ccaattttgg acaactgatg 180
ggaagttggt tagcttgtct atgtggcaat atctatattg taggactaaa tcagttagaa 240
gatgtagcaa ttgatcaaat taataagccc catttgccca ttgctgcagg agaattttcc 300
ccacaacaag gaaaatggat tgtgggaatt acaggaattt tagcactatt aatcgcagga 360
gggcaaggca cttggttgtt agcaacagtg ggaataagtt tagttatagg tactgcttat 420
tctttgccac ctatgagatt aaaacgcttt cccttttggg ctgcaatttg tatctttact 480
gtgcggggag tagtggttaa tttgggcatc tttctccatt ttagccaaca gaattttatt 540
cctccagaag tgtgggcatt gacactattt attgtggtat ttaccgtggc gatcgccatt 600
tttaaagatg tgccagacat ggagggagat aagcagtatc aaattaccac ttttacccta 660
ttattaggca atcaagcagt ttttaacctc actctttcgg taattacttt ttgttattta 720
gccatgatat ttgcagggat tttttggcta ccaaaagtta actctgtatt tctggtaatt 780
gcccatttaa ttttgttagg attactttgg tggcggagtc gaagtgtaaa tttaggagca 840
aagcaagaaa tagccaactt ttatcaattc atttggaaac tctttttcct agaataccta 900
ctctttcccg ccgcttttct cctttaa 927
<210> 3
<211> 990
<212> DNA
<213> Fusarium-derived gamma-tocopherol methyltransferase gene gamma-TMT (Fusarium tjaetaba)
<400> 3
atggctgcca cagcagtgtc tgaccaaact accgacctga gcaagcaata tgacactcct 60
ctcggcctcg ctcactcaac tatgcaagtc ctcaaggaca gaatcaagct ccattacgat 120
cttgcaagtg actactattt gagtttgtgg ggcgaacata tccatcacgg gtactggcca 180
acaccagagt ctgaagccac gcagacaaag gaggaagcac aggccaatct catccagctc 240
cttctcgaca tttcaaagat tccatccaac agctcggttc ttgatgttgg ctgtggtatc 300
ggcggaacat cgcggtatct cgcttccaaa catggtagct ctgtgacggg catcaccatc 360
tcaagcaaac aggtcgagat cgcaaatcgc ctaaccaaag ctgctctcga agacacctct 420
tcgtctgatg tgtcagatga caatggcttc gccaaactcg gcgagggaaa agtcaaattc 480
ctcgagcttg acgccgagaa gatgggcgac ttcttcagcg accagcaggg caacttcgat 540
gcagtatgga taagtgaagc tcttagccat tttccaaaca aggcattgtt ctttgagaat 600
gtgatgaaag tactcaagcc tgggggcaag ttggtgttag cagattggtt cagggatgaa 660
gacattgacg aaacaacttt catcaatgat atcaagccta tcgaagatgg catgctactc 720
ccacccctct gtacccagca agggtacgtc gatctcgcga agaacgccgg acttacagtc 780
ttgtctgagc caaaggacat cagtcaacaa gtccgaaaaa catgggacat tacttggtca 840
cttgtgcaga acccatccct ttgggcattt gcgtttactc aaaaccggga cggtattgct 900
ttcttgcagt catttcgcgc aatgagacga ggttatgcca atggctcctt tcgctacgct 960
gtcatggcat tttataagga aatggcatga 990

Claims (4)

1. A recombinant schizochytrium is characterized in that the recombinant schizochytrium contains geranylgeranyl diphosphate reductase gene derived from arabidopsis thaliana, homogentisate phytotransferase gene HPT derived from chlorella, and gamma-tocopherol methyltransferase gene gamma-TMT derived from fusarium; the nucleic acid sequence of the geranylgeranyl diphosphate reductase gene derived from arabidopsis thaliana is shown as SEQ ID No.1, the nucleic acid sequence of the gene HPT is shown as SEQ ID No.2, and the nucleic acid sequence of the gene gamma-TMT is shown as SEQ ID No. 3.
2. The method of constructing a recombinant schizochytrium limacinum of claim 1, comprising the steps of:
(1) Construction of recombinant plasmid containing geranylgeranyl diphosphate reductase Gene, HPT Gene and Gamma-TMT Gene derived from Arabidopsis thaliana
Obtaining sequences of geranylgeranyl diphosphate reductase gene from arabidopsis thaliana, homogentisate phytotransferase gene HPT from chlorella and gamma-tocopherol methyltransferase gene gamma-TMT from fusarium, synthesizing and adding enzyme cutting sites, and connecting EGFP gene at the tail end of the sequences; the synthetic fragment and the plasmid pBluZeo-18S are respectively subjected to enzyme digestion and connection to construct recombinant plasmids pBluZeo-18S-AtGGPR, pBluZeo-GaHPT-18S and pBluZeo-Ft gamma-TMT-18S, the obtained recombinant plasmids are subjected to heat shock to transform escherichia coli competent cells, positive transformants are screened, and sequencing is carried out to verify correctness;
(2) Schizochytrium limacinum transformed by recombinant plasmid
After the enzyme digestion linearization treatment of the recombinant plasmid, taking 10 mul, adding 100 mul of schizochytrium limacinum ATCC20888 competent cells, uniformly mixing, transferring to a precooled electric rotating cup, standing on ice for 30min, then performing electric shock, immediately adding 1mL of precooled seed culture medium containing 1M sorbitol after the electric shock is finished, gently mixing uniformly, transferring to 4mL of precooled seed culture medium containing 1M sorbitol, and culturing at 28 ℃ and 200rpm for 2h; then uniformly coating 300 mu L of bacterial liquid on a solid plate containing 50 mu g/mL bleomycin, placing the solid plate in an incubator at 28 ℃ for culturing for 24h, and performing PCR verification and correct verification on the obtained single colony extracted genome;
(3) Construction of recombinant Schizochytrium ATCC-20889
And (3) carrying out enzyme digestion connection on the AtGGPR, gaHPT and Ft gamma _ TMT fragments in the recombinant plasmids pBluZeo-AtGGPR-18S, pBluZeo-GaHPT-18S and pBluZeo-Ft gamma-TMT-18S which are successfully verified in the step (2), then connecting the fragments to the plasmid pBluZeo-18S to obtain the recombinant plasmid pBluZeo-AtGGPR-GaHPT-Ft gamma _ TMT-18S, and after enzyme digestion linearization treatment, transferring the electric shock method into schizochytrium to obtain the recombinant strain ATCC-20889.
3. Use of the recombinant schizochytrium limacinum of claim 1 or 2 for the production of tocopherols.
4. The use of claim 3, wherein the obtained recombinant Schizochytrium limacinum is cultured in a seed culture medium in a constant temperature shaker at 180-220rpm and 27-29 ℃ for 48-72h, and then inoculated into a fermentation medium with 3-6% of inoculum size, and cultured in a constant temperature shaker at 180-220rpm and 27-29 ℃ for 3-5d;
the seed culture medium contains: 40g/L glucose, 2g/L yeast extract, 10g/L sodium glutamate and KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、CaCl2·2H2O 1g/L、 KCl 2g/L、MgSO4·7H2O 5g/L、FeCl30.1g/L；
The fermentation medium contains: glucose 40g/L, yeast2g/L of ointment, 10g/L of sodium glutamate and KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、(NH4)2SO4 6g/L、KCl 2g/L、MgSO4· 7H2O 5g/L、FeCl3 0.1g/L。
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