CN113930351A

CN113930351A - Application of GGH gene in increasing yield of tocopherol, recombinant yarrowia lipolytica for high yield of tocotrienol and application of recombinant yarrowia lipolytica

Info

Publication number: CN113930351A
Application number: CN202111291644.7A
Authority: CN
Inventors: 孟永宏; 苟元元; 郭建琦; 牛永洁; 杨璐
Original assignee: Shaanxi Healthful Biological Engineering Co ltd
Current assignee: Shaanxi Healthful Biological Engineering Co ltd
Priority date: 2021-11-03
Filing date: 2021-11-03
Publication date: 2022-01-14
Anticipated expiration: 2041-11-03
Also published as: CN113930351B

Abstract

The invention provides application of expression of geranylgeranyl diphosphate reductase (GGH) gene in improving the yield of tocopherol of recombinant yarrowia lipolytica. The invention also provides a recombinant yarrowia lipolytica for high yield of tocotrienols and a construction method thereof, wherein the recombinant yarrowia lipolytica expresses geranylgeranyl diphosphate reductase (GGH) genes. The invention constructs a tocopherol heterologous synthesis way in yarrowia lipolytica to obtain the recombinant yarrowia lipolytica with high tocopherol yield, wherein the tocopherol yield can reach 423.7 mu g/g DCW, the content of gamma-tocopherol is 238.5 mu g/g DCW, the content of alpha-tocopherol is 185.2 mu g/g DCW, and the method is obviously superior to the prior art.

Description

Application of GGH gene in increasing yield of tocopherol, recombinant yarrowia lipolytica for high yield of tocotrienol and application of recombinant yarrowia lipolytica

Technical Field

The invention belongs to the technical field of metabolic engineering and combinatorial biology, and particularly relates to a recombinant bacterium for improving the yield of tocopherol and a construction method and application thereof.

Technical Field

Tocopherols (tocopherols) belong to one of the vitamin e (vitamin e). It is composed of a phenolic cyclic head and a fatty acid tail with a saturated aliphatic side chain, and has very important effect on the health of human body. Tocopherols are divided into 4 types according to the position and number of methyl groups on the hydroquinone group: α, β, γ and δ, among which a-tocopherol is more easily absorbed by the body, and therefore, α -tocopherol is considered to be the most active substance. Tocopherol is an important antioxidant, has the effects of scavenging free radicals and stopping lipid chain oxidation reaction, has positive effects on cardiovascular diseases, cancers and the like, and also can improve immunity, resist aging and promote fertility.

At present, the extraction process of natural tocopherol is various, and mainly comprises a chemical treatment method, a biological extraction method and a method for separating and purifying high-concentration tocopherol by distillation and extraction. Since natural tocopherol is superior to synthetic chemical products in biological activity and safety, natural tocopherol is generally extracted from deodorized distillate of vegetable oil at home and abroad, and the foreign extraction processes mainly comprise esterification (ester exchange) -distillation, saponification-extraction, hydrolysis-distillation, esterification-supercritical extraction and the like, so that the technology is relatively mature and the extraction efficiency is high. The domestic production mainly adopts the esterification method of deodorized distillate of vegetable oil. The prior methods mainly have the problems of low yield, low purity, large waste, easy environmental pollution, expensive equipment, complex operation, poor quality and the like. The rapid development of biotechnology provides a new way for the production of natural tocopherol, the cloning of key enzyme genes for product synthesis is solved by a bioengineering technical means, and the content of the natural tocopherol in plants can be effectively improved by gene regulation and high-efficiency expression.

At present, the tocopherol biosynthesis pathway is already clear, and related important genes are successfully cloned. Researchers overexpress the tomato GGR gene in tomato and found that the alpha-tocopherol content in leaves was increased by 33.4% -38.6% (Liu H, Liu J, ZHao M, Chen J S. overexpression of ShCHL P in ligands Growth and secretion genes to salt, osmotic, and oxidative stress [ J ]. Plant Growth Regulation 2015,77(2): 211-); in addition, researchers have overexpressed barley HGGT in Arabidopsis thaliana, resulting in a large accumulation of gamma-tocotrienol, with a 10-15 fold increase in total tocopherol and trienol (CAHOON E B, HALL S E, RIPP K G, et al. Metabolic design of vitamin E biosynthesis in plants for tocotrienol production and secreted antioxidant content [ J ]. Nat Biotechnology, 2003,21(9): I082-7.); and overexpression of Arabidopsis MPBQMT and γ -TMT in soybean to increase the α -tocopherol ratio to 90% -95% (VAN EENENNAAM A L, LINCOLN K, DURRETT T P, et al. Research domestically led has constructed a tocotrienol heterologous synthesis pathway in saccharomyces cerevisiae, successfully expressed key genes in the tocotrienol synthesis pathway, but essential gene GGH gene in the tocopherol synthesis pathway cannot be expressed, thus unable to construct the tocopherol heterologous synthesis pathway. The patent constructs a tocopherol heterologous synthesis way in yarrowia lipolytica based on microbial fermentation, screens a GGH gene which can be successfully expressed by gene mining, and screens a recombinant strain with the highest tocopherol yield.

Disclosure of Invention

The invention aims to solve the technical problem of low yield of the existing method for producing tocopherol, and provides a recombinant bacterium capable of producing tocopherol, a construction method and application of the recombinant bacterium.

The idea of the invention is to construct a tocopherol heterologous synthetic approach in yarrowia lipolytica by screening a tocopherol synthetic gene which can be successfully expressed in the yarrowia lipolytica, and screening the recombinant yarrowia lipolytica with high tocopherol yield by gene mining.

The invention provides application of expression of geranylgeranyl diphosphate reductase (GGH) gene in improving the yield of tocopherol of recombinant yarrowia lipolytica.

Preferably, the geranylgeranyl diphosphate reductase (GGH) gene is selected from the group consisting of a HaGGH gene derived from sunflower (whose sequence is shown in SEQ ID No. 1), an AhGGH gene derived from peanut (whose sequence is shown in SEQ ID No. 2), a CbGGH gene derived from capsicum (whose sequence is shown in SEQ ID No. 3), and an OeGGH gene derived from olive (whose sequence is shown in SEQ ID No. 4).

The geranylgeranyl diphosphate reductase of the invention is selected from rape, sunflower, peanut, arabidopsis thaliana, barley, tomato, potato, pepper, papaya trefoil, dodder, pineapple, olea europaea, salvia miltiorrhiza and aquilaria sinensis, and is verified to be only GGH genes from sunflower (HaGGH, the sequence of which is shown in SEQ ID No. 1), peanut (AhGGH, the sequence of which is shown in SEQ ID No. 2), capsicum (CbGGH gene, the sequence of which is shown in SEQ ID No. 3) and olea europaea (OeGGH gene, the sequence of which is shown in SEQ ID No. 4) can realize effective expression.

Based on this, the present invention also provides a recombinant yarrowia lipolytica for high tocotrienol production, characterized in that the recombinant yarrowia lipolytica expresses a geranylgeranyl diphosphate reductase (GGH) gene.

Wherein the geranylgeranyl diphosphate reductase (GGH) gene is selected from the group consisting of a HaGGH gene derived from sunflower, an AhGGH gene derived from peanut, a CbGGH gene derived from capsicum, and an OeGGH gene derived from olive.

As a preferred embodiment, the recombinant yarrowia lipolytica further expresses 4-hydroxyphenylpyruvate dioxygenase (HPPD), Homogentisate Phytotransferase (HPT), 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT), Tocopherol Cyclase (TC) and gamma-tocopherol methyltransferase (gamma-TMT) genes. The sequences are shown as SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 respectively.

In the invention, the 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene is derived from blue algae, the Homogentisate Phytotransferase (HPT) gene is derived from blue algae, the 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT) gene is derived from Arabidopsis thaliana, the Tocopherol Cyclase (TC) gene is derived from blue algae, and the gamma-tocopherol methyltransferase (gamma-TMT) gene is derived from Arabidopsis thaliana.

The invention also provides a construction method of the recombinant yarrowia lipolytica, which comprises the following steps:

(1) construction of recombinant bacteria containing tocopherol biosynthesis pathway genes

Obtaining and synthesizing a 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene derived from blue-green algae, a Homogentisate Phytotransferase (HPT) gene derived from blue-green algae, a 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT) gene derived from arabidopsis thaliana, a Tocopherol Cyclase (TC) gene derived from blue-green algae and a gamma-tocopherol methyltransferase (gamma-TMT) gene derived from arabidopsis thaliana, connecting EGFP genes at the tail ends of the HPPD, HPPT, MPBQMT, TC and gamma-TMT sequences respectively, carrying out enzyme digestion and connection on the synthesized fragments and a vector ploxpura3loxp to obtain recombinant plasmids ploxpura3loxp-HPPD, ploxpura3loxp-HPT, ploxpura3 loxp-BQMT, ploxpura3loxp-TC and ploxpura3 loxp-gamma-TMT, carrying out linearization on the recombinant plasmids, transferring the recombinant plasmids into a ploxpura 3-plof-PLOx-HPPD-PLOx strain engineering yeast strain by a yeast engineering method for realizing linearization, and transforming the recombinant plasmids, The recombinant strain is verified to be correct by colony PCR (polymerase chain reaction) through Polf-ploxpura3loxp-HPT, Polf-ploxpura3loxp-MPBQMT, Polf-ploxpura3loxp-TC and Polf-ploxpura3 loxp-gamma _ TMT;

(2) construction of recombinant Strain containing geranylgeranyl diphosphate reductase Gene GGH

Obtaining and synthesizing geranylgeranyl diphosphate reductase gene sequences from sunflower (HaGGH), peanut (AhGGH), hot pepper (CbGGH) and olive (OeGGH), respectively connecting EGFP genes at the tail ends of the sequences, carrying out enzyme digestion and connection on a synthesized fragment and a vector ploxpura3loxP to obtain recombinant plasmids ploxpura3loxp-HaGGH, ploxpura3loxp-AhGGH, ploxpura3loxp-CbGGH and ploxpura3loxp-OeGGH, carrying out enzyme digestion linearization on the recombinant plasmids, transferring the recombinant plasmids into engineering bacteria Polf of yarrowia lipolytica by adopting a yeast transformation kit method to obtain recombinant strains Polf-ploxpura3loxp-HaGGH, Polf-ploxpura3loxp-AhGGH, Polf-ploxpura3loxp-CbGGH and Polf-ploxpura3loxp-OeGGH, and carrying out PCR (polymerase chain reaction) verification to obtain correct recombinant strains;

(3) construction of recombinant yarrowia lipolytica

After the recombinant plasmids ploxpura3loxp-HPPD, ploxpura3loxp-HPT, ploxpura3loxp-MPBQMT, ploxpura3loxp-TC and ploxpura3 loxp-gamma _ TMT obtained in the step (1) are subjected to enzyme digestion linearization, 5 recombinant plasmids are transferred into the recombinant strains Polf-ploxpura3loxp-HaGGH, Polf-ploxpura3loxp-AhGGH, Polf-ploxpura3loxp-CbGGH and Polf-ploxpura3loxp-OeGGH by adopting a yeast transformation kit method, and the recombinant yarrowia lipolytica is respectively obtained.

In the present invention, the resulting recombinant yarrowia lipolytica was cultured on SD-LEU medium, on which a single colony grown was a highly tocotrienol-producing recombinant yarrowia lipolytica, to remove ura3 marker.

Culturing the recombinant strain obtained by the invention in 50ml YPD liquid culture medium in a constant temperature shaking table at 220rpm and 30 ℃ for 96h, then centrifuging 5ml fermentation liquor, collecting thalli, grinding and breaking cells, extracting twice by using 2m1 acetone, centrifuging, taking supernatant, filtering, and carrying out HPLC analysis.

Acetone is used as an extraction solvent, tocopherol is extracted from fermentation liquor, the yield of the tocopherol is measured, and the expression of a HaGGH gene derived from sunflower, an AhGGH gene derived from peanut, a CbGGH gene derived from hot pepper and an OeGGH gene derived from olea europaea is confirmed to improve the yield of the tocopherol of the recombinant yarrowia lipolytica yeast, wherein the effect of a recombinant strain expressing the OeGGH gene derived from olea europaea is most remarkable.

According to the invention, a tocopherol synthetic gene capable of being successfully expressed in the yarrowia lipolytica yeast is screened, a tocopherol heterogenous synthetic approach is constructed in the yarrowia lipolytica yeast, and the recombinant yarrowia lipolytica yeast with high tocopherol yield is screened through gene mining. The recombinant bacterium Polf-ploxpura3 loxp-HPPD-HPT-MPBQMT-TC-gamma-TMT-OeGGH for expressing the OeGGH gene from the olive is adopted to carry out fermentation culture to synthesize the tocopherol, the yield of the tocopherol can reach 423.7 mu g/g DCW, wherein the content of the gamma-tocopherol is 238.5 mu g/g DCW, and the content of the alpha-tocopherol is 185.2 mu g/g DCW, which is obviously superior to the prior art. The invention constructs a way for heterologously expressing geranylgeranyl diphosphate reductase gene GGH in yarrowia lipolytica and synthesizing tocopherol.

Drawings

FIG. 1 is a tocopherol biosynthesis pathway;

FIG. 2 is a comparison of tocopherol yields of the recombinant bacteria.

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

The present invention relates to the following media:

LB culture medium: peptone 1%, sodium chloride 1%, yeast powder 0.5%, and agar powder 1.7% in solid culture medium, and sterilizing with high pressure steam at 121 deg.C for 20 min.

YPD medium: 2% of glucose, 1% of yeast powder, 2% of peptone and 1.7% of agar powder, and sterilizing the mixture for 20min by high-pressure steam at 121 ℃.

YPD liquid medium: 2% of glucose, 1% of yeast powder and 2% of peptone, and sterilizing for 20min by high-pressure steam at 121 ℃.

SD-LEU medium: glucose 2%, (NH4)₂SO₄0.5 percent of agar, 0.17 percent of YNB, 0.2 percent of Drop-out mix synthetic minus leucoil w/o yeast nitro gene base, 2.5 percent of agar powder is additionally added into the solid culture medium, and the mixture is sterilized by high-pressure steam at 121 ℃ for 20 min.

The present invention relates to the following gene sequences:

TABLE 1 tocopherol synthesis genes from cyanobacteria and Arabidopsis thaliana

TABLE 2 sources of geranylgeranyl diphosphate reductase genes

The following examples relate to recombinant strains whose genotypes are respectively:

the invention relates to the following primers:

TABLE 3 primer information

Example 1

1. Cloning and expression of genes in tocopherol biosynthesis pathway

The method comprises the steps of obtaining a tocopherol synthesis gene sequence (shown in table 1) from NCBI (national center for Biotechnology organization) derived from blue algae and Arabidopsis thaliana, carrying out optimized synthesis, connecting EGFP gene at the end of the optimized sequence, carrying out enzyme digestion and connection on a synthesized fragment and a vector ploxpura3loxp respectively to obtain recombinant plasmids ploxpura3loxp-HPPD, ploxpura3loxp-HPT, ploxpura3loxp-S6GGH, ploxpura3loxp-MPBQMT, ploxpura3loxp-TC and ploxpura3 loxp-gamma _ TMT.

The double enzyme digestion system is carried out according to the instruction of Takara restriction enzyme, DNA gel recovery processing is carried out on the system after enzyme digestion, and the instruction of Axygen kit is carried out. The cleaved fragments and plasmids were ligated by using T4 DNA ligase, and the ligation system (10. mu.l) is shown in Table 1 below. After 30min of connection at 22 ℃, 10 mul of the connection product is added into an escherichia coli competent solution, after 15min of ice placement, 90s of heat shock at 42 ℃, and is rapidly placed in an ice bath for 3min, 1ml of LB liquid culture medium is added, 45min of recovery is carried out under a shaking table at 37 ℃, then centrifugation is carried out, part of supernatant is discarded, a proper amount of supernatant is taken and coated on a corresponding resistant LB plate, a 37 ℃ incubator is placed for 15h, plasmids are extracted, and plasmid extraction is carried out according to the instructions of the Axygen corresponding kit.

TABLE 4 connection System

After the recombinant plasmid is subjected to enzyme digestion linearization, the recombinant plasmid is transferred into yarrowia lipolytica engineering bacteria polf by adopting a yeast transformation kit method to obtain recombinant strains 1, 2, 3, 4, 5 and 6, and a colony PCR (polymerase chain reaction) mode is adopted as a verification method.

The colony PCR process was as follows:

and (3) PCR reaction system:

PCR procedure:

whether the gene can be expressed in the host or not is preliminarily judged by qualitatively observing the fluorescence of the recombinant strain through a fluorescence microscope, and the method comprises the following steps:

(1) and (3) flaking, namely picking a proper amount of single bacterial colony from a YPD plate cultured for 3 days, fully and uniformly mixing in 200 flash distilled water, putting 10 flash points on a glass slide, and covering the glass slide with a cover glass.

(2) Microscopic examination, namely placing the prepared sheet on a fluorescence microscope for observation, finding out a clear yeast body visual field in a bright visual field, then turning on blue light exciting light, turning off an illuminating lamp, and observing whether fluorescence exists or not in a dark visual field.

The results showed that all of the recombinant strains 1, 2, 4, 5, 6 were fluorescent, while the recombinant strain 3 was not fluorescent, indicating that the hydroxyphenyl pyruvate dioxygenase, homogentisate phytotransferase and tocopherol cyclase genes derived from Cyanobacteria (cyanobacterium J007), and the 2-methyl-6-phytylbenzoquinone methyltransferase and gamma-tocopherol methyltransferase genes derived from arabidopsis thaliana were normally expressed in the host, whereas the geranylgeranyl diphosphate reductase gene (shown in SEQ ID No. 10) derived from Synechococcus sp.65ay640) could not be expressed in the host.

2. Geranylgeranyl diphosphate reductase gene GGH excavation

GGH gene sequences of rape (BnGGH), sunflower (HaGGH), peanut (AhGGH), arabidopsis thaliana (AtGGH), barley (HvGGH), tomato, potato, hot pepper, papaya trifoliate, dodder, pineapple, olive, salvia miltiorrhiza and water ivy are obtained from NCBI, optimized synthesis is carried out, and the EGFP gene is connected to the end of the optimized sequence. The synthesized fragment and the vector ploxpura3loxp are respectively subjected to enzyme digestion and connection to obtain recombinant plasmids ploxpura3loxp-BnGGH, ploxpura3loxp-HaGGH, ploxpura3loxp-AhGGH, … and ploxpura3 loxp-TsGGH. The enzyme digestion connection method is the same as the method for cloning and expressing the tocopherol biosynthesis pathway gene.

After the recombinant plasmid is subjected to enzyme digestion linearization, the recombinant plasmid is transferred into yarrowia lipolytica engineering bacteria polf by adopting a yeast transformation kit method to obtain recombinant strains 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, and the verification method adopts a colony PCR mode.

Whether the gene can be expressed in the host or not is preliminarily judged by observing the existence of fluorescence of the recombinant bacteria, and the method is the same as the method. The results showed that all recombinant strains 8, 9, 14, and 18 were fluorescent, while all other recombinant strains were non-fluorescent, indicating that only the sunflower-derived HaGGH gene, peanut-derived AhGGH gene, pepper-derived CbGGH gene, and olive-derived OeGGH gene were normally expressed in the host, while none of the other-derived GGH genes were expressed in the host.

3. Construction of recombinant strains

After the recombinant plasmids ploxpura3loxp-HPPD, ploxpura3loxp-HPT, ploxpura3loxp-MPBQMT, ploxpura3loxp-TC and ploxpura3 loxp-gamma _ TMT obtained in the previous step are subjected to enzyme cutting linearization, 5 recombinant plasmids are transferred into the recombinant strains Polf-ploxpura3loxp-HaGGH, Polf-ploxpura3loxp-AhGGH, Polf-ploxpura3loxp-CbGGH and Polf-ploxpura3loxp-OeGGH by a yeast transformation kit method, and recombinant bacteria 21, 22, 23 and 24 are respectively obtained.

Screening the recombinant strains capable of producing tocopherol by detecting the content of tocopherol in the recombinant strains 21, 22, 23 and 24, and further screening the recombinant strains capable of producing tocopherol at high yield.

The detection sample is prepared by placing 1m1SD fermentation liquid in 1.5ml centrifuge tube, centrifuging at 12000rpm for 5min, placing the supernatant in a new centrifuge tube, adding 40ml glacial acetic acid, and filtering with 0.22 μm water system pinhole filter head.

HPLC detection conditions comprise mobile phase of 0.01M KHzPOa solution (A) and methanol (B); the proportion of 90 percent of A to 10 percent of B; the flow rate is 0.8 ml/min; the detection wavelength is 290 nm; 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 system 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 shown in FIG. 2, the total tocopherol content of the recombinant strain 24(Polf-ploxpura3 loxp-HPPD-HPT-MPBQMT-TC-gamma-TMT-OeGGH) was found to be the highest and 423.7. mu.g/g DCW by comparing the tocopherol contents of the respective recombinant strains, wherein the gamma-tocopherol content reached 238.5. mu.g/g DCW and the alpha-tocopherol content reached 185.2. mu.g/g DCW; the recombinant strain 21 has the highest alpha-tocopherol content of 276.8 mug/g DCW, which is far higher than gamma-tocopherol content. It was preliminarily concluded that the olea derived GGH gene favors the synthesis of total tocopherol, while the sunflower derived GGH gene favors the synthesis of alpha-tocopherol.

Example 2:

the application of the recombinant bacterium in the embodiment 1 in the preparation of tocopherol comprises the following specific steps:

the recombinant strain obtained in example 1 was cultured in 50ml of YPD liquid medium at 220rpm for 96 hours at 30 ℃ in a constant temperature shaking table, 5ml of the fermentation broth was centrifuged to collect the cells, which were then ground and disrupted, extracted twice with 2m1 acetone, centrifuged to collect the supernatant, filtered and analyzed by HPLC.

As shown in Table 5 below, the recombinant strain 24 expressing the 4-hydroxyphenylpyruvate dioxygenase gene HPPD, homogentisate phytotransferase HPT and tocopherol cyclase gene TC derived from Cyanobacteria (Cyanobacter J007), and the 2-methyl-6-phytobenzoquinone methyltransferase MPBQMT and gamma-tocopherol methyltransferase gene gamma-TMT derived from Arabidopsis thaliana (Arabidopsis thaliana), and the GGH gene derived from Olea europaea, had the highest tocopherol content, amounting to 423.7. mu.g/g DCW, where the α -tocopherol content was 185.2. mu.g/g DCW and the gamma-tocopherol content was 238.5. mu.g/g DCW.

TABLE 5 content of various tocotrienols in fermentation broths of various recombinant strains

Sequence listing

<110> Shaanxi Haas Schff bioengineering GmbH

Application of <120> GGH gene in increasing yield of tocopherol, recombinant yarrowia lipolytica for high yield of tocotrienol and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1485

<212> DNA

<213> HaGGH gene derived from sunflower (Helianthus annuus)

<400> 1

atggagaccc ggagcttggt tgtttcgatg aacactaatt tctcttccta tgagctctct 60

cttcctgcat cgcctctcac tcgctcactt gctccgttcc gatcggctaa actagggtcc 120

cgctccattt ctagggtttc ggcgtcgatc tccactccga acagtgaacc ttcttcctcc 180

ggcaaggatg ctgctatctc tgtgaaaccc gtttacgtcc cgacgccgcc caatcgcgac 240

ctccggactc ctcacagtgg ataccatttc gatggaacag cacggaagtt cttcgaggga 300

tggtatttca gggtttcgat tccggagaag aaggagagtt tttgctttat gtattctgtg 360

gagaatccag cgtttcgtaa gagattgtca ccattggaag tggctctcta tggacccaga 420

ttcactggtg tcggtgccca gattcttgga gctaatgata aatacatatg ccaatacaca 480

caagagtctc ataacttctg gggagataga catgagctag ttttggggaa tactttcagt 540

gctgtgccag gcgctagatc tccaagcaag gaggttccac cagaggaatt caacagaaga 600

gtgtccgaag ggttccaagt taccccattt tggcatcaag gtcacatatg cgatgatggc 660

aggactgact acgcggaaac tgtgaaatct gctcggtggg agtatagtac ccgtcctgtt 720

tacggttggg gtgatgttgg ggccaaacaa aagtctactg caggctggcc tgcagctttt 780

cctgtatttg agcctcattg gcagatatgc atggcaggag gcctttcaac agggtggata 840

gaatggggtg gtgaaaggtt tgaatttcgg gatgcacctt cttattcaga aaagaattgg 900

ggtggaggct tccctagaaa atggttttgg gtccagtgta atgtctttga aggagcaagt 960

ggagaagttg ctttgaccgc tgctggcggg ttgaggcaat tgcctggatt gaccgagacc 1020

tatgaaaatg ctgcactggt ttgtgtacac tttgatggaa aattgtacga gtttgtccca 1080

tggaatggtg ttgttagatg ggacatgtct ccatggggtt attggtatat gactgcagag 1140

aacgaaaccc atatggtgga actagaggca agaacaaacg aagcgggtac acctctgcgt 1200

gcgcctacga cagaagctgg actagctacg gcttgcagag atagttgtta cggtgaattg 1260

aagttgcaga tatgggaacg gctatatgat ggaagtaaag gcaaggtaat actggagaca 1320

aagagctcga tggcagcagt ggagatagga ggaggaccgt ggtttgggac atggaaagga 1380

gatacgagca acacacctga gctactcaaa cggtctcttc aggtcccatt ggatcttgaa 1440

accgtctttg gttgggtccc tttcttcaag ccaccgggtc tgtaa 1485

<210> 2

<211> 1362

<212> DNA

<213> AhGGH gene derived from peanut (Arachis hypogaea)

<400> 2

atggcggcaa aaattccaac tttttcacca ttttcactga acacaaaaac caaaagctca 60

aaatccaaat tgcatgttaa caaactcacc atcatagcct ccaaatctac tcacccgtcc 120

atcgccggca ggaagctccg cgcggcggtg atcggtggcg gcccagccgg atcctccgcc 180

gcggaggccc tggcagctgg aggcgtcgag acatttctct tcgagcgcaa cccgccgtcc 240

gcggccaaac cctgcggcgg cgcaatccct ctctgcatgc tcgacgagtt ctccatccct 300

ctccacctgg tcgaccgcca cgtcacacgc atgcgcatct tctctccttc caacatcgcc 360

gtcgatttcg gcaaaaccct aaaatccaac gagttcatcg ccatgctccg ccgcgaggtc 420

ctcgactctt tcctccgttc acgcgccgca tccgccggtg ccaccgtcat ctccgccctc 480

gtcacggccg tcgacctacc tccatcgccg accgctccct acaccataca ctacaccgta 540

cagaacacct accggcggag gcttgccgtc gacgtcgtga tcggcgccga cggagcaaac 600

agccgcgtcg caaaatcaat cggcgccggg gactacacct gcgcaatcgc gttccaggag 660

aggatcagat tgccggacga gaaaatggcg cattatgaaa atctcgccga gatgtacgta 720

ggcaacgacg tatctcccga tttctacggt tgggtgtttc ccaaatgtga ccacgtggca 780

gtgggcactg gtactgtgcg ctcgaagcag ggtattaagt tgctccagag agggatcagg 840

gatagagtcc gtgacaagat caacggtgga aaattgatca aggtagaggc gcaccctatt 900

ccagagcacc cacgtccagt gagagtcaga ggacgcgtgg cactcgttgg tgacgcagca 960

ggctacgtca ccaagtgttc cggcgaagga atatacttcg ccgcgaaatc gggccgagtt 1020

tgcggaaacg ccgtggttaa ggcatcagag gggggtctta aaatgatcga cgaacatgat 1080

ctaagaaggg agtatctgaa aatttgggat ggggaatata ctagtatgtt ccggtttttg 1140

gatctgttgc agagggtttt ctacggtagt aacgctgcta aggaggcatt ggtggagctt 1200

tgtggggatg agtatgttca aagaatgact tttgagagtt acttgtataa gaagttagct 1260

aaagggagag ttttggatga tgctaagatg gttatgaaca ctattgggag tttggtgaag 1320

tgtaacattt tagggagaaa aatggaaggt ttgataatat ag 1362

<210> 3

<211> 1395

<212> DNA

<213> Capsicum-derived CbGGH gene (Capsicum baccatum)

<400> 3

atggcttcca ttgctatcaa aaccttcacc ggtctccgtc aatcctcgcc ggaaaacaac 60

accattactc tatctaaacc cctcctttcc ggccaacctc accgtaggtt acgtatcaat 120

gcttcaaaat ccagcccaag agtcaccggc cgcaacctcc gtgtcgcggt ggttggcggt 180

ggtcctgctg ggggcgccgc cgctgaaaca ctcgctaaag gcggaattga aacgttctta 240

atagagcgta aaatggacaa ctgcaaaccc tgcggtggtg ctattccgct ttgtatggtg 300

ggggaattcg atcttcctct tgatatcatc gacagaaaag ttaccaagat gaagatgatt 360

tctccatcca acgttgctgt agatatcgga cagacattaa agcctcacga gtatatcggt 420

atggtgcgcc gcgaagtact cgatgcttac ctccgtggcc gtgctgccga cgccggagct 480

gccgttctca atggcttgtt tctcaaaatg gacatgccga aagccgcaaa tgcaccttac 540

gttcttcact acacatcgta cgattccaaa actaacggcg ccggcgagaa acgtactctc 600

gaagttgacg ccgttatagg tgctgacggc gcaaattccc gtgtggcgaa atccataaac 660

gcaggtgact acgagtacgc cattgcattc caagaacgta tcaaaatttc ggacgataag 720

atgaagtatt acgagaattt agctgaaatg tacgtcggag atgacgtttc ccctgatttt 780

tacggttggg ttttccccaa atgtgaccat gttgctgttg gcaccggcac agttactcac 840

aaagctgaca ttaagaagtt ccagctagca acaaggctcc gagccgattc gaaaatcacc 900

ggtgggaaaa tcatccgtgt tgaagctcac ccaattcccg aacacccaag gccaaaaaga 960

ttgcaagaca gagttgcctt agtcggagat gcagcagggt acgtaaccaa atgctccggc 1020

gaaggtatct acttcgctgc gaagagcgga cgtatgtgtg cggaagcaat tgttgaagga 1080

tcagaaaatg ggaagagaat gattgatgag agtgatctga ggaagtactt ggagaaatgg 1140

gacaagacat attggccaac atacaaggtt cttgatatat tgcagaaggt attttacagg 1200

tcgaatccag cgagggaagc ttttgtagag atgtgcgcag atgagtatgt gcagaagatg 1260

acatttgata gctatttgta caagaaagtg gcaccaggga accccattga agacttgaag 1320

cttgctgtga atactattgg aagtttggtg agggctaatg cactaagaag ggaaatggac 1380

aagctcagtg tataa 1395

<210> 4

<211> 1389

<212> DNA

<213> Oliggh gene (Olea europaea) derived from Olea europaea

<400> 4

atggcctcaa ttgcccttaa atccttcttc ggactccgtc aaaccacatc ggagaacaaa 60

tccatagttc tatcaaaacc aaccacccaa acccaccgta aattccgtat aaatgcctca 120

aaatccagcc caagagtcac cggacggaac ctcagagttg cagtggtggg aggtggcccg 180

gctggtggcg ccgcagcgga gacactcgct aaaggtggta tagagacgat cctcatcgaa 240

cgtaagcttg acaactgcaa gccatgtggt ggtgctattc cactttgtat ggtgggggaa 300

tttgacttgc cactggatat catcgatcgg agagtcacga aaatgaagat gatttctcca 360

tctaatgtgg ctgttgacat tggccagacc cttaaacccc acgagtatat tgggatggtc 420

cgccgtgaag tcctcgacgc ctacctccgc gaccgagcag ccaccgccgg agctaccgta 480

attaacggtc tcttgttgaa aatggacctg cccaaatcca caggttcacc ctataagtta 540

cattacacgg attacaacgc caaaaccgga ggcgccggcg agaaaaagac aatggaggct 600

gatgccgtca tcggcgccga cggtgctaat tcccgagtgg caaagaacat aaacgcaggt 660

gaatacgagt acgccattgc atttcaagaa cgcatcaaaa tctcagatga caaaatgaag 720

tattacgaaa atctagctga aatgtacgta ggcgatgacg tttcacctga tttctacggc 780

tgggttttcc caaaatgtga tcatgtagct gtcggcacag gcacagtaac ccacaaaggc 840

gacatcaaga aactccaaat tgctacaaga ttgagagcca gggacaaaat tgaaggtgga 900

aaaatcataa gagtggaggc acatcctata ccagagcatc cccggccaag gcgtgtgctc 960

gaccgagtag cgctagtcgg agatgcagcc gggtacgtca caaaatgctc cggtgagggg 1020

atatattttg ctgccaagag tggaagaatg tgtgcagagg caatagttga agggtcagaa 1080

aatgggataa aaatggtgga tgagagtgat ttgaggaagt atttagagaa atgggacaaa 1140

acatattggc ccacatacaa agtactggat gttcttcaga aggtatttta caggtcaaat 1200

ccagcaaggg aggcatttgt agaaatgtgt gctgatgaat atgtgcaaaa aatgacattt 1260

gatagctatt tgtacaagaa agtggtgcct gggaatccac ttgacgactt gaaattggct 1320

gtgaatacaa ttgggagttt ggtgagggca aatgcactaa ggagggagat ggagaagcta 1380

agtgtatga 1389

<210> 5

<211> 1134

<212> DNA

<213> HPPD gene (cyanobacterium) derived from Cyanobacteria

<400> 5

atgcacatcg atcacgtcca tttctacatc aaagatgcac cgcgatcgcg cgattggttt 60

gtcaaagcga tgaattttca agatcgcgga tggtggcgtt cccgtcatac ccagaccggg 120

tgggtgagta gcggtccggt gaatctgttg ctgtcttcgc cgctaacccc cgagagtccg 180

tggtttgact ttgcccgatc gcatcctccc ggagtgatgg atctggcgtt tcgggtggac 240

gatctcgaag gggcgatcga tcgcgcgatc gcggcggggg cgcaagtcct cgaacctgcg 300

cgccaatctc gggacgatcg cggggggctc aaatgggcga gggttgcggg ttgggggtcg 360

ttgtctcata ctctggtcga acgtcgggga cggactgagg ggagatttcc gcaactgggg 420

gagttgaacg tcgatcgaga tcgggtcgcg tctgcgttcg cgaagccagt ccaaaggagt 480

caggagaatc gcgtcgagga tggggacgga gaagacccgt taaacctatt agggatcgat 540

catgtggtgt tgaacgtgaa atcgggggag ttggaggcgg cttgcgatta ttatcagaag 600

agttttgggt tcgagcgaca acagagtttt acgatccgaa cggggcgatc ggggttatat 660

tctcaggtat tggttcatcc cggaggtcag atcgaattac cgattaacga accgacctcg 720

gacaattcgc aaattcaaga gtttttagag tgcaatggcg gttcggggat tcaacatatc 780

gccttgagaa ccgatcgcct cgtcgagacg atcgcccgtt ggcgccgtcg gggagtctcg 840

tttatcgacg ttccggcgac ctactacgcg ggtttggcaa gacggtgtcc ggtcgagctg 900

tccgaggaga cctggggggc gatcgccgcg caagggatct tggtcgattg gcaggatgag 960

gtatcggatg ccttgttatt gcaaaccttt acccagccaa ttttcgaccg tccgaccttt 1020

tttttcgagg cgatcgagcg ctaccgaggg gcactcggct ttggggaagg gaattttcaa 1080

gccttattcg aggcgatcga acgggcgcag atcgagcgtt cgcgcccggg ataa 1134

<210> 6

<211> 945

<212> DNA

<213> Cyanobacteria-derived HPT gene (cyanobacterium)

<400> 6

atgaatggtt ctttattatc tttgaatgga aatttgtacg ccttttggaa gttttctcgt 60

ccgcatacaa tcatcggaac gacattaagt gtatggggat tgtacgcgat cgcgatcgcc 120

ctcaccggga gttcgatcgc ccctgaaaac ttattttatg ctagcttaac ttggctggcg 180

tgtttgtgtg gaaatatcta cattgtcggc ttaaatcaac tcgaagatat cgaaatcgat 240

cgcattaata agccccattt accgatcgcc tccggagaat ttacaatcaa tcgaggacgg 300

gcaattgttg ccatcactgg agtgagtgcg atcgcgatcg ccttgcttca aggtccttgg 360

ttattcgcta cggtcggaat tagcattgcc ctcggtacgg cgtattcttt accgccaatt 420

cgtttaaaac ggttcccgtt ttgggcatct ttctgtattt acaccgttcg cgggattatt 480

gttaatttag ggctgttttt acaccatcac tggctgctaa ctcatccgga aaacttaacc 540

ggagaagttt tcgatattcc tctcgcagtg tgggcgttaa ccctatttat tttaattttt 600

acatttgcga tcgccatttt caaagatatc cctgacattg aaggcgacaa acaatataat 660

atcaccacat ttacgattcg cctcggtgcc cccgccgtct tcaatcttgc ccgatggacg 720

ataacggtgg cttacttcgc cattttgtta ggcggtttct ttttatttat cagtgtcaat 780

cccatttttc taatcgcttc ccatcttttc gccctcggtt tactgtggtg gcgaacacaa 840

aaagtggatt taaaagagcg atcgcaaatt gccagttgct atcaatttat ttggaaactc 900

ttttttctgg aatatctgat ttttcccgcc gcctgttggt tttaa 945

<210> 7

<211> 1017

<212> DNA

<213> MPBQMT gene (Arabidopsis thaliana)

<400> 7

atggcctctt tgatgctcaa cggggccatt accttcccca aaggtttagg ttcccctggt 60

tccaatttgc atgccagatc gattcctcgg ccgaccttac tctcagttac ccgaacctcc 120

acacctagac tctcggtggc tactagatgc agcagcagca gcgtgtcgtc ttcccggcca 180

tcggcgcaac ctaggttcat tcagcacaag aaggaggctt actggttcta caggttctta 240

tccatcgtat acgaccatgt catcaatcct gggcattgga ccgaggatat gagagacgac 300

gctcttgagc cagcggatct cagccatccg gacatgcgag tggtcgatgt cggcggcgga 360

actggtttca ctactctggg catagtcaag acagtgaagg ccaagaatgt gaccattctg 420

gaccagtcgc cacatcagct ggccaaagca aagcaaaagg agccgttgaa agaatgcaag 480

atcgtcgagg gagatgctga ggatcttcct tttccaaccg attatgctga cagatacgtt 540

tctgctggaa gcattgagta ctggccggac ccgcagaggg gaataaggga agcgtacagg 600

gttctcaaga tcggtggcaa agcgtgtctc atcggccctg tctacccaac cttctggctc 660

tctcgcttct tttctgatgt ctggatgctc ttccccaagg aggaagagta cattgagtgg 720

ttcaagaatg ccggtttcaa ggacgttcag ctcaagagga ttggccccaa gtggtaccgt 780

ggtgttcgca ggcacggcct tatcatggga tgttctgtca ctggtgttaa acctgcctcc 840

ggtgactctc ctctccagct tggtccaaag gaagaggacg tagagaagcc tgtcaacaac 900

cccttctcct tcttgggacg cttcctcctg ggaactctag cagctgcctg gtttgtgtta 960

atccctatct acatgtggat caaggatcag atcgttccca aagaccaacc catctga 1017

<210> 8

<211> 1074

<212> DNA

<213> Cyanobacteria-derived TC gene (cyanobacterium)

<400> 8

atgtctgact ggaaaaaagt gcaaacaccc catagcggct atcactggga cgggagcgat 60

cgccgttttt ttgaaggttg gtattatcgg gtcacccttc cggagattaa ccagaccttc 120

gccttcatgt actccatcga agatcctggc ggagggtcgc cctacagtgg cggcggcgcc 180

caaatcctcg ggcccgacga cggctatctc tgtcggacct ttgccgatgt cggacaattt 240

tgggcgacgg cggatcgttt ggggttgggc cattggaaag cccacgacgg cagctttgca 300

ccgcaatggt tagaaccgga aatattcgat cgccacgtcc gcgaaggcta tcaagccacg 360

gcaacctggc accaaggtaa actcgaagat cccggaaccg gaaaatcttg tcgctggcaa 420

tatgcgatcg cgccaatata cggctggggc gatcgcgacg acgtccagcg atcgagtggc 480

ggattactct catttttgcc catttttgaa cctggatggc aaattttgat ggctcacggt 540

tgggccaccg ggtggatcga atggaatggt aagcgttaca cctttgaccg ggtccccgct 600

tacggcgaaa aaaattgggg ggggtcgttc ccggaaaaat ggttttggct caactgcaat 660

agcttcgacg gcgaaccgga tttagcctta accgcaggcg ggggaaggcg cggcgtcttg 720

tggtggatgg agtccgtcgc cttggtgggg attcattatc gcgatcgctt ttacgaattt 780

gcaccgtgga acgcccgcat ccattggcgg gtggaaccgt ggggaaaatg gatcgtgacc 840

gcgtgcaacg acgacctcga agttaaactg gtcggcacca cgacacgacc gggaaccccg 900

ctaagggcgc ccacacggga aggcttggtc tacgtctgtc gggatacgat ggcaggtcag 960

ttgcgcttgg aaatgcgatc gcgccttgac ggaaccatct tgctcgaagc gacgagttcc 1020

tcctgtggcg tagaagtcgg cggcggaccg tggacggaca cctggatcgg ctga 1074

<210> 9

<211> 1047

<212> DNA

<213> Gamma-TMT Gene derived from Arabidopsis thaliana (Arabidopsis thaliana)

<400> 9

atgaaagcaa ctctagcagc accctcttct ctcacaagcc tcccttatcg aaccaactct 60

tctttcggct caaagtcatc gcttctcttt cggtctccat cctcctcctc ctcagtctct 120

atgacgacaa cgcgtggaaa cgtggctgtg gcggctgctg ctacatccac tgaggcgcta 180

agaaaaggaa tagcggagtt ctacaatgaa acttcgggtt tgtgggaaga gatttgggga 240

gatcatatgc atcatggctt ttatgaccct gattcttctg ttcaactttc tgattctggt 300

cacaaggaag ctcagatccg tatgattgaa gagtctctcc gttttgccgg tgttactgat 360

gaagaggagg agaaaaagat aaagaaagta gtggatgttg ggtgtgggat tggaggaagc 420

tcaagatatc ttgcctctaa atttggagct gaatgcattg gcattactct cagccctgtt 480

caggccaaga gagccaatga tctcgcggct gctcaatcac tcgctcataa ggcttccttc 540

caagttgcgg atgcgttgga tcagccattc gaagatggaa aattcgatct agtgtggtcg 600

atggagagtg gtgagcatat gcctgacaag gccaagtttg taaaagagtt ggtacgtgtg 660

gcggctccag gaggtaggat aataatagtg acatggtgcc atagaaatct atctgcgggg 720

gaggaagctt tgcagccgtg ggagcaaaac atcttggaca aaatctgtaa gacgttctat 780

ctcccggctt ggtgctccac cgatgattat gtcaacttgc ttcaatccca ttctctccag 840

gatattaagt gtgcggattg gtcagagaac gtagctcctt tctggcctgc ggttatacgg 900

actgcattaa catggaaggg ccttgtgtct ctgcttcgta gtggtatgaa aagtattaaa 960

ggagcattga caatgccatt gatgattgaa ggttacaaga aaggtgtcat taagtttggt 1020

atcatcactt gccagaagcc actctaa 1047

<210> 10

<211> 1221

<212> DNA

<213> S6GGH gene (Synechococcus sp) derived from Synechococcus

<400> 10

ttgagcctga gagttgctgt tgttgggggt ggcccagccg gagcctctgc cgccgaagtt 60

ctagcccaag ccggaatcga gacctttttg tttgagcgga agctagacaa cgccaagccc 120

tgcgggggcg ctattcccct ctgtatggtc tcagagttcg acctccctga ggagattatc 180

gaccgcaaag tgcgcaagat gaagatgatc tccccttcca actacgaggt ggatatcgcc 240

ctcgcaaagg aaaatgagta tatcggcatg tgccgccgcg agatcctgga tgccttcttg 300

cgcaaccgcg ctcgccaaaa aggggcggag ctgatccacg gcaaggtgat gcatctggaa 360

atcccccaaa atggccggga tccctacatc ctgcactaca gcgatttttc cgacggctca 420

aatcggggag taccccgttc gctggcggtg gatctggtga tcggggcaga tggctttcac 480

tccaaggtgg cagaagccat cggtgccggc gactacaact atgctctggc ctttcaggag 540

cggattcggc tcccggacga caaaatggcc tactacgagg agcgggcaga gatgtacgtg 600

ggggacgatg tttctccgga cttctatgcc tgggtgttcc ccaagtgcga ccacgtggcg 660

gtgggcaccg gcaccatgaa ggccaaccaa gcccagatcc aaaagttgca ggccgggatc 720

cgcgcccgcg ctgccgagcg gattcggggc ggccaggtga tcaaggtgga agcccatccg 780

atcccagagc atccgcgtcc gcgccgtgtg gtgggtcggg cagccctggt gggagatgcg 840

gcaggctatg taaccaagtc cagcggcgag gggatctatt ttgccgccaa gtcgggccgc 900

atgtgcgccg agaccatcgt cgagacctcc gagggcggga agcgcatccc cagcgaagaa 960

gatctcaagc tgtacctcaa gcgctgggat cgccaatacg gcctaaccta caaggttttg 1020

gacattttgc aacgggtgtt ctaccgttcc aacgccaccc gcgaggcctt tgtggagatg 1080

tgcgctgacc gggatgtgca gcggatgacc tttgacagct acctctacaa gacggtggtg 1140

cccatgaacc cttgggtgca gttcaagatc acggttaaga ctctaggcag cctgctgcga 1200

gggaacgccc tggcacctta g 1221

Claims

1. Application of expression of geranylgeranyl diphosphate reductase (GGH) gene in improving yield of tocopherol of recombinant yarrowia lipolytica yeast.

2. Use according to claim 1, characterized in that the geranylgeranyl diphosphate reductase (GGH) gene is selected from the group consisting of the HaGGH gene derived from sunflower, the AhGGH gene derived from peanut, the CbGGH gene derived from capsicum, and the OeGGH gene derived from olea europaea.

3. A recombinant yarrowia lipolytica for high tocotrienol production, characterized in that said recombinant yarrowia lipolytica expresses a geranylgeranyl diphosphate reductase (GGH) gene.

4. The recombinant yarrowia lipolytica of claim 3, characterized in that said geranylgeranyl diphosphate reductase (GGH) gene is selected from the group consisting of the HaGGH gene derived from sunflower, the AhGGH gene derived from peanut, the CbGGH gene derived from capsicum, and the OeGGH gene derived from olive.

5. The recombinant yarrowia lipolytica of claim 3, characterized in that said recombinant yarrowia lipolytica further expresses 4-hydroxyphenylpyruvate dioxygenase (HPPD), Homogentisate Phytotransferase (HPT), 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT), Tocopherol Cyclase (TC) and γ -tocopherol methyltransferase (γ -TMT) genes.

6. The recombinant yarrowia lipolytica of claim 5, characterized in that said 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene is derived from cyanobacteria, Homogentisate Phytotransferase (HPT) gene is derived from cyanobacteria, 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT) gene is derived from Arabidopsis thaliana, Tocopherol Cyclase (TC) gene is derived from cyanobacteria, and γ -tocopherol methyltransferase (γ -TMT) gene is derived from Arabidopsis thaliana.

7. The method for constructing recombinant yarrowia lipolytica according to any one of claims 3-5, comprising the steps of:

Obtaining and synthesizing a 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene derived from blue-green algae, a Homogentisate Phytotransferase (HPT) gene derived from blue-green algae, a 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT) gene derived from arabidopsis thaliana, a Tocopherol Cyclase (TC) gene derived from blue-green algae and a gamma-tocopherol methyltransferase (gamma-TMT) gene derived from arabidopsis thaliana, connecting EGFP genes at the tail ends of the HPPD, HPPT, MPBQMT, TC and gamma-TMT sequences respectively, carrying out enzyme digestion and connection on the synthesized fragments and a vector ploxpura3loxp to obtain recombinant plasmids ploxpura3loxp-HPPD, ploxpura3loxp-HPT, ploxpura3 loxp-BQMT, ploxpura3loxp-TC and ploxpura3 loxp-gamma-TMT respectively, carrying out enzyme digestion and linearization on the recombinant plasmids, respectively transferring the recombinant plasmids into a poliomyelip-pPolf-HPPD strain by a yeast engineering method of a yeast transformed yeast to obtain the recombinant pPolopyf-3 lox-pF-TMT, The recombinant strain is verified to be correct by colony PCR (polymerase chain reaction) through Polf-ploxpura3loxp-HPT, Polf-ploxpura3loxp-MPBQMT, Polf-ploxpura3loxp-TC and Polf-ploxpura3 loxp-gamma _ TMT;

(3) construction of recombinant yarrowia lipolytica

8. The method of constructing a recombinant yarrowia lipolytica of claim, further comprising the step of (4) removing the ura3 marker:

the obtained recombinant yarrowia lipolytica is cultured on SD-LEU culture medium, and a single colony grown on the SD-LEU culture medium is the recombinant yarrowia lipolytica with high tocotrienol yield.

9. Use of the recombinant yarrowia lipolytica of any one of claims 3-5 for the production of tocotrienols.