CN113755356A

CN113755356A - A genetically engineered bacterium that secretes extracellular tocotrienol and its application

Info

Publication number: CN113755356A
Application number: CN202111213334.3A
Authority: CN
Inventors: 于洪巍; 叶丽丹; 焦学
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2021-12-07

Abstract

The invention discloses a genetically engineered bacterium that secretes tocotrienol extracellularly and its application. A genetically engineered bacterium that secretes tocotrienol extracellularly, comprising a chassis strain and an imported gene transferred into the chassis strain, the chassis strain is an engineering strain producing tocotrienol, and the imported gene includes a source of Phaffia rhodozyma The geranylgeranyl pyrophosphate synthase gene mutant CrtE03M and the mitochondrial NADH kinase encoding gene POS5. The yield of tocotrienol is increased by increasing the content of the tocotrienol precursor GGPP and the supply of the cofactor NADPH.

Description

Gene engineering bacterium for extracellularly secreting tocotrienol and application thereof

Technical Field

The invention relates to the field of genetic engineering and metabolic engineering, in particular to a genetic engineering bacterium for extracellularly secreting tocotrienol and application thereof.

Background

Tocotrienols (Tocotrienols) are important components of vitamin E, are excellent antioxidants, have multiple functions of cholesterol reduction, neuroprotection, radiation protection, cancer resistance and the like, and have great application potential in the industries of food, feed, pharmacy, cosmetics and the like. Due to the lack of a chemical synthesis way, the supply of tocotrienols at present depends on plant extraction, but the problems of farmland occupation, long growth period, easy influence of climate change and the like exist. In recent years, the biosynthesis of tocotrienols using engineered microbial cell factories has received much attention. In 2008, delta-tocotrienol was synthesized for the first time in E.coli with a yield of 15. mu.g/g of dry cell mass (DCW). In 2020, the biosynthesis of delta-tocotrienol is realized in Saccharomyces cerevisiae, and the yield is 4.1 mg/L. In the same year, through metabolic modification of saccharomyces cerevisiae (S.cerevisiae), a strain YS-M5 is constructed, and the yield of the tocotrienol in a shake flask reaches 7.6mg/g DCW. However, tocotrienols were intracellular products in these works and are not conducive to industrial production.

Tocotrienols are lipophilic compounds that, due to their hydrophobic nature, readily adhere to cell membranes and exert stress on yeast cells. In addition, the storage capacity of the cell to accommodate lipophilic products is limited by the space available in the cell membrane. This problem can be solved if the product can be secreted into the culture medium, while also greatly simplifying downstream processing. During the fermentation process of the monoterpene and the sesquiterpene, the secretion of the product can be obviously promoted by adding an organic phase for in-situ extraction, the toxic action of the product on cells is relieved, and the yield can be improved under most conditions. On the basis of in situ extraction, overexpression of a suitable transporter can further facilitate extracellular transport of the product. The Pleiotropic Drug Resistance (PDR) family of proteins are known for their detoxification function in prokaryotic and eukaryotic cells and have been shown to be involved in the extracellular transport of a variety of hydrophobic compounds, such as carotenoids.

However, to date, there have been no reports on tocotrienol transporters, either in photosynthetic organisms that naturally produce tocotrienols or in engineered microorganisms such as yeast. Therefore, how to realize and promote the secretory synthesis of tocotrienols is a problem to be solved by those skilled in the art.

Disclosure of Invention

The invention aims to provide a genetically engineered bacterium capable of extracellularly secreting and producing tocotrienol and application thereof, and the efficient microbial heterologous synthesis of the tocotrienol is realized by an extracellularly secreting production method.

In order to achieve the purpose, the invention provides a genetically engineered bacterium for extracellular secretion of tocotrienols, which comprises a chassis strain and an introduced gene transferred into the chassis strain, wherein the chassis strain is an engineered strain for producing tocotrienols, and the introduced gene comprises a geranylgeranyl pyrophosphate (GGPP) synthase gene mutant CrtE03M and a mitochondrial NADH kinase coding gene POS5 which are derived from Phaffia rhodozyma.

Preferably, the GeneBank number of the geranylgeranyl pyrophosphate synthase gene mutant CrtE03M is DQ016502.1, and the GeneBank number of the mitochondrial NADH kinase coding gene POS5 is NM-001184002.1.

Preferably, the Chassis strain is Saccharomyces cerevisiae YS-M5.

The gene introduced into the gene engineering bacteria for extracellular tocotrienol secretion provided by the invention also comprises a coding gene of at least one of the following proteins:

(1) multidrug-resistant transcription factors Pdr1p and Pdr3 p;

(2) multidrug resistance family transporters Pdr10p, Pdr11p and Yol075 cp.

Preferably, the coding gene of the multidrug resistance transcription factor Pdr1p is GeneBank number NM _001180878.1, the coding gene of the multidrug resistance transcription factor Pdr3p is GeneBank number NM _001178245.1, the coding gene of the multidrug resistance family transporter Pdr10p is GeneBank number NM _001183748.1, the coding gene of the multidrug resistance family transporter Pdr10p is GeneBank number NM _001179363.1, and the coding gene of the multidrug resistance family transporter Yol075cp is GeneBank number NM _ 001183329.2. The invention introduces multidirection resistance transcription factor coding genes Pdr1p and Pdr3p or transporter coding genes Pdr10p, Pdr11p and Yol075cp into the cell of an engineering strain for producing tocotrienol, so that the corresponding transcription regulation factor or transporter is expressed in the engineering strain for producing tocotrienol, participates in the extracellular secretion of tocotrienol, and constructs a genetic engineering bacterium capable of efficiently secreting tocotrienol.

Specifically, the construction method comprises the following steps:

(1) cloning a coding gene of a multidrug-resistant transcription factor Pdr1P with GeneBank number NM _001180878.1, a coding gene of a multidrug-resistant transcription factor Pdr3P with GeneBank number NM _001178245.1, a coding gene of a multidrug-resistant family transporter Pdr10P with GeneBank number NM _001183748.1, a coding gene of a multidrug-resistant family transporter Pdr11P with GeneBank number NM _001179363.1 or a coding gene of a multidrug-resistant family transporter Yol075cp with GeneBank number NM _001183329.2 into P of an integrated PUMRI-21-DPP1_GAL1At the later multiple cloning site, obtaining recombinant plasmids PUMRI-21-DPP1-PDR1 or PUMRI-21-DPP1-PDR3 or PUMRI-21-DPP1-PDR10 or PUMRI-21-DPP1-PDR11 or PUMRI-21-DPP1-YOL 075C;

(2) the recombinant plasmid PUMRI-21-DPP1-PDR1 or PUMRI-21-DPP1-PDR3 or PUMRI-21-DPP1-PDR10 or PUMRI-21-DPP1-PDR11 or PUMRI-21-DPP1-YOL075C is transformed into an engineering strain for producing tocotrienol, and a recombinant strain integrating the multidirection resistance family protein gene in a chromosome is obtained by screening, namely the genetic engineering strain for efficiently producing the tocotrienol is obtained.

The integrative plasmid PUMRI-21-DPP1 is obtained by cloning a coding gene of DPP1 to the SfiI site of the plasmid PUMRI-21, and can be specifically referred to as a patent with the application number of CN 201510001391.3.

The invention also provides application of the genetic engineering bacteria for efficiently secreting and producing tocotrienols in preparation of tocotrienols.

Specifically, the application comprises the following steps: after the genetic engineering bacteria for efficiently producing the tocotrienols are subjected to amplification culture, the genetic engineering bacteria are inoculated into an YPD liquid culture medium, are subjected to shaking culture, and are subjected to in-situ extraction by adopting different organic phase-to-tocotrienol-producing engineering strains; collecting thalli in the fermentation liquor, extracting tocotrienol after cell disruption, and collecting an organic phase to directly obtain the tocotrienol. For intracellular synthesis of tocotrienols, extraction was performed using an organic solvent.

The invention also provides a method for preparing tocotrienols, and particularly, during fermentation culture, an organic phase is added into a culture medium to serve as an extracting agent for in-situ extraction, after the fermentation culture, thalli in fermentation liquor are collected, cells are crushed and then extracted to obtain intracellular synthesized tocotrienols, and meanwhile, the organic phase is collected to directly obtain extracellular secreted tocotrienols.

Preferably, the organic phase used as extractant is dodecane, sunflower oil or olive oil.

Preferably, the organic phase used as the extractant is olive oil, the addition amount of the olive oil is 5-40% by volume, and the addition time is 0-72h after inoculation.

The invention has the advantages that:

1. increasing the yield of tocotrienols by increasing the GGPP content of the tocotrienol precursor and the supply of the cofactor NADPH;

2. extracellular secretion of tocotrienols is realized by an in-situ extraction method;

3. the extracellular secretion of tocotrienols is promoted by single or combined overexpression of encoding genes of multidirectional drug resistance transcription factors (Pdr1p, Pdr3p) and encoding genes of multidirectional drug resistance family transporters (Pdr10p, Pdr11p, Yol075cp) in an engineering strain for producing tocotrienols, the extracellular secretion production of tocotrienols is realized for the first time, and the application prospect is good.

Drawings

FIG. 1 is a map of yeast integrative plasmid PUMRI-21-DPP1-pGAL1-crtE03M-pGAL10-POS 5.

FIG. 2 is a graph of the intracellular and extracellular tocotrienol production of engineered strains when cultured in two phases with different organic solvents as in situ extractants.

FIG. 3 is a graph showing the effect of the amount and time of olive oil addition on the yield of intracellular and extracellular tocotrienols in the engineered strains.

FIG. 4 is a map of yeast integrative plasmid PUMRI-21-DPP1-pGAL1-PDR11-pGAL10-YOL 075C.

FIG. 5 is a graph showing the effect of overexpression of PDR transcription factor and transporter alone on tocotrienol secretion production.

FIG. 6 is a graph showing the effect of the combination of PDR transporters over expression on the secretory production of tocotrienol.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1

And extracting and detecting the tocotrienols in the fermentation liquor and the organic phase.

1. The specific method for extracting tocotrienols from saccharomyces cerevisiae comprises the following steps:

(1) taking 1mL of yeast fermentation liquor, centrifuging at 12000rpm for 2min, discarding the supernatant, then washing twice with 2mL of distilled water, centrifuging, discarding the supernatant, adding 200 mu L of distilled water, and resuspending the thalli;

(2) adding 500mL grinding beads (half of each zirconia bead with the particle size of 0.1mm and 0.5 mm) into a 2mL centrifuge tube, and transferring all the resuspended bacteria liquid into the 2mL centrifuge tube;

(3) placing the centrifuge tube filled with the beads and the thalli in a full-automatic sample rapid grinding instrument, and grinding for 5min at 65 HZ;

(4) after grinding, placing the centrifugal tube in a 4 ℃ centrifuge at 12000rpm, centrifuging for 10min, and removing a supernatant;

(5) adding 1mL of acetone into the centrifuge tube for extraction, fully and uniformly mixing, and placing for 10min by ultrasound;

(6) after ultrasonic treatment, centrifuging at 12000rpm for 2min, and sucking supernatant into a new 2mL centrifuge tube;

(7) adding 1mL of acetone into the original 2mL of centrifuge tube, fully and uniformly mixing, performing ultrasonic treatment for 10min, centrifuging at 12000rpm for 2min, and sucking the supernatant into the new centrifuge tube in the step (6);

(8) the mixed extracts were filtered through a 0.22 μm organic frit and subjected to HPLC.

2. The specific method for extracting the tocotrienol from the organic phase of the fermentation liquid is as follows:

centrifuging the fermentation liquid at 4000rpm for 5min, collecting the organic phase, diluting the organic phase with acetone by 10-30 times, filtering with 0.22 μm organic filter head, and detecting by HPLC.

HPLC detection conditions are as follows:

and detecting the tocotrienol and the content thereof in the saccharomyces cerevisiae by HPLC. The liquid phase analyzer was Shimadzu LC-20AT, and the column chromatography was C18-H column (4.6X 250mm, 5 μm, Agilent, ZORBAX, SB-C18, America). Gradient elution is adopted, mobile phases are pure water (A) and acetonitrile (B), and the gradient elution program is 0-10min, and is from 30% A/70% B to 10% A/90% B; 10-40min, from 10% A/90% B to 100% A/0% B; 40-80min, 0% A/100% B; 80-81min, from 0% A/100% B to 30% A/70% B. The flow rate was 0.8mL/min, the column temperature was 40 ℃ and the measurement wavelength was 292 nm.

Example 2

And (3) constructing a high-yield tocotrienol strain.

A mutant of a rhodophaffia rhodozyma derived geranylgeranyl pyrophosphate synthase gene (CrtE03M, GeneBank number is DQ016502.1) and a gene encoding mitochondrial NADH kinase (POS5, GeneBank number is NM-001184002.1) are cloned on a yeast integration type plasmid PUMRI-21-DPP1, and the integration type plasmid PUMRI-21-DPP1 is obtained by cloning a gene encoding DPP1 to a SfiI site of the plasmid PUMRI-21, which can be specifically referred to a patent with application number of CN 201510001391.3. The following primers were used:

CrtE03M-F-BamHI：CGGGATCCATGGATTACGCGAACATCCTC，

CrtE03M-R-SalI：ACGCGTCGACTCACAGAGGGATATCGGCTAG，

POS5-R-SpeI：CTAGACTAGTTTAATCATTATCAGTCTGTCTCTTG，

POS5-F-NotI：ATAAGAATGCGGCCGCATGTTTGTCAGGGTTAAATTG。

PCR was performed using a high fidelity enzyme (Prime STARTM HS DNA polymerase) using genomic DNA as a template. The reaction system (50. mu.L) was as follows:

the PCR procedure was as follows:

the gene CrtE03M is cloned into a plasmid PUMRI-21-DPP1 by double enzyme digestion BamH I and Sal I, and the gene POS5 is cloned into the plasmid by double enzyme digestion Spe I and Not I to obtain a constructed plasmid named PUMRI-21-DPP1-pGAL1-crtE03M-pGAL10-POS 5.

The constructed plasmids PUMRI-21-DPP1-pGAL1-crtE03M-pGAL10-POS5 (figure 1) are transferred into saccharomyces cerevisiae YS-M5 on the chassis by a method of LiAc/SS carrier DNA/PEG (High-efficiency conversion using the LiAc/SS carrier DNA/PEG method. Nature Protocols,2007) to construct a High-yield tocotrienol strain YVT 17. The strain was inoculated from a test tube into 50mL YPD medium at an initial OD600 of 0.05 and cultured for 96 hours at 30 ℃ on a shaker at a rotation speed of 220 rpm. After the fermentation is finished, 1mL of fermentation liquor is extracted and then is detected by using High Performance Liquid Chromatography (HPLC), and the yield of the tocotrienol is 19.24mg/g DCW.

The saccharomyces cerevisiae YS-M5 on the chassis is stored in a laboratory, and the construction method is as follows: obtaining a strain YS-11 BY integrating the respective coding gene sequences of a truncated HMG-CoA reductase (tHMG1), a Saccharomyces rhodozyma-derived geranylgeranyl pyrophosphate synthase (CrtE) and an Arabidopsis thaliana-derived codon-optimized p-hydroxyphenylpyruvate dioxygenase (HPPD) into the genome of Saccharomyces cerevisiae BY 4741; integrating coding gene sequences of codon-optimized truncated 2 methyl-6-phytylbenzoquinone methyltransferase (namely tMPBQMT for cutting 51 amino acids from the N end) from arabidopsis thaliana, codon-optimized truncated tocopherol cyclase (namely tTC for cutting 47 amino acids from the N end) from synechocystis sp.PCC6803 from synechocystis, and codon-optimized truncated gamma-tocopherol methyltransferase (namely tTMT for cutting 40 amino acids from the N end) from arabidopsis thaliana into a strain YS-11 to obtain a strain YS-15C; overexpresses coding genes of speed-limiting enzymes SyHPT, tTC and tTMT in the strain YS-15C to obtain a strain YS-245C; in the strain YS-245C, a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase mutant (Aro 4) free from feedback inhibition by tyrosine was introduced^K229L) And chorismate mutase mutant (Aro 7)^G141S) And transketolase (TKL1) and tyrosine-free feedback inhibitor derived from Zymomonas mobilisThe respective coding genes of the prepared prephenate dehydrogenase (TyrC) and the respective coding genes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (Aro3) and phenylpyruvate decarboxylase (Aro10) are knocked out to obtain a strain YS-M1; in the strain YS-M1, YPL062W gene and mutant (CrtE03M) encoding gene overexpressing geranylgeranyl pyrophosphate synthetase derived from Phaffia rhodozyma were deleted to construct a strain YS-M3; in the strain YS-M3, the strain YS-M4 was obtained by further overexpressing the gene tTC and the gene tTMT; in the strain YS-M4, the promoter P was overexpressed_IDIControlled tMPBQMT gene, promoter P_GAL1The SAM2 gene is controlled, and YPL062W gene and Ty4 gene are knocked out simultaneously to obtain a strain YS-M5. The specific construction method is referred to in the literature (Fermentative production of Vitamin E toxins in Saccharomyces cerevisiae under cooled-shock-raised temperature control. Nat Commun, 2020).

Example 3

Optimizing the in-situ extraction conditions of the tocotrienol.

The addition of 5% (v/v) dodecane or sunflower oil or olive oil to the medium confirmed that olive oil was an ideal tocotrienol extractant by analyzing the tocotrienol production and secretion ratio of the engineered strain (fig. 2). In order to further promote the secretion of tocotrienols, the addition amount and the addition time of olive oil were optimized, 5%, 10%, 20%, 30%, 40% (v/v) olive oil was added respectively 24h after inoculation or 5% (v/v) olive oil was added at different time periods as an in situ extractant, and a two-phase fermentation system of the strain YVT17 was established. After culturing for 96h, the fermentation liquor is collected by centrifugation, and the organic phase is directly diluted by 10-30 times by acetone and then is subjected to HPLC detection. The results showed that the extracellular secretion ratio of tocotrienols reached 56.12%, and the total tocotrienol production increased by 5.51% compared to monophasic culture (fig. 3). Therefore, the optimized in situ extraction conditions are: the addition amount of olive oil is 5%, and the addition time is 24h after inoculation.

Example 4

Construction of a tocotrienol-secreting synthetic strain.

1. The saccharomyces cerevisiae genome is used as a template, and the following primers are used:

PDR1-F-BamHI：CGGGATCCATGCGAGGCTTGACACC，

PDR1-R-SalI：ACGCGTCGACAACTTTTATCTATACAAACGTAT，

PDR3-F-BamHI：CGGGATCCATGAAAGTGAAGAAATCAACTAGATCAA，

PDR3-R-SalI：ACGCGTCGACTTGCGTTTTCATAAGAAGGGATATGAAG，

PDR5-F-XhoI：CCCTCGAGATGCCCGAGGCCAAGCTT，

PDR5-R-NheI：CTAGCTAGCCTATTATTTCTTGGAGAGTTTACCGTTCT，

PDR10-F-SalI：ACGCGTCGACATGTTGCAAGCGCCCTCAA，

PDR10-R-NheI：CTAGCTAGCAATTATTTCTTTAATTTTTTGCTTTTCTTTGGAAC，

PDR11-F-XhoI：CCGCTCGAGATGTCTCTTTCCAAATATTTTAATCCAATTC，

PDR11-R-SacII：TCCCCGCGGTTATACGCTTTGTTCGTTTGGATTATG，

PDR12-F-XhoI：CCGCTCGAGATGTCTTCGACTGACGAACATATTG，

PDR12-R-SacII：TCCCCGCGGTTATTTCTTCGTGATTTTATTTTCGTCAC，

SNQ2-F-BamHI：CGGGATCCATGAGCAATATCAAAAGCACGCA，

SNQ2-R-SalI：ACGCGTCGACTTACTGCTTCTTTTTCCTTATGTTTTTAAT，

YOR1-F-BamHI：CGGGATCCATGTCTATAGAGACCCTTTATGACG，

YOR1-R-SalI：ACGCGTCGACTTAACTTCTGTTCTCGAAATCATTTTCCA，

YOL075C-F-XhoI：CCGCTCGAGATGTCACAGCAGGAGAATGG，

YOL075C-R-SacII：TCCCCGCGGTCACCATTTTATCCACTCCAATTTTG，

AUS1-F-XhoI：CCGCTCGAGATGTCAATTTCAAAGTACTTCACTC，AUS1-R-SacII：TCCCCGCGGTTAGTTCTGTACAGGCTTCTTCC，

YOL075C-F-NotI：AAGGAAAAAAGCGGCCGCATGTCACAGCAGGAGAATGG，

YOL075C-R-SpeI：CTAGACTAGTTCACCATTTTATCCACTCCAATTTTG，

to clone genes encoding multidrug resistance transcription factors (Pdr1p (GeneBank No.: NM-001180878.1), Pdr3p (GeneBank No.: NM-001178245.1)) and genes encoding multidrug resistance family transporters (Pdr5p (GeneBank No.: NM-001183572.3), Pdr10p (GeneBank No.: NM-001183748.1), Pdr11p (GeneBank No.: NM-001179363.1), Pdr12p (GeneBank No.: NM-001183872.1), Aus1p (GeneBank No.: NM-001183329.2), Yol075cp (GeneBank No.: NM-001183430.1), Yor1p (GeneBank No.: NM-001181410.3), Snq2p (GeneBank No.: NM-001180319.1));

2. the target gene is cloned on an integrated PUMRI-21-DPP vector plasmid by utilizing a PCR technology and a double enzyme digestion method, and Pdr1, Pdr3, Pdr5, Pdr10, Pdr11, Pdr12, Aus1, 075, Yor1, 2 are expressed under a GAL promoter to obtain recombinant plasmids PUMRI-21-DPP-PDR, PUMRI-21-DPP-PDR, PUMRI-21-DPP-PDR, PUMRI-21-DPP-PDR, PUMRI-21-DPP-PDR, PUMRI-21-DPP-PDR, PUMRI-21-DPP-YOL 075, PUMRI-21-DPP-AUS, PUMRI-21-DPP-YOR and PUMRI-21-DPP-SNQ connected with the target gene.

3. The above-constructed plasmids were integrated into the High-yielding tocotrienol strain YVT17 constructed in example 2 by the method of LiAc/SS carrier DNA/PEG (High-efficiency layer transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols,2007), and coated on an amino acid-deficient SD plate; the strains YVT17: PDR1, YVT17: PDR3, YVT17: PDR5, YVT17: PDR10, YVT17: PDR11, YVT17: PDR12, YVT17: YOL075C, YVT17: AUS1, YVT17: YOR1, YVT17: SNQ2 with correct integration are obtained by PCR verification.

4. The correct transporter overexpression strain constructed in step 3 was cultured in two phases under the in situ extraction conditions optimized in example 3.HPLC analysis shows that YVT17: PDR11 and YVT17: YOL075C shows better tocotrienol transport efficiency (figure 5), and the secretion ratio of tocotrienol is 65.58% and 65.7%, respectively.

Example 5

The combined expression transporter further promotes the secretion of tocotrienols.

1. Transporter proteins Pdr11p and Yol075cp with high extracellular transport efficiency of tocotrienol are respectively placed under promoters GAL1 and GAL10 to construct an integration plasmid PUMRI-21-DPP1-PDR11-YOL075C (figure 4); the well-constructed plasmids are integrated into the high-yield tocotrienol strain YVT17 constructed in the example 2 by using a LiAc/SS carrier DNA/PEG method and coated on an amino acid-deficient SD plate; the correct integrated strain YVT17, PDR11-YOL075C, was verified by PCR.

2. The correct transporter strain constructed in step 1 was cultured in two phases under the in situ extraction conditions optimized in example 3.HPLC analysis shows that YVT17: PDR11-YOL075C shows higher tocotrienol transport efficiency (FIG. 6) than YVT17: PDR11 and YVT17: YOL075C, and the secretion rate of tocotrienol reaches 73.66%.

Sequence listing

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Claims

1. a genetically engineered bacterium of extracellular secretion of tocotrienol, comprising chassis bacterial strain and the import gene transferred into chassis bacterial strain, it is characterized in that, described chassis bacterial strain is the engineering bacterial strain that produces tocotrienol, and described import gene Including the geranylgeranyl pyrophosphate synthase gene mutant CrtE03M and the mitochondrial NADH kinase encoding gene POS5 derived from Phaffia rhodozyma.

2. the genetically engineered bacteria of extracellular secretion of tocotrienols as claimed in claim 1, is characterized in that, the GeneBank number of described geranylgeranyl pyrophosphate synthase gene mutant CrtE03M is DQ016502.1, mitochondrial The GeneBank number of the NADH kinase-encoding gene POS5 is NM_001184002.1.

3. The genetically engineered bacteria for extracellular secretion of tocotrienols as claimed in claim 1, wherein the chassis strain is Saccharomyces cerevisiae YS-M5.

4. the genetically engineered bacteria of extracellular secretion of tocotrienol as claimed in claim 1, is characterized in that, described import gene also comprises the coding gene of following at least one protein:

(1) The pleiotropic drug resistance transcription factors Pdr1p and Pdr3p;

(2) Multidirectional drug resistance family transporters Pdr10p, Pdr11p and Yol075cp;

Among them, the GeneBank number of the gene encoding the pleiotropic drug resistance transcription factor Pdr1p is NM_001180878.1, the GeneBank number of the gene encoding the pleiotropic drug resistance transcription factor Pdr3p is NM_001178245.1, and the encoding gene of the pleiotropic drug resistance family transporter Pdr10p The GeneBank number of the gene is NM_001183748.1, the GeneBank number of the gene encoding the pluripotent drug resistance family transporter Pdr10p is NM_001179363.1, and the GeneBank number of the gene encoding the pluripotent drug resistance family transporter Yol075cp is NM_001183329.2.

5. The application of the genetically engineered bacteria that secretes extracellular tocotrienol as claimed in any one of claims 1 to 4 in the preparation of tocotrienol.

6 . A method for preparing tocotrienol, characterized in that, the genetically engineered bacteria that secretes extracellular tocotrienol according to any one of claims 1 to 4 are fermented and cultured, and the tocotrienol is obtained by extraction.

7. The method for preparing tocotrienol as claimed in claim 6, characterized in that, during fermentation and cultivation, an organic phase is added in the culture medium as an extractant for in-situ extraction, and after fermentation and cultivation, bacteria in the fermentation broth are collected. After the cells are broken, the intracellular tocotrienol is obtained by extraction, and the organic phase is collected at the same time to directly obtain the extracellular tocotrienol.

8. The method for preparing tocotrienol as claimed in claim 7, wherein the organic phase as the extraction agent is dodecane, sunflower oil or olive oil.

9. the method for preparing tocotrienol as claimed in claim 8 is characterized in that, the organic phase as extraction agent is olive oil, and the addition of olive oil is 5%～40% by volume, and the addition time is 0-72h after inoculation.

10. The method for preparing tocotrienol as claimed in claim 9, wherein the addition amount of olive oil is 5% by volume, and the addition time is 24h after inoculation.