CN110423732A - A kind of enzyme expressed in saccharomyces cerevisiae and high yield α-and γ-tocotrienols genetic engineering bacterium and its construction method - Google Patents

Abstract

The invention discloses a kind of key enzyme expressed in saccharomyces cerevisiae and high yield α-and γ-tocotrienols genetic engineering bacterium and its construction method.The present invention by gene cloning and codon optimization, obtain five can in saccharomyces cerevisiae successful expression enzyme, respectively HPPD, HPT, MPBQMT, TC and γ-TMT；And this five enzymes are gradually integrated into saccharomyces cerevisiae genome, further pass through excision chloroplast transit peptides and overexpression rate-limiting enzyme, building obtains high yield α-and γ -- the gene engineering microzyme strain of tocotrienols, name are as follows: YS-356c, deposit number are as follows: CCTCC NO:M2019572.Its total tocotrienols yield reaches 2.09mg/g dry cell weight.Engineered strain of the invention fermented can be cultivated, and α-and γ-tocotrienols are directly obtained from thallus, have good market prospects and open application value.

Description

An enzyme expressed in Saccharomyces cerevisiae and a gene for high α- and γ-tocotrienol production Engineering bacteria and its construction method

technical field

The invention relates to the fields of genetic engineering and microorganisms, in particular to an enzyme expressed in Saccharomyces cerevisiae and a genetically engineered bacterium with high production of α- and γ-tocotrienol and a construction method thereof.

Background technique

Vitamin E is an important fat-soluble compound, including tocopherol and tocotrienol, which is an essential vitamin for maintaining normal metabolism and function of the body. Natural vitamin E only exists in photosynthetic organisms. Humans and animals cannot synthesize it by themselves and can only ingest it from the outside world. Vitamin E products on the market are mainly α-tocopherol, but recent studies have shown that compared to tocopherol, tocotrienol has the effect of lowering cholesterol (tocotrienol is unique), and better anti-oxidation, anti-cancer , anti-inflammatory, cardioprotective and neuroprotective functions. However, the sources of tocotrienols are scarce, and the commercialized natural tocotrienols are mainly extracted from oil palm fruit. Due to their low content, the separation is difficult and the yield is limited, resulting in high production costs. However, chemically synthesized tocotrienols cannot be commercially produced due to complex steps and many by-products. Therefore, the heterologous synthesis of tocotrienols by microorganisms is an efficient and promising method.

In 2008, researchers from the University of Stuttgart in Germany realized the synthesis of δ-tocotrienol in Escherichia coli by introducing genes from Arabidopsis and cyanobacteria, but the yield was very low, only 15 μg/g dry weight of cells, and δ- Tocotrienols are unmethylated forms of tocotrienols and their applications are limited. So far, the heterologous biosynthesis of the more powerful and widely applicable α- or γ-tocotrienols has not been achieved. The invention provides a method for heterologously synthesizing α- and γ-tocotrienols with safe and efficient Saccharomyces cerevisiae as a cell factory, provides a new idea and method for industrial production of tocotrienols, and solves the problem of low output and production problems. high cost problem.

Contents of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide a key enzyme that can be expressed in Saccharomyces cerevisiae and a genetically engineered bacterium that can produce α- and γ-tocotrienol and its construction method, and use plant or blue-green algae vitamin The key enzymes in the synthesis pathway of E are templates. Through gene cloning and codon optimization, five enzymes that can be successfully expressed in S. - Genetically engineered yeast strains of tocotrienols, and further construct a high-yielding strain by excising chloroplast transit peptides and overexpressing rate-limiting enzymes, which can produce α- and γ-tocopherols safely, efficiently, with high yields, and at low cost Trienols.

In order to achieve the above object, the present invention adopts the following technical solutions:

An enzyme that can be expressed in Saccharomyces cerevisiae, including: 4-hydroxyphenylpyruvate dioxygenase (HPPD); homogentisate phytotransferase (HPT); 2-methyl-6-phytylbenzoquinone Methyltransferase (MPBQMT); Tocopherol Cyclase (TC); γ-Tocopheryl Methyltransferase (γ-TMT).

The aforementioned enzyme expressed in Saccharomyces cerevisiae, after codon optimization, the obtained sequence:

The nucleotide sequence of 4-hydroxyphenylpyruvate dioxygenase (HPPD) is shown in SEQ ID NO: 1;

The nucleotide sequence of homogentisate phytotransferase (HPT) is shown in SEQ ID NO: 2;

The nucleotide sequence of 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT) is shown in SEQ ID NO: 3;

The nucleotide sequence of tocopherol cyclase (TC) is shown in SEQ ID NO: 4;

The nucleotide sequence of γ-tocopheryl methyltransferase (γ-TMT) is shown in SEQ ID NO:5.

A genetically engineered bacterium for high-yielding α- and γ-tocotrienol, the new strain is named: Saccharomyces cerevisiae YS-356c, and the preservation number is: CCTCC NO: M2019572.

A method for constructing a genetically engineered bacterium with high production of α- and γ-tocotrienol, characterized in that enzymes that can be expressed in Saccharomyces cerevisiae: 4-hydroxyphenylpyruvate dioxygenase (HPPD); Acid phytyltransferase (HPT); 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT); Tocopherol cyclase (TC); γ-tocopherol methyltransferase (γ-TMT) , and gradually introduced into Saccharomyces cerevisiae strains to obtain the genetically engineered strain YS-16 producing α- and γ-tocotrienol.

A method for constructing a genetically engineered bacterium with high production of α- and γ-tocotrienol, characterized in that enzymes that can be expressed in Saccharomyces cerevisiae: 4-hydroxyphenylpyruvate dioxygenase (HPPD); Acid phytyltransferase (HPT); 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT); Tocopherol cyclase (TC); γ-tocopherol methyltransferase (γ-TMT) , and gradually introduced into Saccharomyces cerevisiae strains to obtain a genetically engineered strain YS-16 producing α- and γ-tocotrienol; and excised the chloroplast transit peptides of the three enzymes MPBQMT, TC and γ-TMT, and then overexpressed the rate-limiting Enzymes HPT, TC and γ-TMT, and obtained a genetically engineered strain YS-356c that can produce high α- and γ-tocotrienol.

The aforementioned method for constructing genetically engineered bacteria with high α- and γ-tocotrienol production uses P _Gal1 or P _Gal10 as a promoter, and gradually integrates foreign genes into the chromosome of the host bacteria; the host bacteria is Saccharomyces cerevisiae.

In the aforementioned method for constructing genetically engineered bacteria with high α- and γ-tocotrienol production, the modified enzyme is obtained by excising the chloroplast transit peptides of the three enzymes MPBQMT, TC and γ-TMT. The amino acid sequence of MPBQMT is as follows: SEQ ID NO: 6, TC as shown in SEQ ID NO: 8, γ-TMT as shown in SEQ ID NO: 10; its nucleotide sequence: MPBQMT as shown in SEQ ID NO: 7, TC as shown in SEQ ID NO : shown in 9, γ-TMT as shown in SEQ ID NO: 11; then overexpress rate-limiting enzymes HPT (SEQ ID NO: 2), TC (SEQ ID NO: 9) and γ-TMT (SEQ ID NO: 11 ) to obtain a genetically engineered strain YS-356c that can produce high α- and γ-tocotrienol.

The benefits of the present invention are:

The present invention uses the key enzyme gene sequence in the vitamin E synthesis pathway of plants or cyanobacteria as a template, through gene cloning and codon optimization, to obtain five enzymes that can be successfully expressed in Saccharomyces cerevisiae, and then gradually integrate these five enzymes into the knockout On the chromosome of Saccharomyces cerevisiae YS40 with the GAL80 gene removed, the engineered strain YS-16 was obtained, which achieved the heterologous synthesis of α- and γ-tocotrienol through biological fermentation for the first time, and its total tocotrienol production reached 243.3 μg/g stem cell Heavy;

The present invention further excises the chloroplast transit peptide of the three enzymes MPBQMT, TC and γ-TMT, and then overexpresses the rate-limiting enzymes HPT, TC and γ-TMT to obtain a genetically engineered strain of high-yield α- and γ-tocotrienol YS-356c, its total tocotrienol yield reached 2085.3μg/g cell dry weight, compared with strain YS-16, the yield increased by 7.57 times, which has a certain market prospect and application value;

The genetically engineered bacteria constructed by the invention can realize the production of α- and γ-tocotrienol safely and efficiently.

Description of drawings

Fig. 1 is the construction method of a kind of embodiment of genetic engineering bacterial strain YS-356c of the present invention;

Fig. 2 is the HPLC detection spectrum of the tocotrienol bacterial strain of the product of embodiment 1 of the present invention;

Fig. 3 is the mass spectrogram (positive ion mode) of the gamma-tocotrienol output of embodiment 1 of the present invention;

Fig. 4 is the mass spectrogram (negative ion mode) of the gamma-tocotrienol output of embodiment 1 of the present invention;

Fig. 5 is the mass spectrogram (positive ion mode) of the α-tocotrienol produced in Example 1 of the present invention;

Fig. 6 is the mass spectrogram (negative ion mode) of α-tocotrienol produced in Example 1 of the present invention;

Fig. 7 is a comparison chart of HPLC peak diagrams of engineering strains of two embodiments of the present invention.

Detailed ways

The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

A method for constructing a genetically engineered bacterium with high production of α- and γ-tocotrienol, comprising the following contents:

1. Cloning and expression of key enzymes in the biosynthetic pathway of tocotrienols,

Using the key enzyme gene sequences in the vitamin E synthesis pathway of plants or cyanobacteria as templates, through gene cloning and codon optimization, five enzymes that can be successfully expressed in Saccharomyces cerevisiae were obtained;

They are:

4-hydroxyphenylpyruvate dioxygenase (HPPD),

Homogentisate phytotransferase (HPT),

2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT),

tocopherol cyclase (TC),

γ-tocopheryl methyltransferase (γ-TMT);

4-hydroxyphenylpyruvate dioxygenase (HPPD), homogentisate phytotransferase (HPT), 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT), tocopherol cyclase ( TC), gamma-tocopherol methyltransferase (gamma-TMT), its acquisition process is:

1. Extract plant RNA and reverse transcribe it into cDNA, or extract cyanobacteria genomic DNA;

2. Clone the target gene,

Using cDNA or genomic DNA as a template, high-fidelity enzymes are used for PCR amplification;

The target fragment of the PCR product and the vector plasmid are double-digested with restriction endonucleases;

The digested fragments and plasmids were ligated using T4 DNA ligase;

After the ligation product was transformed into E. coli competent cell solution, it was spread on the resistant plate culture;

Pick out the positive clones on the resistant plate;

After the positive clones were inoculated and cultured, the plasmids were extracted, and the recombinant plasmids were further verified by DNA sequencing and sequence comparison;

3. Optimize the codons of the target gene sequence,

Through the codon optimization website, Saccharomyces cerevisiae was used as the host for codon optimization, the optimized gene sequence was chemically synthesized, and the corresponding fragment was connected to the pUMRI vector plasmid to obtain a recombinant plasmid with the target gene;

4. Linearize the recombinant plasmid with the target gene and integrate it into the chromosome of Saccharomyces cerevisiae,

Firstly, the recombinant plasmid was digested with SfiI endonuclease to obtain a linearized fragment, which was then chemically transformed into Saccharomyces cerevisiae competent cells, spread on G418 resistant plates and cultured, and PCR was verified to obtain correctly integrated strains.

5. Detection of the product of the integrated strain to determine the expression of the corresponding enzyme.

2. Connect the five enzymes HPPD, HPT, MPBQMT, TC and γ-TMT to the pUMRI vector plasmid with _pGal1 or _pGal10 as the promoter respectively, and use the endonuclease sfiI to recombine the plasmid After enzyme digestion and linearization, it was gradually integrated into the chromosome of Saccharomyces cerevisiae YS40, which knocked out the GAL80 gene, through chemical transformation, and a genetically engineered yeast strain YS-16 producing α- and γ-tocotrienol was constructed.

3. On the basis of the previously constructed genetically engineered yeast strain YS-16, the chloroplast transit peptides of the three enzymes MPBQMT, TC and γ-TMT were further excised, and then the rate-limiting enzymes HPT, TC and γ-TMT were overexpressed to obtain a Genetically engineered strain YS-356c producing α- and γ-tocotrienols;

The name of the strain is: Saccharomyces cerevisiae YS-356c, Saccharomyces cerevisiae YS-356c, the preservation number is: CCTCC NO: M2019572, the preservation unit is: China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China; The date is: July 19, 2019.

The method for excising the chloroplast transit peptides of MPBQMT, TC and γ-TMT three enzymes is:

According to the chloroplast transit peptide prediction website, the lengths of the transit peptides of the three enzymes MPBQMT, TC and γ-TMT were determined to be 51aa, 47aa and 40aa, respectively;

According to the predicted cleavage site of the chloroplast transit peptide, locate the corresponding DNA sequence, redesign the primers, excise the DNA sequence of the corresponding transit peptide by PCR, and then connect it to the plasmid vector by enzyme digestion and enzyme ligation. Then transformed into the corresponding yeast strains.

The method for overexpressing the rate-limiting enzymes HPT, TC and γ-TMT is as follows: on the basis of a single-copy strain, at different integration sites, use P _Gal1 or P _Gal10 as a promoter to increase a copy of the rate-limiting enzyme.

A preparation method of α- or γ-tocotrienol is prepared in the fermentation culture of genetic engineering strain YS-16 or high-yield strain YS-356c. The strain was placed in YPD liquid medium supplemented with 0.1% (w/v) tyrosine, cultured with shaking at 30°C and 220 rpm for 96 hours, the fermentation broth was centrifuged to remove the supernatant, and the obtained cells were ground and broken, ethyl acetate After separation and extraction, the organic phase is dried to obtain α- and γ-tocotrienol, which have good application prospects.

△The following experiments verify that genetically engineered bacteria can achieve efficient synthesis of α- and γ-tocotrienol

The experimental process is:

Step 1, cloning and expression of key genes in the biosynthetic pathway of tocotrienols.

Using the key enzyme gene sequences in the vitamin E synthesis pathway of plants or cyanobacteria as templates, through gene cloning and further codon optimization, five enzymes that can be successfully expressed in Saccharomyces cerevisiae were obtained;

They are:

4-hydroxyphenylpyruvate dioxygenase (HPPD), SEQ ID NO: 1;

Homogentisate phytotransferase (HPT), SEQ ID NO: 2;

2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT), SEQ ID NO: 3;

Tocopherol cyclase (TC), SEQ ID NO: 4;

Gamma-tocopherol methyltransferase (gamma-TMT), SEQ ID NO:5.

Five enzymes that can be successfully expressed in Saccharomyces cerevisiae were obtained through the following specific steps:

1.1 Obtain key enzyme gene sequences in the vitamin E synthesis pathway,

By extracting plant RNA and reverse-transcribing it into cDNA, the key enzyme gene sequences in the vitamin E synthesis pathway were obtained;

Specific steps are as follows:

(1) Weigh about 100 mg of plant tissue, put it into a frozen mortar, add an appropriate amount of liquid nitrogen, fully grind, and repeat 3 times to grind into powder.

(2) Add 1ml RNAiso, homogenate, and transfer to RNase-free centrifuge tube.

(3) Place at room temperature for 5 minutes to fully lyse.

(4) Centrifuge at 12000rpm for 5min, transfer the supernatant to a new 1.5ml centrifuge tube.

(5) Add 200 μl (1/5 RNAiso Plus volume) of chloroform, vortex and mix, and let stand at room temperature for 5 minutes.

(6) Centrifuge at 12000g for 15min at 4°C, and transfer the supernatant to a new 1.5ml centrifuge tube.

(7) Add 0.5-1 times the volume of RNAiso Plus isopropanol, mix well, and let stand at room temperature for 10 minutes.

(8) Centrifuge at 12,000 g for 10 min at 4°C, discard the supernatant, and the RNA is a white precipitate that settles at the bottom of the tube.

(9) Add an equal volume of 75% ethanol to RNAiso Plus to wash the precipitate, shake the centrifuge tube gently, and suspend the precipitate.

(10) Centrifuge at 7500g for 5min at 4°C, discard the supernatant and keep the precipitate.

(11) Dry at room temperature for 5-10 minutes. Note: The RNA sample should not be too dry, otherwise it will be difficult to dissolve.

(12) Add 50 μl DEPC-treated water to dissolve, take 3 μl to run nucleic acid gel for verification.

The above-mentioned RNA samples verified by gel running were reverse-transcribed using the PrimeScript ^TM 1st Strand cDNA Synthesis Kit kit. For details, please refer to the product manual. Take 3 μL of reverse-transcribed cDNA samples, run nucleic acid gel verification, and place them in a -80°C refrigerator as a template for the next step of PCR.

1.2 Extraction of cyanobacteria genomic DNA,

Place Synechocystis sp. PCC6803 in 50ml BG-11 liquid medium, shake at 30°C and 120rpm until logarithmic growth phase, take 5ml of algae liquid and centrifuge at 12000rpm, discard the supernatant, and use the Biospin Fungal Genomic DNA Extraction Kit from BioFlux Company Extract Synechocystis DNA, see the product manual for details.

1.3 Cloning of the target gene,

Using cDNA or genomic DNA as a template, high-fidelity enzyme (Prime STAR ^TM HS DNA polymerase) was used for PCR amplification. The reaction system (50μl) is as follows:

The PCR program is as follows:

The target fragment of the PCR product and the integrated pUMRI series plasmid are double-digested with Takara restriction endonuclease. The double-digestion system follows the instructions of Takara restriction endonuclease. After digestion, the system is subjected to DNA gel recovery treatment. The specific steps Follow the instructions of the Axygen kit.

The digested fragments and plasmids were ligated using T4 DNA ligase, and the ligation system (10 μl) was as follows:

After 30 minutes of connection at 22°C, 10 μl of the ligation product was added to the E. coli competent cell solution, placed on ice for 15 minutes, then heat-shocked at 42°C for 90 seconds, and quickly placed in an ice bath for 3 minutes, and 1ml of LB liquid medium was added. Resuscitate on a shaker at 37°C for 45min, then centrifuge, discard part of the supernatant, apply an appropriate amount to the corresponding resistant LB plate, and place in a 37°C incubator for 15h.

In order to select positive clones on the resistant plate, first randomly pick 5-10 single clones from the plate to a centrifuge tube containing 400 μl of the corresponding resistant LB medium, culture at 37°C for 3 hours, in 10 μl PCR system , take 0.5 μl of bacterial liquid as a template, and use Easy Taq DNA polymerase for PCR verification. The amplification system (10 μl) is as follows:

The PCR program is as follows:

After the PCR of the bacterial solution, carry out nucleic acid electrophoresis verification, inoculate the positive cloned bacteria into 5ml LB test tubes with corresponding resistance, at 30°C, 220rpm, culture overnight for 12-16h, then carry out plasmid extraction, which is extracted according to the corresponding Axygen reagent After extraction, the recombinant plasmids were further verified by DNA sequencing and sequence comparison.

1.4 Codon optimization of the target gene,

The corresponding amino acid sequence of the target gene was uploaded to the codon optimization website www.jcat.de, and the codon optimization was carried out with Saccharomyces cerevisiae as the host. The optimized gene sequence was chemically synthesized by Shanghai Jierui Bioengineering Co., Ltd. And the corresponding fragments were connected to pUMRI series plasmids.

1.5 Integrate the recombinant plasmid with the target gene into the Saccharomyces cerevisiae chromosome,

It should be noted that the selection of pUMRI series plasmids for vector plasmids is only a preferred embodiment. The pUMRI series plasmids are a set of gene assembly tools for Saccharomyces cerevisiae chromosome integration previously constructed by our research group (GenBank: KM216412; KM216413; KM216415). The plasmid has URA3 and KanMX dual selection markers, and its structure is: homologous segment upstream of the chromosome - direct repeat sequence - KanMX expression cassette - URA3 expression cassette - direct repeat sequence - multiple cloning site - homologous region downstream of the chromosome part. The KanMX gene can encode kanamycin resistance for the screening of clones in Escherichia coli, and can also encode geneticin resistance for selection of Saccharomyces cerevisiae on the G418 resistance plate. These plasmids contain Sfi I restriction sites at the junction of the upstream and downstream homology arms, which are linearized by Sfi I restriction.

Enzyme digestion system (30ul): 26μl plasmid, 3μl 10×QuickCut Buffer, 1μl Sfi I endonuclease.

Digestion conditions: water bath at 50°C for 2 hours.

The digested product was cleaned with a PCR cleaning kit and used for the Saccharomyces cerevisiae transformation experiment.

Competent preparation of Saccharomyces cerevisiae: Pick a single yeast colony from the YPD plate and inoculate it into a 5ml YPD test tube, culture overnight at 30°C, 220rpm, transfer 2% of the inoculum to a 50ml YPD shaker flask, culture at 30°C, 220rpm4 -5h, OD ₆₀₀ grows to about 2.0.

Saccharomyces cerevisiae transformation steps:

(1) Heat the ssDNA in a metal bath at 100°C for 5 minutes, then quickly put it in ice and cool it for later use.

(2) Take 45ml of competent yeast liquid in a 50ml sterilized centrifuge tube, centrifuge at 4000rpm, 20°C for 5min, and discard the supernatant.

(3) Wash once with 20ml sterile water, discard the supernatant by centrifugation, resuspend with 1ml sterile water, dispense 100μl per tube into 1.5ml sterilized centrifuge tubes, and discard the supernatant by centrifugation.

(4) Add the following transformation systems (360 μl) in turn to the centrifuge tube:

(5) Mix well and place in a metal bath at 42°C for 40 minutes.

(6) Centrifuge at 12000rpm for 30s, discard the supernatant, and add 1ml YPD liquid medium to resuscitate for 2h.

(7) Centrifuge at 12000rpm for 1min, discard the supernatant, add 1ml of sterile water to resuspend, take 100ul and spread on the corresponding resistance plate.

Step 2, the five enzymes HPPD, HPT, MPBQMT, TC and γ-TMT are gradually integrated into the chromosome of Saccharomyces cerevisiae YS40, which has knocked out the GAL80 gene, to construct a gene producing α- and γ-tocotrienol Engineering yeast strain YS-16, as Example 1;

The specific steps are as follows:

2.1 Construction of synthetic pathway

The five enzymes HPPD, HPT, MPBQMT, TC and γ-TMT, with _PGal1 or _PGal10 as the promoter, were connected to pUMRI series plasmids by enzyme digestion, and the recombinant plasmid Sfi I was digested and linearized Finally, it was gradually integrated into the chromosome of Saccharomyces cerevisiae YS40 through chemical transformation, and a genetically engineered strain YS-16 producing α- and γ-tocotrienol was constructed.

2.2 Cultivation of engineering bacteria

Pick a yeast single colony on the YPD plate and put it in a 5ml YPD liquid test tube, culture overnight at 30°C and 220rpm in a constant temperature shaker, then transfer to 250ml of 50ml YPD liquid medium with 0.1% (W/V) tyrosine added In the Erlenmeyer shake flask, the initial OD ₆₀₀ in the shake flask was set to 0.05, and placed in a constant temperature shaker at 30° C. at 220 rpm for 96 hours.

2.3 Product extraction

(1) Take 5ml of yeast fermentation broth, centrifuge at 4000rpm for 5min, discard the supernatant, then wash once with 5ml of distilled water, discard the supernatant by centrifugation, add 200μL of distilled water, and resuspend the bacteria.

(2) Add 500ml of grinding beads (0.1mm and half of 0.5mm zirconia beads) into a 2ml centrifuge tube, and then transfer all the resuspended bacterial solution to a 2ml centrifuge tube.

(3) Place the centrifuge tube containing the beads and bacteria in an automatic sample rapid grinder at 65HZ, and grind for 5 minutes.

(4) After grinding, add 800 μl of acetone to the centrifuge tube for extraction, mix thoroughly, and place in ultrasonic for 10 min.

(5) After ultrasonication, centrifuge at 4000rpm for 30s, suck the supernatant into a new 2ml centrifuge tube, then add 1ml acetone to the original 2ml centrifuge tube, reextract once, mix thoroughly, and sonicate for 10min.

(6) Without centrifugation, all the turbid liquid is transferred to the new centrifuge tube of step (5), so that about 2 ml of turbid extract is obtained.

(7) Centrifuge at 12000rpm for 5min, take 1ml of the supernatant into a new 1.5ml centrifuge tube, filter it with a 0.22μm organic filter, and wait for HPLC detection.

2.4 Tocotrienol HPLC analysis conditions

Mobile phase: acetonitrile (A) and pure water (B)

Ratio: 70%A/30%B

Chromatographic column: Agilent, ZORBAX, SB-C18 (4.6×250mm)

Flow rate: 0.8ml/min

Detection wavelength: 292nm (ultraviolet detector)

Gradient elution conditions: 0–10min: 70%A/30%B—90%A/10%B

10–40min: 90%A/10%B—100%A/0%B

40–70min: 100%A/0%B

70–80min: 100%A/0%B—70%A/30%B

2.5 LC-MS analysis conditions

Liquid phase conditions:

Mobile phase: methanol (A) and pure water (B)

Ratio: 70%A/30%B

Chromatographic column: Agilent, ZORBAX, SB-C18 (4.6×250mm)

Flow rate: 0.8ml/min

Detection wavelength: 292nm (ultraviolet detector)

Gradient elution conditions: 0–10min: 70%A/30%B—90%A/10%B

10–40min: 90%A/10%B—100%A/0%B

40–70min: 100%A/0%B

70–80min: 100%A/0%B—70%A/30%B

Mass Spectrometry Conditions:

ESI ionization; atomizer pressure: 25psi; drying gas flow rate: 8L/min; drying gas temperature: 220°C; capillary pressure: 4500V.

The product of Example 1 was analyzed and detected by high-performance liquid chromatography (HPLC), and the obtained product peaks corresponded to the standard products γ-tocotrienol and α-tocotrienol, and further combined with mass spectrometry detection, the results are shown in Figure 2 , confirm that its products are γ-tocotrienol and α-tocotrienol, after making a standard curve, determine its yield, wherein the output of γ-tocotrienol is 172 μg/g cell dry weight, α-tocotrienol The yield of phenols was 71.3 μg/g dry weight of cells, and the yield of total tocotrienols was 243.3 μg/g dry weight of cells. The results of liquid phase mass spectrometry are shown in Figures 3, 4, 5, and 6.

Step 3, by excising the chloroplast transit peptides of the three enzymes MPBQMT, TC and γ-TMT to improve their catalytic activity, the nucleotide sequences of the modified enzymes are respectively as SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11; and then overexpress the rate-limiting enzymes HPT (SEQ ID NO: 2), TC (SEQ ID NO: 9) and γ-TMT (SEQ ID NO: 11), to obtain a high-yielding α- and γ- Genetically engineered bacteria YS-356C of tocotrienol, as embodiment 2;

The specific steps are as follows:

3.1 Prediction and excision of chloroplast transit peptides

According to the chloroplast transit peptide design website (http://www.cbs.dtu.dk/services/ChloroP/), import the amino acid sequences of MPBQMT, TC and γ-TMT respectively. After submission, if cTP is displayed as Y, it means the fragment There is a chloroplast transit peptide sequence, cTP-length is displayed as the length of the amino acid sequence of the chloroplast transit peptide, and cTP-score refers to the fraction of the cleavage site. The higher the score, the greater the possibility, but sometimes cTP-length predicts The length is not necessarily accurate, and several points with relatively high cTP-score can be selected as alternative splicing sites. The specific effect needs to be further compared with the catalytic activity to finally determine the optimal chloroplast transit peptide splicing site. , the lengths of the transit peptides of these three enzymes were determined to be 51aa, 47aa and 40aa, respectively.

According to the predicted cleavage site of the chloroplast transit peptide, locate the corresponding DNA sequence, redesign the primers, excise the DNA sequence of the corresponding transit peptide by PCR, and then connect it to the plasmid vector by enzyme digestion and enzyme ligation. Then transformed into the corresponding yeast strains.

MPBQMT amino acid sequence SEQ ID NO: 6;

MPBQMT nucleotide sequence such as SEQ ID NO: 7;

The amino acid sequence of tocopherol cyclase (TC) is shown in SEQ ID NO: 8;

The nucleotide sequence of tocopherol cyclase (TC) is shown in SEQ ID NO:9.

The amino acid sequence of γ-tocopherol methyltransferase (γ-TMT) is shown as SEQ ID NO: 10;

The nucleotide sequence of γ-tocopherol methyltransferase (γ-TMT) is shown in SEQ ID NO:11.

3.2 Overexpression of the rate-limiting enzyme increases product yield;

On the basis of single-copy strains, at different integration sites, with P _Gal1 or P _Gal10 as the promoter, one copy of the rate-limiting enzyme is added for overexpression, and the key limit enzyme is determined by overexpressing different combinations of key enzymes. speed step, and obtain an optimal overexpression combination, respectively, the rate-limiting enzymes HPT (SEQ ID NO: 2), TC (SEQ ID NO: 9) and γ-TMT (SEQ ID NO: 11), in a single copy The three rate-limiting enzymes were overexpressed on the strain, and a high-yielding yeast engineering strain YS-356c was obtained. Compared with the original strain YS-16, the total tocotrienol production was increased by 7.54 times, reaching 2.09mg/g dry cell weight , wherein the output of γ-tocotrienol was 1407.9 μg/g dry cell weight, and that of α-tocotrienol was 677.4 μg/g dry cell weight.

The experimental results of embodiment 1 and embodiment 2 are compared, and the HPLC peak diagram contrast is as shown in Figure 7:

Result analysis:

The genetically engineered strain YS-16 realized the heterologous synthesis of α-tocotrienol and γ-tocotrienol by biological fermentation for the first time; and the optimized high-yielding strain YS-356c, its yield increased by 7- 8 times, with certain market prospects and application value.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the above-mentioned embodiments do not limit the present invention in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

sequence listing

<110> Zhejiang University

<120> Enzyme expressed in Saccharomyces cerevisiae and genetically engineered bacteria with high α- and γ-tocotrienol production and its construction method

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tctgctaagt ctgacttgtc tactggtaac atggttcacg cttcttactt gttgacttct 360

ggtgacttga gattcttgtt cactgctcca tactctccat ctttgtctgc tggtgaaatc 420

aagccaacta ctactgcttc tatcccatct ttcgaccacg gttcttgtag atctttcttc 480

tcttctcacg gtttgggtgt tagagctgtt gctatcgaag ttgaagacgc tgaatctgct 540

ttctctatct ctgttgctaa cggtgctatc ccatcttctc caccaatcgt tttgaacgaa 600

gctgttacta tcgctgaagt taagttgtac ggtgacgttg ttttgagata cgtttcttac 660

aaggctgaag acactgaaaa gtctgaattc ttgccaggtt tcgaaagagt tgaagacgct 720

tcttctttcc cattggacta cggtatcaga agattggacc acgctgttgg taacgttcca 780

gaattgggtc cagctttgac ttacgttgct ggtttcactg gtttccacca attcgctgaa 840

ttcactgctg acgacgttgg tactgctgaa tctggtttga actctgctgt tttggcttct 900

aacgacgaaa tggttttgtt gccaatcaac gaaccagttc acggtactaa gagaaagtct 960

caaatccaaa cttacttgga acacaacgaa ggtgctggtt tgcaacactt ggctttgatg 1020

tctgaagaca tcttcagaac tttgagagaa atgagaaaga gatcttctat cggtggtttc 1080

gacttcatgc catctccacc accaacttac taccaaaact tgaagaagag agttggtgac 1140

gttttgtctg acgaccaaat caaggaatgt gaagaattgg gtatcttggt tgacagagac 1200

gaccaaggta ctttgttgca aatcttcact aagccattgg gtgacagacc aactatcttc 1260

atcgaaatca tccaaagagt tggttgtatg atgaaggacg aagaaggtaa ggcttaccaa 1320

tctggtggtt gtggtggttt cggtaagggt aacttctctg aattgttcaa gtctatcgaa 1380

gaatacgaaa agactttgga agctaagcaa ttggttggtt aa 1422

<210> 2

<211> 927

<212>DNA

<213> Artificial Sequence

<400> 2

atggctacta ttcaagcttt ttggagattt tctaggccac atactattat tggtactact 60

ttgtctgttt gggctgttta tttgttgact attttgggtg atggtaactc tgttaactca 120

ccagcttctt tggacttggt tttcggtgct tggttggctt gcttgttggg taacgtctac 180

attgttggat tgaatcaatt gtgggatgtt gatattgaca gaattaacaa acctaatttg 240

ccattagcta acggtgattt ttcaatagct caaggtagt ggattgttgg tttgtgcggt 300

gtcgcttctt tggctattgc ttggggtttg ggtttgtggt tgggtttgac tgttggtatt 360

tctttaatta ttggtactgc ttattctgtt ccaccagtta gattgaaaag attctctttg 420

ttagctgcat tgtgtatttt aactgttaga ggtattgttg ttaatttggg tttattcttg 480

ttctttagaa ttggtttggg ttatccacca actttgataa ctccaatttg ggttttaact 540

ttgtttattt tggttttcac tgttgcaatt gctattttca aggatgttcc agatatggaa 600

ggtgatagac aattcaaaat tcaaacattg aattgcaaa ttggtaaaca aaatgtcttc 660

agaggtactt taattttgtt gacaggttgttatttggcta tggctatttg gggtttgtgg 720

gctgctatgc ctttgaacac tgctttcttg attgtttctc atttgtgttt gttggctttg 780

ttgtggtgga ggtctaggga tgttcacttg gaatctaaaa ctgaaattgc ttcattctat 840

caatttattt ggaaattgtt ctttttagaa tatttgttgt acccartggc tttgtggtta 900

ccaaattttt ctaatactat tttctaa 927

<210> 3

<211> 1017

<212>DNA

<213> Artificial Sequence

<400> 3

atggcttctt tgatgttgaa cggtgctatc actttcccaa agggtttggg ttctccaggt 60

tctaacttgc acgctagatc tatcccaaga ccaactttgt tgtctgttac tagaacttct 120

actccaagat tgtctgttgc tactagatgt tcttcttctt ctgtttcttc ttctagacca 180

tctgctcaac caagattcat ccaacacaag aaggaagctt actggttcta cagattcttg 240

tctatcgttt acgaccacgt tatcaaccca ggtcactgga ctgaagacat gagagacgac 300

gctttggaac cagctgactt gtctcaccca gacatgagag ttgttgacgt tggtggtggt 360

actggtttca ctactttggg tatcgttaag actgttaagg ctaagaacgt tactatcttg 420

gaccaatctc cacaccaatt ggctaaggct aagcaaaagg aaccattgaa ggaatgtaag 480

atcgttgaag gtgacgctga agacttgcca ttcccaactg actacgctga cagatacgtt 540

tctgctggtt ctatcgaata ctggccagac ccacaaagag gtatcagaga agcttacaga 600

gttttgaaga tcggtggtaa ggcttgtttg atcggtccag tttacccaac tttctggttg 660

tctagattct tctctgacgt ttggatgttg ttcccaaagg aagaagaata catcgaatgg 720

ttcaagaacg ctggtttcaa ggacgttcaa ttgaagagaa tcggtccaaa gtggtacaga 780

ggtgttagaa gacacggttt gatcatgggt tgttctgtta ctggtgttaa gccagcttct 840

ggtgactctc cattgcaatt gggtccaaag gaagaagacg ttgaaaagcc agttaacaac 900

ccattctctt tcttgggtag attcttgttg ggtactttgg ctgctgcttg gttcgttttg 960

atcccaatct acatgtggat caaggaccaa atcgttccaa aggaccaacc aatctaa 1017

<210> 4

<211> 1467

<212>DNA

<213> Artificial Sequence

<400> 4

atggaaatca gatctttgat cgtttctatg aacccaaact tgtcttcttt cgaattgtct 60

agaccagttt ctccattgac tagatctttg gttccattca gatctactaa gttggttcca 120

agatctatct ctagagtttc tgcttctatc tctactccaa actctgaaac tgacaagatc 180

tctgttaagc cagtttaacgt tccaacttct ccaaacagag aattgagaac tccaacactct 240

ggttaccact tcgacggtac tccaagaaag ttcttcgaag gttggtactt cagagtttct 300

atcccagaaa agagagaatc tttctgtttc atgtactctg ttgaaaaccc agctttcaga 360

caatctttgt ctccattgga agttgctttg tacggtccaa gattcactgg tgttggtgct 420

caaatcttgg gtgctaacga caagtacttg tgtcaatacg aacaagactc tcacaacttc 480

tggggtgaca gacacgaatt ggttttgggt aacactttct ctgctgttcc aggtgctaag 540

gctccaaaca aggaagttcc accagaagaa ttcaacagaa gagtttctga aggtttccaa 600

gctactccat tctggcacca aggtcacatc tgtgacgacg gtagaactga ctacgctgaa 660

actgttaagt ctgctagatg ggaatactct actagaccag tttacggttg gggtgacgtt 720

ggtgctaagc aaaagtctac tgctggttgg ccagctgctt tcccagtttt cgaaccacac 780

tggcaaatct gtatggctgg tggtttgtct actggttgga tcgaatgggg tggtgaaaga 840

ttcgaattca gagacgctcc atcttactct gaaaagaact ggggtggtgg tttcccaaga 900

aagtggttct gggttcaatg taacgttttc gaaggtgcta ctggtgaagt tgctttgact 960

gctggtggtg gtttgagaca attgccaggt ttgactgaaa cgtacgaaaa cgctgctttg 1020

gtttgtgttc actacgacgg taagatgtac gaattcgttc catggaacgg tgttgttaga 1080

tgggaaatgt ctccatgggg ttactggtac atcactgctg aaaacgaaaa ccacgttgtt 1140

gaattggaag ctagaactaa cgaagctggt actccattga gagctccaac tactgaagtt 1200

ggtttggcta ctgcttgtag agactcttgt tacggtgaat tgaagttgca aatctgggaa 1260

agattgtacg acggttctaa gggtaaggtt atcttggaaa ctaagtcttc tatggctgct 1320

gttgaaatcg gtggtggtcc atggttcggt acttggaagg gtgacacttc taacactcca 1380

gaattgttga agcaagcttt gcaagttcca ttggacttgg aatctgcttt gggtttggtt 1440

ccattcttca agccaccagg tttgtaa 1467

<210> 5

<211> 1047

<212>DNA

<213> Artificial Sequence

<400> 5

atgaaggcta ctttggctgc tccatcttct ttgacttctt tgccatacag aactaactct 60

tctttcggtt ctaagtcttc tttgttgttc agatctccat cttcttcttc ttctgtttct 120

atgactacta ctagaggtaa cgttgctgtt gctgctgctg ctacttctac tgaagctttg 180

agaaagggta tcgctgaatt ctacaacgaa acttctggtt tgtgggaaga aatctggggt 240

gaccacatgc accacggttt ctacgaccca gactcttctg ttcaattgtc tgactctggt 300

cacaaggaag ctcaaatcag aatgatcgaa gaatctttga gattcgctgg tgttactgac 360

gaagaagaag aaaagaagat caagaaggtt gttgacgttg gttgtggtat cggtggttct 420

tctagatact tggcttctaa gttcggtgct gaatgtatcg gtatcacttt gtctccagtt 480

caagctaaga gagctaacga cttggctgct gctcaatctt tggctcacaa ggcttctttc 540

caagttgctg acgctttgga ccaaccattc gaagacggta agttcgactt ggtttggtct 600

atggaatctg gtgaacacat gccagacaag gctaagttcg ttaaggaatt ggttagagtt 660

gctgctccag gtggtagaat catcatcgtt acttggtgtc acagaaactt gtctgctggt 720

gaagaagctt tgcaaccatg ggaacaaaac atcttggaca agatctgtaa gactttctac 780

ttgccagctt ggtgttctac tgacgactac gttaacttgt tgcaatctca ctctttgcaa 840

gacatcaagt gtgctgactg gtctgaaaac gttgctccat tctggccagc tgttatcaga 900

actgctttga cttggaaggg tttggtttct ttgttgagat ctggtatgaa gtctatcaag 960

ggtgctttga ctatgccatt gatgatcgaa ggttacaaga agggtgttat caagttcggt 1020

atcatcactt gtcaaaagcc attgtaa 1047

<210> 6

<211> 288

<212> PRT

<213> Artificial Sequence

<400> 6

Met Ser Ser Ser Ser Val Ser Ser Ser Arg Pro Ser Ala Gln Pro Arg Phe

1 5 10 15

Ile Gln His Lys Lys Glu Ala Tyr Trp Phe Tyr Arg Phe Leu Ser Ile

20 25 30

Val Tyr Asp His Val Ile Asn Pro Gly His Trp Thr Glu Asp Met Arg

35 40 45

Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro Asp Met Arg Val

50 55 60

Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu Gly Ile Val Lys

65 70 75 80

Thr Val Lys Ala Lys Asn Val Thr Ile Leu Asp Gln Ser Pro His Gln

85 90 95

Leu Ala Lys Ala Lys Gln Lys Glu Pro Leu Lys Glu Cys Lys Ile Val

100 105 110

Glu Gly Asp Ala Glu Asp Leu Pro Phe Pro Thr Asp Tyr Ala Asp Arg

115 120 125

Tyr Val Ser Ala Gly Ser Ile Glu Tyr Trp Pro Asp Pro Gln Arg Gly

130 135 140

Ile Arg Glu Ala Tyr Arg Val Leu Lys Ile Gly Gly Lys Ala Cys Leu

145 150 155 160

Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg Phe Phe Ser Asp

165 170 175

Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp Phe Lys

180 185 190

Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg Ile Gly Pro Lys Trp

195 200 205

Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met Gly Cys Ser Val Thr

210 215 220

Gly Val Lys Pro Ala Ser Gly Asp Ser Pro Leu Gln Leu Gly Pro Lys

225 230 235 240

Glu Glu Asp Val Glu Lys Pro Val Asn Asn Pro Phe Ser Phe Leu Gly

245 250 255

Arg Phe Leu Leu Gly Thr Leu Ala Ala Ala Trp Phe Val Leu Ile Pro

260 265 270

Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys Asp Gln Pro Ile

275 280 285

<210> 7

<211> 867

<212>DNA

<213> Artificial Sequence

<400> 7

atgtcttctt ctgtttcttc ttctagacca tctgctcaac caagattcat ccaacacaag 60

aaggaagctt actggttcta cagattcttg tctatcgttt acgaccacgt tatcaaccca 120

ggtcactgga ctgaagacat gagagacgac gctttggaac cagctgactt gtctcaccca 180

gacatgagag ttgttgacgt tggtggtggt actggtttca ctactttggg tatcgttaag 240

actgttaagg ctaagaacgt tactatcttg gaccaatctc cacaccaatt ggctaaggct 300

aagcaaaagg aaccattgaa ggaatgtaag atcgttgaag gtgacgctga agacttgcca 360

ttcccaactg actacgctga cagatacgtt tctgctggtt ctatcgaata ctggccagac 420

ccacaaagag gtatcagaga agcttacaga gttttgaaga tcggtggtaa ggcttgtttg 480

atcggtccag tttacccaac tttctggttg tctagattct tctctgacgt ttggatgttg 540

ttcccaaagg aagaagaata catcgaatgg ttcaagaacg ctggtttcaa ggacgttcaa 600

ttgaagagaa tcggtccaaa gtggtacaga ggtgttagaa gacacggttt gatcatgggt 660

tgttctgtta ctggtgttaa gccagcttct ggtgactctc cattgcaatt gggtccaaag 720

gaagaagacg ttgaaaagcc agttaacaac ccattctctt tcttgggtag attcttgttg 780

ggtactttgg ctgctgcttg gttcgttttg atcccaatct acatgtggat caaggaccaa 840

atcgttccaa aggaccaacc aatctaa 867

<210> 8

<211> 442

<212> PRT

<213> Artificial Sequence

<400> 8

Met Ala Ser Ile Ser Thr Pro Asn Ser Glu Thr Asp Lys Ile Ser Val

1 5 10 15

Lys Pro Val Tyr Val Pro Thr Ser Pro Asn Arg Glu Leu Arg Thr Pro

20 25 30

His Ser Gly Tyr His Phe Asp Gly Thr Pro Arg Lys Phe Phe Glu Gly

35 40 45

Trp Tyr Phe Arg Val Ser Ile Pro Glu Lys Arg Glu Ser Phe Cys Phe

50 55 60

Met Tyr Ser Val Glu Asn Pro Ala Phe Arg Gln Ser Leu Ser Pro Leu

65 70 75 80

Glu Val Ala Leu Tyr Gly Pro Arg Phe Thr Gly Val Gly Ala Gln Ile

85 90 95

Leu Gly Ala Asn Asp Lys Tyr Leu Cys Gln Tyr Glu Gln Asp Ser His

100 105 110

Asn Phe Trp Gly Asp Arg His Glu Leu Val Leu Gly Asn Thr Phe Ser

115 120 125

Ala Val Pro Gly Ala Lys Ala Pro Asn Lys Glu Val Pro Pro Glu Glu

130 135 140

Phe Asn Arg Arg Val Ser Glu Gly Phe Gln Ala Thr Pro Phe Trp His

145 150 155 160

Gln Gly His Ile Cys Asp Asp Gly Arg Thr Asp Tyr Ala Glu Thr Val

165 170 175

Lys Ser Ala Arg Trp Glu Tyr Ser Thr Arg Pro Val Tyr Gly Trp Gly

180 185 190

Asp Val Gly Ala Lys Gln Lys Ser Thr Ala Gly Trp Pro Ala Ala Phe

195 200 205

Pro Val Phe Glu Pro His Trp Gln Ile Cys Met Ala Gly Gly Leu Ser

210 215 220

Thr Gly Trp Ile Glu Trp Gly Gly Glu Arg Phe Glu Phe Arg Asp Ala

225 230 235 240

Pro Ser Tyr Ser Glu Lys Asn Trp Gly Gly Gly Phe Pro Arg Lys Trp

245 250 255

Phe Trp Val Gln Cys Asn Val Phe Glu Gly Ala Thr Gly Glu Val Ala

260 265 270

Leu Thr Ala Gly Gly Gly Leu Arg Gln Leu Pro Gly Leu Thr Glu Thr

275 280 285

Tyr Glu Asn Ala Ala Leu Val Cys Val His Tyr Asp Gly Lys Met Tyr

290 295 300

Glu Phe Val Pro Trp Asn Gly Val Val Arg Trp Glu Met Ser Pro Trp

305 310 315 320

Gly Tyr Trp Tyr Ile Thr Ala Glu Asn Glu Asn His Val Val Glu Leu

325 330 335

Glu Ala Arg Thr Asn Glu Ala Gly Thr Pro Leu Arg Ala Pro Thr Thr

340 345 350

Glu Val Gly Leu Ala Thr Ala Cys Arg Asp Ser Cys Tyr Gly Glu Leu

355 360 365

Lys Leu Gln Ile Trp Glu Arg Leu Tyr Asp Gly Ser Lys Gly Lys Val

370 375 380

Ile Leu Glu Thr Lys Ser Ser Met Ala Ala Val Glu Ile Gly Gly Gly

385 390 395 400

Pro Trp Phe Gly Thr Trp Lys Gly Asp Thr Ser Asn Thr Pro Glu Leu

405 410 415

Leu Lys Gln Ala Leu Gln Val Pro Leu Asp Leu Glu Ser Ala Leu Gly

420 425 430

Leu Val Pro Phe Phe Lys Pro Pro Gly Leu

435 440

<210> 9

<211> 1329

<212>DNA

<213> Artificial Sequence

<400> 9

atggcttcta tctctactcc aaactctgaa actgacaaga tctctgttaa gccagtttac 60

gttccaactt ctccaaacag agaattgaga actccaacact ctggttacca cttcgacggt 120

actccaagaa agttcttcga aggttggtac ttcagagttt ctatcccaga aaagagagaa 180

tctttctgtt tcatgtactc tgttgaaaac ccagctttca gacaatcttt gtctccattg 240

gaagttgctt tgtacggtcc aagattcact ggtgttggtg ctcaaatctt gggtgctaac 300

gacaagtact tgtgtcaata cgaacaagac tctcacaact tctggggtga cagacacgaa 360

ttggttttgg gtaacacttt ctctgctgtt ccaggtgcta aggctccaaa caaggaagtt 420

ccaccagaag aattcaacag aagagtttct gaaggtttcc aagctactcc attctggcac 480

caaggtcaca tctgtgacga cggtagaact gactacgctg aaactgttaa gtctgctaga 540

tgggaatact ctactagacc agtttacggt tggggtgacg ttggtgctaa gcaaaagtct 600

actgctggtt ggccagctgc tttcccagtt ttcgaaccac actggcaaat ctgtatggct 660

ggtggtttgt ctactggttg gatcgaatgg ggtggtgaaa gattcgaatt cagagacgct 720

ccatcttact ctgaaaagaa ctggggtggt ggtttcccaa gaaagtggtt ctgggttcaa 780

tgtaacgttt tcgaaggtgc tactggtgaa gttgctttga ctgctggtgg tggtttgaga 840

caattgccag gtttgactga aacttacgaa aacgctgctt tggtttgtgt tcactacgac 900

ggtaagatgt acgaattcgt tccatggaac ggtgttgtta gatgggaaat gtctccatgg 960

ggttactggt acatcactgc tgaaaacgaa aaccacgttg ttgaattgga agctagaact 1020

aacgaagctg gtactccatt gagagctcca actactgaag ttggtttggc tactgcttgt 1080

agagactctt gttacggtga attgaagttg caaatctggg aaagattgta cgacggttct 1140

aagggtaagg ttatcttgga aactaagtct tctatggctg ctgttgaaat cggtggtggt 1200

ccatggttcg gtacttggaa gggtgacact tctaacactc cagaattgtt gaagcaagct 1260

ttgcaagttc cattggactt ggaatctgct ttgggtttgg ttccattctt caagccacca 1320

ggtttgtaa 1329

<210> 10

<211> 308

<212> PRT

<213> Artificial Sequence

<400> 10

Met Thr Thr Thr Arg Gly Asn Val Ala Val Ala Ala Ala Ala Thr Ser

1 5 10 15

Thr Glu Ala Leu Arg Lys Gly Ile Ala Glu Phe Tyr Asn Glu Thr Ser

20 25 30

Gly Leu Trp Glu Glu Ile Trp Gly Asp His Met His His Gly Phe Tyr

35 40 45

Asp Pro Asp Ser Ser Val Gln Leu Ser Asp Ser Gly His Lys Glu Ala

50 55 60

Gln Ile Arg Met Ile Glu Glu Ser Leu Arg Phe Ala Gly Val Thr Asp

65 70 75 80

Glu Glu Glu Glu Lys Lys Ile Lys Lys Val Val Asp Val Gly Cys Gly

85 90 95

Ile Gly Gly Ser Ser Arg Tyr Leu Ala Ser Lys Phe Gly Ala Glu Cys

100 105 110

Ile Gly Ile Thr Leu Ser Pro Val Gln Ala Lys Arg Ala Asn Asp Leu

115 120 125

Ala Ala Ala Gln Ser Leu Ala His Lys Ala Ser Phe Gln Val Ala Asp

130 135 140

Ala Leu Asp Gln Pro Phe Glu Asp Gly Lys Phe Asp Leu Val Trp Ser

145 150 155 160

Met Glu Ser Gly Glu His Met Pro Asp Lys Ala Lys Phe Val Lys Glu

165 170 175

Leu Val Arg Val Ala Ala Pro Gly Gly Arg Ile Ile Ile Val Thr Trp

180 185 190

Cys His Arg Asn Leu Ser Ala Gly Glu Glu Ala Leu Gln Pro Trp Glu

195 200 205

Gln Asn Ile Leu Asp Lys Ile Cys Lys Thr Phe Tyr Leu Pro Ala Trp

210 215 220

Cys Ser Thr Asp Asp Tyr Val Asn Leu Leu Gln Ser His Ser Leu Gln

225 230 235 240

Asp Ile Lys Cys Ala Asp Trp Ser Glu Asn Val Ala Pro Phe Trp Pro

245 250 255

Ala Val Ile Arg Thr Ala Leu Thr Trp Lys Gly Leu Val Ser Leu Leu

260 265 270

Arg Ser Gly Met Lys Ser Ile Lys Gly Ala Leu Thr Met Pro Leu Met

275 280 285

Ile Glu Gly Tyr Lys Lys Gly Val Ile Lys Phe Gly Ile Ile Thr Cys

290 295 300

Gln Lys Pro Leu

305

<210> 11

<211> 927

<212>DNA

<213> Artificial Sequence

<400> 11

atgactacta ctagaggtaa cgttgctgtt gctgctgctg ctacttctac tgaagctttg 60

agaaagggta tcgctgaatt ctacaacgaa acttctggtt tgtgggaaga aatctggggt 120

gaccacatgc accacggttt ctacgaccca gactcttctg ttcaattgtc tgactctggt 180

cacaaggaag ctcaaatcag aatgatcgaa gaatctttga gattcgctgg tgttactgac 240

gaagaagaag aaaagaagat caagaaggtt gttgacgttg gttgtggtat cggtggttct 300

tctagatact tggcttctaa gttcggtgct gaatgtatcg gtatcacttt gtctccagtt 360

caagctaaga gagctaacga cttggctgct gctcaatctt tggctcacaa ggcttctttc 420

caagttgctg acgctttgga ccaaccattc gaagacggta agttcgactt ggtttggtct 480

atggaatctg gtgaacacat gccagacaag gctaagttcg ttaaggaatt ggttagagtt 540

gctgctccag gtggtagaat catcatcgtt acttggtgtc acagaaactt gtctgctggt 600

gaagaagctt tgcaaccatg ggaacaaaac atcttggaca agatctgtaa gactttctac 660

ttgccagctt ggtgttctac tgacgactac gttaacttgt tgcaatctca ctctttgcaa 720

gacatcaagt gtgctgactg gtctgaaaac gttgctccat tctggccagc tgttatcaga 780

actgctttga cttggaaggg tttggtttct ttgttgagat ctggtatgaa gtctatcaag 840

ggtgctttga ctatgccatt gatgatcgaa ggttacaaga agggtgttat caagttcggt 900

atcatcactt gtcaaaagcc attgtaa 927

Claims (7)

1. the enzyme that one kind can be expressed in saccharomyces cerevisiae characterized by comprising 4- hydroxyphenylpyruvate dioxygenase (HPPD)；Alcapton phytyl transferase (HPT)；2- methyl -6- phytyl benzoquinone methyl transferase (MPBQMT)；Tocopherol cyclase (TC)；Gama-tocopherol methyl transferase (γ-TMT).

2. a kind of enzyme expressed in saccharomyces cerevisiae according to claim 1, which is characterized in that after codon optimization, The sequence of acquisition:

The nucleotide sequence of 4- hydroxyphenylpyruvate dioxygenase (HPPD) is as shown in SEQ ID NO:1；

The nucleotide sequence of alcapton phytyl transferase (HPT) is as shown in SEQ ID NO:2；

The nucleotide sequence of 2- methyl -6- phytyl benzoquinone methyl transferase (MPBQMT) is as shown in SEQ ID NO:3；

The nucleotide sequence of tocopherol cyclase (TC) is as shown in SEQ ID NO:4；

The nucleotide sequence of gama-tocopherol methyl transferase (γ-TMT) is as shown in SEQ ID NO:5.

3. a kind of high yield α-and γ-tocotrienols genetic engineering bacterium, which is characterized in that the name of novel bacterial are as follows: wine brewing ferment Female YS-356c, Saccharomyces cerevisiae YS-356c, deposit number are as follows: CCTCC NO:M2019572.

4. the construction method of a kind of high yield α-and γ-tocotrienols genetic engineering bacterium, which is characterized in that can make wine The enzyme expressed in yeast: 4- hydroxyphenylpyruvate dioxygenase (HPPD)；Alcapton phytyl transferase (HPT)；2- methyl -6- phytyl Benzoquinone methyl transferase (MPBQMT)；Tocopherol cyclase (TC)；Gama-tocopherol methyl transferase (γ-TMT), gradually imports Into Wine brewing yeast strain, obtains and produce α-and γ-tocotrienols engineering strain YS-16.

5. the construction method of a kind of high yield α-and γ-tocotrienols genetic engineering bacterium, which is characterized in that can make wine The enzyme expressed in yeast: 4- hydroxyphenylpyruvate dioxygenase (HPPD)；Alcapton phytyl transferase (HPT)；2- methyl -6- phytyl Benzoquinone methyl transferase (MPBQMT)；Tocopherol cyclase (TC)；Gama-tocopherol methyl transferase (γ-TMT), gradually imports Into Wine brewing yeast strain, obtains and produce α-and γ-tocotrienols engineering strain YS-16；And cut off MPBQMT, TC and The chloroplast transit peptides of three enzymes of γ-TMT obtain a plant height and produce α-and γ-life after expression rate-limiting enzyme HPT, TC and γ-TMT Educate the genetic engineering bacterium YS-356c of trienol.

6. a kind of high yield α-according to claim 4 or 5 and γ-tocotrienols construction of genetic engineering method, It is characterized in that, with P_Gal1Or P_Gal10For promoter, foreign gene is gradually integrated into host strain chromosome；The host strain is Saccharomyces cerevisiae.

7. a kind of high yield α-according to claim 5 and γ-tocotrienols construction of genetic engineering method, feature It is, by cutting off MPBQMT, the chloroplast transit peptides of three enzymes of TC and γ-TMT obtain improved enzyme, amino acid sequence Column: MPBQMT is as shown in SEQ ID NO:6, and TC is as shown in SEQ ID NO:8, and γ-TMT is as shown in SEQ ID NO:10；Its core Nucleotide sequence: MPBQMT is as shown in SEQ ID NO:7, and TC is as shown in SEQ ID NO:9, γ-TMT such as SEQ ID NO:11 institute Show；After expression rate-limiting enzyme HPT (SEQ ID NO:2), TC (SEQ ID NO:9) and γ-TMT (SEQ ID NO:11) are obtained One plant height produces α-and γ-tocotrienols genetic engineering bacterium YS-356C.