CN110423732B

CN110423732B - Enzyme expressed in saccharomyces cerevisiae, genetic engineering bacteria for high yield of alpha-and gamma-tocotrienols and construction method thereof

Info

Publication number: CN110423732B
Application number: CN201910749343.0A
Authority: CN
Inventors: 于洪巍; 沈斌; 叶丽丹
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-08-14
Filing date: 2019-08-14
Publication date: 2022-01-07
Anticipated expiration: 2039-08-14
Also published as: CN110423732A

Abstract

The invention discloses a key enzyme expressed in saccharomyces cerevisiae, a genetic engineering bacterium for high yield of alpha-and gamma-tocotrienol and a construction method thereof. Five enzymes which can be successfully expressed in saccharomyces cerevisiae are obtained through gene cloning and codon optimization, wherein the five enzymes are respectively HPPD, HPT, MPBQMT, TC and gamma-TMT; gradually integrating the five enzymes into a saccharomyces cerevisiae genome, further constructing and obtaining the genetic engineering yeast strain with high alpha-and gamma-tocotrienols yield by excising chloroplast transit peptide and over-expressing rate-limiting enzyme, and naming as: YS-356c, accession number: CCTCC NO: and M2019572. The total yield of tocotrienols reached 2.09mg/g dry cell weight. The engineering strain of the invention can be fermented and cultured to directly obtain alpha-and gamma-tocotrienols from thalli, and has good market prospect and application value.

Description

Enzyme expressed in saccharomyces cerevisiae, genetic engineering bacteria for high yield of alpha-and gamma-tocotrienols and construction method thereof

Technical Field

The invention relates to the field of genetic engineering and microorganisms, in particular to an enzyme expressed in saccharomyces cerevisiae, a genetic engineering bacterium for high yield of alpha-and gamma-tocotrienols and a construction method thereof.

Background

Vitamin E is an important fat-soluble compound, including tocopherols and tocotrienols, and is an essential vitamin for maintaining the normal metabolism and function of the body. Natural vitamin E is only present in photosynthetic organisms, and humans and animals cannot synthesize themselves, but can only be ingested from the outside. Vitamin E products on the market are mainly alpha-tocopherol, but recent studies have shown that tocotrienols have cholesterol-lowering efficacy (tocotrienol is unique) and better antioxidant, anticancer, anti-inflammatory, cardioprotective and neuroprotective functions than tocopherol. However, the source of tocotrienols is rare, and the commercial natural tocotrienols are mainly extracted from the oil palm fruit, and the production cost is high due to the low content, the high separation difficulty and the limited yield. The chemically synthesized tocotrienols cannot be commercially produced due to the complex steps and more byproducts. Therefore, the heterologous synthesis of tocotrienols using microorganisms is an efficient and promising approach.

In 2008, researchers at stuttgart university in germany achieved the synthesis of delta-tocotrienol in escherichia coli by introducing genes derived from arabidopsis thaliana and blue algae, but the yield was very low, only 15 μ g/g of cell dry weight, and the application of delta-tocotrienol was limited because delta-tocotrienol was in the form of unmethylated tocotrienol. To date, heterologous biosynthesis of alpha-or gamma-tocotrienols with greater functionality and broader application has not been achieved. The invention provides a method for heterogeneously synthesizing alpha-and gamma-tocotrienols by taking safe and efficient saccharomyces cerevisiae as a cell factory, provides a new thought and method for industrial production of the tocotrienols, and solves the problems of low yield and high production cost at present.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a key enzyme capable of being expressed in saccharomyces cerevisiae, a genetically engineered bacterium for high yield of alpha-and gamma-tocotrienols and a construction method thereof.

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

an enzyme that can be expressed in Saccharomyces cerevisiae comprising: 4-hydroxyphenylpyruvate dioxygenase (HPPD); homogentisate Phytotransferase (HPT); 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT); tocopherol Cyclase (TC); gamma-tocopherol methyltransferase (gamma-TMT).

The sequence obtained after codon optimization of the enzyme expressed in Saccharomyces cerevisiae was:

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

the nucleotide sequence of Homogentisate Phytotransferase (HPT) is shown in SEQ ID NO: 2 is shown in the specification;

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

the nucleotide sequence of the Tocopherol Cyclase (TC) is shown as SEQ ID NO: 4 is shown in the specification;

the nucleotide sequence of gamma-tocopherol methyltransferase (gamma-TMT) is shown in SEQ ID NO: 5, respectively.

A genetically engineered bacterium for high yield of alpha-and gamma-tocotrienol, the new strain is named as: saccharomyces cerevisiae YS-356c, with the preservation number: CCTCC NO: and M2019572.

A construction method of a genetic engineering bacterium for high yield of alpha-and gamma-tocotrienols is characterized in that an enzyme capable of being expressed in saccharomyces cerevisiae: 4-hydroxyphenylpyruvate dioxygenase (HPPD); homogentisate Phytotransferase (HPT); 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT); tocopherol Cyclase (TC); and (3) gradually introducing gamma-tocopherol methyltransferase (gamma-TMT) into the saccharomyces cerevisiae strain to obtain a genetic engineering strain YS-16 for producing alpha-and gamma-tocotrienols.

A construction method of a genetic engineering bacterium for high yield of alpha-and gamma-tocotrienols is characterized in that an enzyme capable of being expressed in saccharomyces cerevisiae: 4-hydroxyphenylpyruvate dioxygenase (HPPD); homogentisate Phytotransferase (HPT); 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT); tocopherol Cyclase (TC); gradually introducing gamma-tocopherol methyltransferase (gamma-TMT) into a saccharomyces cerevisiae strain to obtain a genetic engineering strain YS-16 for producing alpha-and gamma-tocotrienols; and removing chloroplast transit peptide of MPBQMT, TC and gamma-TMT, and overexpressing HPT, TC and gamma-TMT to obtain a genetically engineered bacterium YS-356c with high alpha-and gamma-tocotrienol yield.

The construction method of the genetic engineering bacteria for high yield of the alpha-and gamma-tocotrienols uses P_Gal1Or P_Gal10Gradually integrating exogenous genes into a host bacterium chromosome as a promoter; the host bacteria is Saccharomyces cerevisiae.

The construction method of the genetically engineered bacteria for high yield of the alpha-and gamma-tocotrienols is characterized in that the modified enzymes are obtained by excising chloroplast transit peptides of MPBQMT, TC and gamma-TMT enzymes, and the amino acid sequences of the modified enzymes are as follows: MPBQMT is shown as SEQ ID NO: 6, TC is shown as SEQ ID NO: 8, and gamma-TMT is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence thereof is as follows: MPBQMT is shown as SEQ ID NO: 7, TC is shown as SEQ ID NO: 9, gamma-TMT is shown as SEQ ID NO: 11 is shown in the figure; and then over-expressing the rate-limiting enzymes HPT (SEQ ID NO: 2), TC (SEQ ID NO: 9) and gamma-TMT (SEQ ID NO: 11) to obtain a genetically engineered bacterium YS-356c with high alpha-and gamma-tocotrienols yield.

The invention has the advantages that:

the invention takes a key enzyme gene sequence in a synthesis path of vitamin E of plants or blue algae as a template, obtains five enzymes which can be successfully expressed in saccharomyces cerevisiae through gene cloning and codon optimization, gradually integrates the five enzymes on a saccharomyces cerevisiae YS40 chromosome with GAL80 gene knocked out to obtain an engineering strain YS-16, realizes the heterologous synthesis of alpha-and gamma-tocotrienols through a biological fermentation method for the first time, and the total tocotrienol yield reaches 243.3 mu g/g of cell dry weight;

the invention further excises chloroplast transit peptides of MPBQMT, TC and gamma-TMT three enzymes, and then overexpresses the rate-limiting enzymes HPT, TC and gamma-TMT to obtain a genetically engineered bacterium YS-356c with high yield of alpha-and gamma-tocotrienols, wherein the total tocotrienol yield reaches 2085.3 mu g/g dry cell weight, and compared with the strain YS-16, the yield is improved by 7.57 times, thus the genetically engineered bacterium has certain market prospect and application value;

the gene engineering bacteria constructed by the invention can safely and efficiently realize the production of alpha-and gamma-tocotrienol.

Drawings

FIG. 1 is a construction method of one embodiment of the genetically engineered strain YS-356c of the invention;

FIG. 2 is an HPLC detection profile of a tocotrienol strain of the product of example 1 of the present invention;

FIG. 3 is a mass spectrum (positive ion mode) of gamma-tocotrienol produced in example 1 of the present invention;

FIG. 4 is a mass spectrum (negative ion mode) of gamma-tocotrienol produced in example 1 of the present invention;

FIG. 5 is a mass spectrum (positive ion mode) of alpha-tocotrienol produced in example 1 of this invention;

FIG. 6 is a mass spectrum (negative ion mode) of alpha-tocotrienol produced in example 1 of the present invention;

FIG. 7 is a comparison of HPLC peak profiles of engineered strains of two examples of the invention.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

A construction method of a genetic engineering bacterium for high yield of alpha-and gamma-tocotrienols comprises the following steps:

firstly, cloning and expressing key enzymes in the biosynthesis pathway of tocotrienols,

taking a key enzyme gene sequence in a plant or blue algae vitamin E synthesis path as a template, and obtaining five enzymes which can be successfully expressed in saccharomyces cerevisiae through gene cloning and codon optimization;

the method comprises the following steps:

4-hydroxyphenylpyruvate dioxygenase (HPPD),

homogentisate Phytotransferase (HPT),

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

a Tocopherol Cyclase (TC),

gamma-tocopherol methyltransferase (gamma-TMT);

4-hydroxyphenylpyruvate dioxygenase (HPPD), Homogentisate Phytotransferase (HPT), 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT), Tocopherol Cyclase (TC), gamma-tocopherol methyltransferase (gamma-TMT), obtained by:

1. extracting plant RNA and reverse transcribing into cDNA, or extracting blue algae genome DNA;

2. the target gene is cloned, and the cloning is carried out,

carrying out PCR amplification by using cDNA or genome DNA as a template and adopting high-fidelity enzyme;

carrying out double enzyme digestion on the target fragment of the PCR product and the vector plasmid by restriction endonuclease;

connecting the digested fragments and plasmids by using T4DNA ligase;

transforming the ligation product into an escherichia coli competent cell solution, and coating the escherichia coli competent cell solution on a resistant plate for culture;

selecting positive clones on the resistant plate;

inoculating and culturing the positive clone bacteria, extracting plasmids, further carrying out DNA sequencing verification on recombinant plasmids, and comparing sequences;

3. optimizing the codons of the target gene sequence,

performing codon optimization by using saccharomyces cerevisiae as a host through a codon optimization website, chemically synthesizing an optimized gene sequence, and connecting a corresponding fragment to a pUMRI vector plasmid to obtain a recombinant plasmid connected with a target gene;

4. the recombinant plasmid connected with the target gene is integrated on a saccharomyces cerevisiae chromosome after linearization,

firstly, carrying out enzyme digestion on recombinant plasmids by using SfiI endonuclease to obtain linearized fragments, then chemically converting the linearized fragments into saccharomyces cerevisiae competent cells, coating the saccharomyces cerevisiae competent cells on a G418 resistant plate for culture, and carrying out PCR verification to obtain correctly integrated strains.

5. And detecting products of the integrated strains, and determining the expression of corresponding enzymes.

Two, using five enzymes of HPPD, HPT, MPBQMT, TC and gamma-TMT as P_Gal1Or P_Gal10For promoter, the recombinant plasmid is connected to pUMRI vector plasmid through enzyme digestion and enzyme ligation respectivelyAfter the restriction linearization of the dicer sfiI, the dicer sfiI is gradually integrated on a Saccharomyces cerevisiae YS40 chromosome with GAL80 gene knocked out through chemical transformation to construct a genetically engineered yeast strain YS-16 producing alpha-and gamma-tocotrienols.

Thirdly, on the basis of the gene engineering yeast strain YS-16 constructed in the previous stage, further excising chloroplast transit peptides of MPBQMT, TC and gamma-TMT enzymes, and then overexpressing the rate-limiting enzymes HPT, TC and gamma-TMT to obtain a gene engineering bacterium YS-356c with high alpha-and gamma-tocotrienol yield;

the name of the strain is as follows: saccharomyces cerevisiae YS-356c, with the preservation number: CCTCC NO: m2019572, the collection unit is: china Center for Type Culture Collection (CCTCC), address: wuhan university in Wuhan, China; the preservation date is as follows: 7/19/2019.

The method for excising chloroplast transit peptides of three enzymes including MPBQMT, TC and gamma-TMT comprises the following steps:

determining the lengths of the transit peptides of the MPBQMT, TC and gamma-TMT enzymes as 51aa, 47aa and 40aa respectively according to a chloroplast transit peptide prediction website;

and (3) positioning to a corresponding DNA sequence according to the predicted chloroplast transit peptide cleavage site, redesigning a primer, excising the DNA sequence of the corresponding transit peptide in a PCR (polymerase chain reaction) mode, connecting to a plasmid vector in a restriction enzyme-linked mode, and then transforming to a corresponding yeast strain.

The method for over-expressing the rate-limiting enzymes HPT, TC and gamma-TMT comprises the following steps: on the basis of a single copy strain, at different integration sites, with P_Gal1Or P_Gal10One copy of the rate-limiting enzyme is added as a promoter.

A process for preparing alpha-or gamma-tocotrienol from the fermented culture of the genetically engineered strain YS-16 or high-yield strain YS-356 c. The strain is placed in YPD liquid culture medium added with 0.1% (w/v) tyrosine, shaking culture is carried out for 96h at 30 ℃ and 220rpm, the supernatant is removed by centrifugation of fermentation liquor, the obtained thalli are ground and crushed, ethyl acetate is separated and extracted, and an organic phase is dried to obtain alpha-and gamma-tocotrienol, so that the application prospect is good.

The efficient synthesis of alpha-and gamma-tocotrienols by the genetically engineered bacteria is verified by experiments below delta

The experimental process is as follows:

cloning and expressing key genes in a tocotrienol biosynthesis pathway.

Taking a key enzyme gene sequence in a plant or blue algae vitamin E synthesis path as a template, and obtaining five enzymes which can be successfully expressed in saccharomyces cerevisiae through gene cloning and further codon optimization;

the method comprises the following steps:

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 by the following specific steps:

1.1 obtaining key enzyme gene sequence in vitamin E synthetic pathway,

obtaining a key enzyme gene sequence in a vitamin E synthetic route by extracting plant RNA and carrying out reverse transcription on the plant RNA into cDNA;

the method comprises the following specific steps:

(1) weighing plant tissue about 100mg, placing into a frozen mortar, adding appropriate amount of liquid nitrogen, grinding thoroughly, and grinding into powder for 3 times.

(2) Add 1ml of RNAasso, homogenize, and transfer to RNase-free centrifuge tubes.

(3) Standing at room temperature for 5min to allow it to fully lyse.

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

(5) Add 200. mu.l (1/5 volumes of RNAioso Plus) chloroform, mix well with shaking, and let stand at room temperature for 5 min.

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

(7) Adding 0.5-1 times of RNAioso Plus volume of isopropanol, mixing, and standing at room temperature for 10 min.

(8) Centrifugation was carried out at 12000g for 10min at 4 ℃ and the supernatant was discarded, leaving RNA as a white precipitate on the bottom of the tube.

(9) Adding 75% ethanol with the same volume as the RNAioso Plus, washing the precipitate, gently shaking the centrifuge tube, and suspending the precipitate.

(10) Centrifuging at 7500g for 5min at 4 deg.C, discarding the supernatant and retaining the precipitate.

(11) Air drying at room temperature for 5-10 min. Note: the RNA sample cannot be dried too much or is difficult to dissolve.

(12) Add 50. mu.l DEPC treated water to dissolve, take 3. mu.l run nucleic acid gel for validation.

The RNA samples after the running gel verification were used as PrimeScript^TMThe 1st Strand cDNA Synthesis Kit is used for reverse transcription, and the specific steps are described in the product specification. Taking 3 mu L of the cDNA sample after reverse transcription, running nucleic acid gel for verification, and placing the cDNA sample in a refrigerator at the temperature of minus 80 ℃ to be used as a next PCR template for later use.

1.2 extracting the genome DNA of the blue algae,

the synechocystis PCC6803 is placed in 50ml BG-11 liquid culture medium, is subjected to shaking culture at 30 ℃ and 120rpm until the logarithmic phase, 5ml of alga liquid is centrifuged at 12000rpm, the supernatant is discarded, and synechocystis DNA is extracted by using a BioFlux Biospin fungus genome DNA extraction kit, wherein the specific steps are described in the product specification.

1.3 cloning of the target gene,

using cDNA or genomic DNA as template and high fidelity enzyme (Prime STAR)^TMHS DNA polymerase) were subjected to PCR amplification. The reaction system (50. mu.l) was as follows:

the PCR procedure was as follows:

and carrying out double enzyme digestion on the target fragment of the PCR product and the integrated pUMRI series plasmid by Takara restriction enzyme, carrying out DNA gel recovery treatment on the system after enzyme digestion according to the instruction of the Takara restriction enzyme in a double enzyme digestion system, and carrying out the specific steps according to the instruction of an Axygen kit.

The digested fragments and plasmids were ligated using T4DNA ligase in the following ligation scheme (10. mu.l):

and (2) connecting at 22 ℃ for 30min, adding 10 mu l of the connection product into an escherichia coli competent cell solution, standing on ice for 15min, thermally shocking at 42 ℃ for 90s, quickly placing in an ice bath for 3min, adding 1ml of LB liquid culture medium, recovering under a shaking table at 37 ℃ for 45min, centrifuging, removing part of supernatant, coating a proper amount of supernatant on a corresponding resistant LB plate, and standing in a 37 ℃ incubator for 15 h.

To select positive clones on resistant plates, first 5-10 single clones were randomly picked from the plates to 400. mu.l of a centrifuge tube containing the corresponding resistant LB medium, incubated at 37 ℃ for 3 hours, and subjected to PCR validation with Easy Taq DNA polymerase using 0.5. mu.l of the broth as a template in a 10. mu.l PCR system, the amplification system (10. mu.l) being as follows:

the PCR procedure was as follows:

and (3) after PCR of the bacterial liquid, carrying out nucleic acid electrophoresis verification, inoculating the positive clone bacteria into a 5ml LB test tube with corresponding resistance, culturing at 30 ℃ and 220rpm overnight for 12-16h, carrying out plasmid extraction according to the instructions of the Axygen corresponding kit, and carrying out further DNA sequencing verification and sequence comparison on recombinant plasmids after extraction.

1.4 codon optimization of the target gene,

the corresponding amino acid sequence of the target gene is uploaded to a codon optimization website www.jcat.de, the codon optimization is carried out by taking saccharomyces cerevisiae as a host, and the optimized gene sequence is chemically synthesized by Shanghai Czeri bioengineering GmbH. And the corresponding fragment was ligated to pUMRI series plasmids.

1.5 integrating the recombinant plasmid connected with the target gene on a saccharomyces cerevisiae chromosome,

it should be noted that: vector plasmid selection the pUMRI series plasmid is only a preferred example, and is a set of gene assembly tools (GenBank: KM 216412; KM 216413; KM216415) constructed before this subject group for chromosomal integration in Saccharomyces cerevisiae. The plasmid has dual selection markers of URA3 and KanMX, and has the structure as follows: chromosome upstream homology segment-forward repeat sequence-KanMX expression cassette-URA 3 expression cassette-forward repeat sequence-multiple cloning site-chromosome downstream homology segment. Wherein the KanMX gene can encode kanamycin resistance for cloning in Escherichia coli, and also can encode geneticin resistance for screening Saccharomyces cerevisiae on G418 resistant plates. These plasmids contain Sfi I restriction sites at the junction of upstream and downstream homology arms, which are linearized by Sfi I restriction.

Enzyme digestion system (30 ul): 26 μ l plasmid, 3 μ l 10 XQuickCut Buffer, 1 μ l Sfi I endonuclease.

Enzyme cutting conditions are as follows: water bath was carried out at 50 ℃ for 2 hours.

And (3) cleaning the product after enzyme digestion by using a PCR (polymerase chain reaction) cleaning kit for a saccharomyces cerevisiae transformation experiment.

Preparing saccharomyces cerevisiae competence: picking single yeast colony from YPD plate, inoculating to 5ml YPD test tube, culturing at 30 deg.C and 220rpm overnight, transferring to 50ml YPD shake flask according to 2%, culturing at 30 deg.C and 220rpm for 4-5 hr, OD₆₀₀Up to about 2.0 a.

Converting saccharomyces cerevisiae:

(1) the ssDNA was heated in a metal bath at 100 ℃ for 5min and then rapidly cooled in ice for further use.

(2) 45ml of competent yeast liquid is taken to be placed in a 50ml sterilized centrifuge tube, centrifuged for 5min at 4000rpm and 20 ℃, and the supernatant is discarded.

(3) Washing with 20ml of sterile water, centrifuging to remove the supernatant, resuspending with 1ml of sterile water, packaging into 1.5ml sterile centrifuge tubes according to 100 mul per tube, and centrifuging to remove the supernatant.

(4) The following chemotrope systems (360. mu.l) were added to the centrifuge tube in sequence:

(5) mixing, and standing in metal bath at 42 deg.C for 40 min.

(6) Centrifuging at 12000rpm for 30s, discarding the supernatant, adding 1ml YPD liquid culture medium, and recovering for 2 h.

(7) Centrifuging at 12000rpm for 1min, discarding the supernatant, adding 1ml of sterile water for resuspension, and taking 100ul of the suspension to be spread on a corresponding resistant plate.

Step two, gradually integrating five enzymes including HPPD, HPT, MPBQMT, TC and gamma-TMT to a Saccharomyces cerevisiae YS40 chromosome with GAL80 gene knocked out, and constructing a genetic engineering yeast strain YS-16 for producing alpha-and gamma-tocotrienols, wherein the genetic engineering yeast strain YS-16 is used as the example 1;

the specific steps are as follows:

2.1 construction of the synthetic pathway

The five enzymes HPPD, HPT, MPBQMT, TC and gamma-TMT are used as P_Gal1Or P_Gal10The promoter is respectively connected to pUMRI series plasmids through enzyme digestion and enzyme ligation, the recombinant plasmid Sfi I is subjected to enzyme digestion and linearization, and is gradually integrated on a saccharomyces cerevisiae YS40 chromosome through chemical transformation, so that the genetic engineering strain YS-16 for producing alpha-and gamma-tocotrienols is constructed.

2.2 cultivation of the engineering bacteria

Single yeast colonies were picked from YPD plates in 5ml YPD liquid tubes at 30 ℃ and 220rpmThe cells were grown overnight in a shaker, transferred to a 250ml triangular flask containing 50ml YPD liquid medium supplemented with 0.1% (W/V) tyrosine, and the initial OD in the flask was set₆₀₀Is 0.05, and is placed in a constant temperature shaker at 30 ℃ and 220rpm for 96 hours.

2.3 product extraction

(1) 5ml of yeast fermentation liquor is taken, centrifuged at 4000rpm for 5min, the supernatant is discarded, then the yeast fermentation liquor is washed once by 5ml of distilled water, centrifuged, the supernatant is discarded, 200 mu L of distilled water is added, and the thalli are resuspended.

(2) A500 ml volume of grinding beads (0.1mm and 0.5mm halves of zirconia beads) was added to a 2ml centrifuge tube, and the resuspended pellet was then transferred to a 2ml centrifuge tube in its entirety.

(3) And (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, 800. mu.l of acetone was added to the centrifuge tube for extraction, mixed well and subjected to ultrasound for 10 min.

(5) And (3) after ultrasonic treatment, centrifuging at 4000rpm for 30s, sucking the supernatant into a new 2ml centrifuge tube, adding 1ml of acetone into the original 2ml centrifuge tube, re-extracting once, fully and uniformly mixing, and performing ultrasonic treatment for 10 min.

(6) The turbid liquid was transferred in its entirety without centrifugation into a new centrifuge tube from step (5), thus obtaining about 2ml of turbid extract.

(7) Centrifuging at 12000rpm for 5min, collecting 1ml supernatant to a new 1.5ml centrifuge tube, filtering with 0.22 μm organic filter head, and detecting by HPLC.

2.4 conditions for HPLC analysis of tocotrienols

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

The proportion is as follows: 70% A/30% B

A chromatographic column: agilent, ZORBAX, SB-C18 (4.6X 250mm)

Flow rate: 0.8ml/min

Detection wavelength: 292nm (ultraviolet detector)

Gradient elution conditions: 0-10 min: 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

2.5LC-MS analysis conditions

Liquid phase conditions:

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

The proportion is as follows: 70% A/30% B

A chromatographic column: agilent, ZORBAX, SB-C18 (4.6X 250mm)

Flow rate: 0.8ml/min

Detection wavelength: 292nm (ultraviolet detector)

Gradient elution conditions: 0-10 min: 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 spectrum conditions:

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

The product of example 1 was analyzed and detected by High Performance Liquid Chromatography (HPLC), the obtained product peaks corresponded to the standard gamma-tocotrienol and alpha-tocotrienol, and further combined with mass spectrometry, as shown in fig. 2, the products were determined to be gamma-tocotrienol and alpha-tocotrienol, and after making a standard curve, the yields were determined, wherein the gamma-tocotrienol yield was 172 μ g/g dry cell weight, the alpha-tocotrienol yield was 71.3 μ g/g dry cell weight, the total tocotrienol yield was 243.3 μ g/g dry cell weight, and the liquid mass spectrometry results are shown in fig. 3, 4, 5, and 6.

And step three, the catalytic activity of the chloroplast transit peptides of MPBQMT, TC and gamma-TMT enzymes is improved by cutting off the chloroplast transit peptides, and the nucleotide sequences of the modified enzymes are respectively shown as SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 is shown in the figure; then, the rate-limiting enzymes HPT (SEQ ID NO: 2), TC (SEQ ID NO: 9) and gamma-TMT (SEQ ID NO: 11) are overexpressed to obtain a genetically engineered bacterium YS-356C with high alpha-and gamma-tocotrienols yield, which is taken as an example 2;

the specific steps are as follows:

3.1 prediction and excision of chloroplast transit peptides

According to chloroplast transit peptide design website (http:// www.cbs.dtu.dk/services/Chlorop /), introducing amino acid sequences of MPBQMT, TC and gamma-TMT respectively, after submission, if the cTP is shown as Y, the fragment is indicated to have a chloroplast transit peptide sequence, the cTP-length is shown as a chloroplast transit peptide amino acid sequence length, the cTP-score refers to the fraction of the cleavage site, the higher the fraction means the higher the possibility, but sometimes the length predicted by the cTP-length is not necessarily accurate, a plurality of points with relatively higher cTP-score can be selected as alternative splicing sites, the specific effect needs to be further compared with catalytic activity to finally determine the optimal chloroplast transit peptide cleavage site, and finally, the transit peptide lengths of the three enzymes are respectively 51aa, 47aa and 40 aa.

MPBQMT amino acid sequence SEQ ID NO: 6;

the nucleotide sequence of MPBQMT is shown as SEQ ID NO: 7;

the amino acid sequence of the Tocopherol Cyclase (TC) is as shown in SEQ ID NO: 8 is shown in the specification;

the nucleotide sequence of the Tocopherol Cyclase (TC) is shown as SEQ ID NO: shown at 9.

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

the nucleotide sequence of gamma-tocopherol methyltransferase (gamma-TMT) is shown as SEQ ID NO: shown at 11.

3.2 overexpression of the rate-limiting enzyme improves the product yield;

on the basis of a single copy strain, at different integration sites, with P_Gal1Or P_Gal10For the promoter, the overexpression is carried out in such a way that one copy of the rate-limiting enzyme is added, and the critical limit is determined by overexpressing different combinations of the critical enzymesQuickly performing the steps, and obtaining an optimal overexpression combination which is respectively a rate-limiting enzyme HPT (SEQ ID NO: 2), a TC (SEQ ID NO: 9) and a gamma-TMT (SEQ ID NO: 11), and overexpressing the three rate-limiting enzymes on a single-copy strain to obtain a high-yield yeast engineering strain YS-356c, wherein the total tocotrienol yield is improved by 7.54 times and reaches 2.09mg/g of cell dry weight compared with the original strain YS-16, wherein the yield of the gamma-tocotrienol is 1407.9 mu g/g of cell dry weight, and the yield of the alpha-tocotrienol is 677.4 mu g/g of cell dry weight.

Comparing the experimental results of example 1 and example 2, the HPLC peak profiles are shown in fig. 7:

and (4) analyzing results:

the genetic engineering strain YS-16 realizes the heterologous synthesis of alpha-tocotrienol and gamma-tocotrienol by a biological fermentation method for the first time; the yield of the optimized high-yield strain YS-356c is improved by 7-8 times compared with that of YS-16, and the optimized high-yield strain YS-356c has certain market prospect and application value.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.

Sequence listing

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<213> Artificial Sequence

<400> 1

atgtgtttgt ctttggcttc tactgctcaa agaaacactc aattcagatc tagagttttg 60

gttttggctg aattggttaa gtctatgggt caccaaaacg ctgctgtttc tgaaaaccaa 120

aaccacgacg acggtgctgc ttcttctcca ggtttcaagt tggttggttt ctctaagttc 180

gttagaaaga acccaaagtc tgacaagttc aaggttaaga gattccacca catcgaattc 240

tggtgtggtg acgctactaa cgttgctaga agattctctt ggggtttggg tatgagattc 300

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 caaggtaggt 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 acattgcaaa ttggtaaaca aaatgtcttc 660

agaggtactt taattttgtt gacaggttgt tatttggcta tggctatttg gggtttgtgg 720

gctgctatgc ctttgaacac tgctttcttg attgtttctc atttgtgttt gttggctttg 780

ttgtggtgga ggtctaggga tgttcacttg gaatctaaaa ctgaaattgc ttcattctat 840

caatttattt ggaaattgtt ctttttagaa tatttgttgt acccattggc 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 cagtttacgt tccaacttct ccaaacagag aattgagaac tccacactct 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 cttacgaaaa 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 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 actccacact 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

1. A genetically engineered bacterium for high yield of alpha-and gamma-tocotrienols is characterized in that the genetically engineered bacterium is named as: saccharomyces cerevisiae (Saccharomyces cerevisiae) YS-356c, accession number: CCTCC NO: and M2019572.