CN111235044B - Recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol, construction method and application - Google Patents

Abstract

The invention discloses a recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol, a construction method and application thereof, wherein the method comprises the following steps: integrating exogenous genes hpd, hpt and vte1 into a genome rDNA locus of a saccharomyces cerevisiae BY4742 strain to obtain a recombinant strain VE 01; the invention integrates thmg1 gene and ggppssa gene at the delta site of the recombinant strain VE01 genome to obtain a recombinant strain VE02. The method for synthesizing the delta-tocotrienol by using the microorganisms is simple and convenient to operate, and can obtain the delta-tocotrienol with high yield.

Description

Recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol, construction method and application

Technical Field

The invention relates to the technical field of biosynthesis, in particular to a recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol and a construction method thereof.

Background

Vitamin e (vitamin e) is a fat-soluble vitamin whose hydrolysate is tocopherol, one of the most important antioxidants. It was discovered as early as 20s in the 20 th century that Evans and his colleagues discovered during the course of their research reproduction that rancid lard could cause infertility in rats. Crystals were isolated in 1936 and artificially synthesized by Swiss chemists in 1938. Vitamin E comprises a group of eight structurally related tocopherols (tocophenols) and tocotrienols (tocotrienols), which were less studied in the early days because tocotrienols have a very low biological activity compared to tocopherols. Recent studies have shown that tocotrienols have functions not possessed by tocopherols, such as neuronal protection and cholesterol lowering effects. And the tocotrienol has strong antitumor activity, and can inhibit tumor cell proliferation, induce tumor cell apoptosis, block cell cycle and inhibit angiogenesis in vitro and in vivo. Has good clinical application prospect. Delta-tocotrienol is an isomer of vitamin E and has recently received much attention due to its potential diverse biomedical applications, such as anticancer activity, prevention of osteoporosis and cholesterol reduction.

To date, according to reports on the market prospects of vitamin E, approximately 80% of vitamin E in the market comes from chemical synthesis and 20% from plant or seed extracts. However, the use of toxic catalysts in the chemical synthesis of vitamin E makes the process environmentally unfriendly and non-sustainable. However, delta-tocotrienols are mainly extracted from oil palm and rice, and little has been studied on the biosynthesis of their microorganisms. Engineered saccharomyces cerevisiae can replace extraction methods in plants as an alternative and promising way to produce delta-tocotrienols.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol.

The second purpose of the invention is to provide a construction method of the recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol.

A third object of the invention is the use of a recombinant strain of saccharomyces cerevisiae for the production of delta-tocotrienol.

The technical scheme of the invention is summarized as follows:

the construction method of the recombinant saccharomyces cerevisiae strain for synthesizing the delta-tocotrienol comprises the following steps: integrating exogenous genes hpd, hpt and vte1 into a genome rDNA locus of a saccharomyces cerevisiae BY4742 strain to obtain a recombinant strain VE 01; integrating a thmg1 gene and a ggppssa gene at a delta site of a recombinant strain VE01 genome to obtain a recombinant strain VE02, wherein the nucleotide sequence of the hpd gene is shown as SEQ ID NO. 7; the nucleotide sequence of the hpt gene is shown as SEQ ID NO. 8; the nucleotide sequence of the vte1 gene is shown as SEQ ID NO. 9; the nucleotide sequence of the thmg1 gene is shown in SEQ ID NO. 10; the nucleotide sequence of the ggppssa gene is shown as SEQ ID NO. 11.

The synthetic delta-tocotrienol recombinant saccharomyces cerevisiae strain constructed by the method.

The application of the recombinant saccharomyces cerevisiae strain for synthesizing the delta-tocotrienol to produce the delta-tocotrienol comprises the following steps: culturing and fermenting the recombinant delta-tocotrienol-synthesizing saccharomyces cerevisiae strain VE02 of claim 2 in a fermentation medium to obtain delta-tocotrienol, wherein the formula of the fermentation medium is 35.66g/L glucose, 24.46g/L peptone, 14.01g/L yeast extract powder and the balance water.

The invention has the advantages that:

the recombinant saccharomyces cerevisiae strain for synthesizing delta-tocotrienol constructed by the invention can be used for synthesizing the delta-tocotrienol again by taking glucose as a carbon source, and can replace a plant extraction method. The method for synthesizing the delta-tocotrienol by using the microorganisms is simple and convenient to operate, and can obtain the delta-tocotrienol with high yield.

Drawings

FIG. 1 is a scheme for the synthesis of delta-tocotrienol in Saccharomyces cerevisiae.

FIG. 2 is a graph of the yield of recombinant s.cerevisiae strain VE01 for the synthesis of delta-tocotrienol.

FIG. 3 is a graph of the yield of recombinant s.cerevisiae strain VE02 for the synthesis of delta-tocotrienol.

FIG. 4 is a graph showing the yield of delta-tocotrienol synthesized by recombinant Saccharomyces cerevisiae strain VE02 after optimization of fermentation conditions.

Detailed Description

The original strain Saccharomyces cerevisiae BY4742 was purchased at ATCC official website in 2016, 9 months (https:// www.atcc.org/products/all/201389. aspx).

The present invention will be further described with reference to the following examples.

Example 1

The construction method of the recombinant saccharomyces cerevisiae strain for synthesizing the delta-tocotrienol comprises the following steps: the expression cassettes of three exogenous genes hpd, hpt and vte1 are integrated to the rDNA locus of a yeast (Saccharomyces cerevisiae BY4742) genome BY using an rDNA locus specificity homologous recombination method to obtain a recombinant strain VE 01.

Integrating the thmg1 gene and the ggppssa gene at the delta site of the recombinant strain VE01 genome to obtain a recombinant strain VE02.

The nucleotide sequence of the hpd gene is shown as SEQ ID NO. 7; the nucleotide sequence of the hpt gene is shown as SEQ ID NO. 8; the nucleotide sequence of the vte1 gene is shown as SEQ ID NO. 9; the nucleotide sequence of the thmg1 gene is shown in SEQ ID NO. 10; the nucleotide sequence of the ggppssa gene is shown as SEQ ID NO. 11.

The method comprises the following specific steps:

(1) the construction method of the recombinant saccharomyces cerevisiae strain VE01 for synthesizing the delta-tocotrienol comprises the following steps:

obtaining amino acid sequences of a Pseudomonas putida KT2440 derived gene hpd, a Synechocystis sp.PCC6803 derived gene hpt and an Arabidopsis thaliana derived gene vte1 from an NCBI database, artificially synthesizing sequences of the three genes through codon optimization, and simultaneously constructing expression cassettes of the three genes,

the hpd gene expression cassette uses Ppgk1 promoter and Tpgk1 terminator (SEQ ID NO.1 and SEQ ID NO.4),

the hpt gene expression cassette uses the Ptef1 promoter and the Ttpi1 terminator (SEQ ID NO.2 and SEQ ID NO.5),

the vte1 gene expression cassette used Ppgk1 promoter and Tcyc1 terminator (SEQ ID NO.1 and SEQ ID NO.6),

and (3) introducing the hpd gene expression cassette, the hpt gene expression cassette and the vte1 gene expression cassette into a saccharomyces cerevisiae rDNA locus by a homologous recombination method to obtain the recombinant saccharomyces cerevisiae strain VE01 for synthesizing the delta-tocotrienol.

(2) The construction method of the recombinant saccharomyces cerevisiae strain VE02 for synthesizing the delta-tocotrienol comprises the following steps:

obtaining gene thmg1 gene of Saccharomyces cerevisiae BY4742 from NCBI database, obtaining the nucleotide sequence (SEQ ID NO.10) BY artificial synthesis,

the amino acid sequence from the Sulfolobus acidocaldarius gene ggppssa was obtained from NCBI database, the nucleotide sequence of this gene (SEQ ID NO.11) was artificially synthesized by codon optimization,

constructing expression cassettes of the two genes,

the expression cassette for the thmg1 gene used Ppgk1 promoter and Tpgk1 terminator (SEQ ID NO.1 and SEQ ID NO.4),

the ggppssa gene expression cassette uses Ptdh1 promoter and Tref 1 terminator (SEQ ID NO.3 and SEQ ID NO.12), and a thamg 1 gene expression cassette and the ggppssa gene expression cassette are introduced into a saccharomyces cerevisiae delta site through a homologous recombination method to obtain a synthetic delta-tocotrienol recombinant saccharomyces cerevisiae strain 02 (see fig. 1).

1. Construction of modules

The homologous arms rDNA-up and rDNA-down are both from the genome of Saccharomyces cerevisiae BY 4742; the selectable marker gene URA3 was derived from plasmid pRS426 (purchased from addge, inc.

The genome of Saccharomyces cerevisiae BY4742 is used as a template, rDNaup-F (SEQ ID NO.13), rDNaup-R-Tcyc1(SEQ ID NO.14), Ttpi 1-rDNaudown-F (SEQ ID NO.15) and rDNaudown-R (SEQ ID NO.16) are used as primers, and the homologous arm rDNA-up (SEQ ID NO.17) and rDNA-down (SEQ ID NO.18) are amplified respectively;

respectively amplifying a promoter Ppgk1(SEQ ID NO.1) and a terminator Tcyc1(SEQ ID NO.6) by taking vte1-Tcyc1-F (SEQ ID NO.19), Tcyc1-R-rDNaup (SEQ ID NO.20), URA3-Ppgk1-F (SEQ ID NO.21) and Ppgk1-R-vte1(SEQ ID NO.22) as primers;

using Tpgk1-Ptef1-F (SEQ ID NO.23), Ptef1-R-hpt (SEQ ID NO.24), hpt-Ttpi1-F (SEQ ID NO.25), Ttpi1-R-rDNAdown (SEQ ID NO.26) as primers to respectively amplify a promoter Ptef1(SEQ ID NO.2) and a terminator Ttpi1(SEQ ID NO. 5);

the plasmid PRS426 is used as a template, Ppgk1-URA3-F (SEQ ID NO.27) and URA3-R-Ppgk1(SEQ ID NO.28) are used as primers, and a marker gene URA3(SEQ ID NO.29) is amplified, wherein the sequence is shown in a sequence table.

The PCR enzyme used in the present invention is Fast Pfu polymerase from holo-gold Biotechnology Ltd. A50. mu.L PCR amplification system was as follows: DNA template, 1. mu.L; 2.5. mu.L of each of the front lead (10. mu.M) and the rear lead (10. mu.M); dNTP (10mM), 5. mu.L; 5 Xpfu Buffer, 10. mu.L; fast Pfu polymerase, 1 μ L; finally, the solution is replenished to 50 mu L by double distilled water. An amplification program is set up on the PCR instrument. The amplification conditions were pre-denaturation at 95 ℃ for 2min (1 cycle); denaturation at 95 ℃ for 20sec, annealing at 55 ℃ for 20sec, and extension at 72 ℃ for 30s (35 cycles); extension at 72 ℃ for 5min (1 cycle).

The 7 amplified fragments are mixed with 5 fragments of artificially synthesized gene fragment hpd (SEQ ID NO.7), hpt gene (SEQ ID NO.8), nucleotide sequence of vte1 gene (SEQ ID NO.9), Ppgk1 promoter (SEQ ID NO.1) and Tpgk1 terminator (SEQ ID NO.4) and spliced into a large fragment by a BM seamless ligation kit (Bomader Biotech Co., Ltd.). The molar ratio of each fragment was 1:1, the total amount of fragments reached 1. mu.g, the ligation volume was as follows, 10. mu.L of DNA fragment, 10. mu.L of 2 × Seamless Cloning Mix. The ligation program was set up on the PCR instrument. The connection conditions were 50min at 50 ℃ and 10min at 16 ℃.

The fragments obtained by PCR, adjusted at a concentration of 500ng, were converted into competent yeast using the LiAc/salmon sperm DNA/PEG method. In the absence of uracil, individual colonies were grown on SC-drop agar plates (20g/L glucose, 2g/L amino acid mixture, 6.7g/L yeast nitrogen base without amino acids, 20g/L agar for solid plates). After incubation at 30 ℃ for 36h, colonies were cultured overnight in yeast protein ept glucose (YPD) medium (20g/L glucose, 20g/L peptone, 10g/L yeast extract, 20g/L agar solid medium) at 30 ℃ at 200 rpm. The recombinant strain VE01 was constructed.

Using a Saccharomyces cerevisiae BY4742 genome as a template, using deltaup-F (SEQ ID NO.30), deltaup-R-LEU (SEQ ID NO.31), Tref 1-deltadown-F (SEQ ID NO.32) and deltadown-R (SEQ ID NO.33) as primers to respectively amplify homologous arms delta-up (SEQ ID NO.34) and delta-down (SEQ ID NO. 35);

LEU-Ppgk1-F (SEQ ID NO.36), Ppgk1-R-thmgr (SEQ ID NO.37), thmg1-Tpgk1-F (SEQ ID NO.38) and Tpgk1-R (SEQ ID NO.39) are used as primers to respectively amplify a promoter Ppgk1(SEQ ID NO.1) and a terminator Tpgk1(SEQ ID NO. 4);

tpgk1-Ptdh1-F (SEQ ID NO.40), Ptdh1-R-ggppssa (SEQ ID NO.41), ggppssa-Tref 1-F (SEQ ID NO.42) and Tref 1-R-deltadown (SEQ ID NO.43) are used as primers to respectively amplify a promoter Ptdh1(SEQ ID NO.3) and a terminator Tref 1(SEQ ID NO. 12);

the plasmid PRS425 is used as a template, and deltaup-LEU-F (SEQ ID NO.44) and LEU-R-Ppgk1(SEQ ID NO.45) are used as primers to amplify a marker gene LEU (SEQ ID NO.46), and the sequence is shown in a sequence table.

The 7 fragments obtained by PCR amplification and the artificially synthesized gene thmg1 and ggppssa gene fragment are spliced into a large fragment by a BM seamless ligation kit (Bomader Biotechnology Co., Ltd.). The molar ratio of each fragment was 1:1, the total amount of fragments reached 1. mu.g, the ligation volume was as follows, 10. mu.L of DNA fragment, 10. mu.L of 2 × Seamless Cloning Mix. The ligation program was set up on the PCR instrument. The connection conditions were 50min at 50 ℃ and 10min at 16 ℃.

The fragments obtained by PCR, adjusted at a concentration of 500ng, were converted into competent yeast using the LiAc/salmon sperm DNA/PEG method. In the absence of uracil, individual colonies were grown on SC-drop agar plates (20g/L glucose, 2g/L amino acid mixture, 6.7g/L yeast nitrogen base without amino acids, 20g/L agar for solid plates). After incubation at 30 ℃ for 36h, colonies were cultured overnight in yeast protein ept glucose (YPD) medium (20g/L glucose, 20g/L peptone, 10g/L yeast extract, 20g/L agar solid medium) at 30 ℃ at 200 rpm. The recombinant strain VE02 was constructed.

2. Fermentation process

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder and the balance of water;

fermentation medium: 35.66g/L glucose, 24.46g/L peptone, 14.01g/L yeast extract powder and the balance of water.

Control group: the recombinant strain VE02 was inoculated into 5mL of seed medium 30oC at 200rpm for 20h, then the 5mL of medium was inoculated into 30mL of fresh seed medium, the density of the strain was adjusted to a final density of 0.1 at 30oC at 200rpm at OD600nm, and the final density was incubated for 72h, and the culture broth was taken for product analysis.

Experimental groups: inoculating the recombinant strain VE02 into 5mL of seed culture medium at 30oC for 200rpm, culturing for 20h, then transferring the 5mL of culture medium into 30mL of fresh fermentation culture medium, adjusting the bacterial density to make the final bacterial density at OD600nm at 0.1, 30oC for 200rpm, culturing for 72h, and taking the culture solution for product analysis.

3. Analysis of the product

Extraction and analysis of delta-tocotrienol: 30mL of the culture solution of the control group and the experimental group, respectively, were centrifuged at 4oC 8000rpm/min for 10 minutes to obtain cell pellets, and washed twice with double distilled water (ddH2O) to remove the residual medium. Then, 7ml of double distilled water was added to suspend the cells, followed by 7ml of acid-washed glass beads (0.4-0.6mm) and 15ml of analytical grade acetone. The supernatant was obtained by shaking at room temperature for 30 minutes with a vortex at 5000rpm and then centrifuging at 4 ℃ for 10 minutes at 8000 rpm. The supernatant was evaporated in a vacuum rotary evaporator and the residue dissolved in acetonitrile (1 mL). Analysis was performed by High Performance Liquid Chromatography (HPLC) using a Waters 2695HPLC instrument with a UV detector. The solution was filtered and injected into an RP18Lichrospher100 analytical column (5 μm, 150X4.6 mm, Merck, Germany) with delta-tocotrienol (purity 98% or more, Toronto research chemical, Canada) as a label. The mobile phase was acetonitrile containing 0.1% v/v trifluoroacetic acid (TFA), total flow rate 0.6mL/min, chromatogram at 292nm detection, column temperature 30oC, see FIG. 2, FIG. 3 and FIG. 4.

Analysis of precursor homogentisate and geranylgeranyl pyrophosphate: 1ml of the culture solution was taken for each of the control group and the experimental group, and centrifuged at 12000rpm for 10 minutes to obtain a supernatant. Diluted 10-fold with ddH2O and filtered using a 0.2 μm inorganic filter. Finally, the same HPLC analysis method as delta-tocotrienol, homogentisic acid (HGA) (purity 98% or more, Sigma-Aldrich, China) was used as the label. The geranylgeranyl pyrophosphate assay is performed by detecting geranylgeranyl pyrophosphate alcohol. We added 1ml of hexane to 1ml of the culture solutions of the control group and the experimental group, respectively, and mixed for 10 minutes at 5000rpm using a vortex. After centrifugation at 12000rpm for 10 minutes, the hexane layer was sampled and 1 μ l was injected by a FULI autosampler into FULI9790 GC equipped with a fused silica capillary column (30m × 0.25mm ID, 0.25mm DB-5MS, J & W Scientific, Folsom, CA). Standard GGOH (purity is more than or equal to 95%; Shanghai Aladdin, China) is used as a control.

4. Results

In this study, we first integrated the three essential genes (hpd, hpt and vte1) into the rDNA site of the genome of the starting strain Saccharomyces cerevisiae BY4742 to give strain VE 01. However, no delta-tocotrienol was detected and detection of the precursor showed that geranylgeranyl pyrophosphate (GGPP) was also not detected, only Homogentisate (HGA) was detected. In order to improve the yield of geranylgeranyl pyrophosphate, the thrmg 1 and ggppssa genes are integrated into the genome of the yeast strain VE01 through delta site integration to obtain a recombinant strain VE02, the recombinant strain VE02 is cultured in a seed culture medium to obtain 1.39mg/L of delta-tocotrienol, and in order to further improve the yield of the delta-tocotrienol, the yield of the delta-tocotrienol is improved (3.56mg/L) by optimizing the adjusted fermentation culture medium, and is about 2.6 times higher than that of the previous culture medium. In conclusion, 3.56mg/L of delta-tocotrienol was finally obtained.

Sequence listing

<110> Tianjin university

<120> delta-tocotrienol synthesized recombinant saccharomyces cerevisiae strain, construction method and application

<160> 46

<170> SIPOSequenceListing 1.0

<210> 1

<211> 713

<212> DNA

<213> Saccharomyces cerevisiae

<400> 1

taacatctgc ataataggca tttgcaagaa ttactcgtga gtaaggaaag agtgaggaac 60

tatcgcatac ctgcatttaa agatgccgat ttgggcgcga atcctttatt ttggcttcac 120

cctcatacta ttatcagggc cagaaaaagg aagtgtttcc ctccttcttg aattgatgtt 180

accctcataa agcacgtggc ctcttatcga gaaagaaatt accgtcgctc gtgatttgtt 240

tgcaaaaaga acaaaactga aaaaacccag acacgctcga cttcctgtct tcctattgat 300

tgcagcttcc aatttcgtca cacaacaagg tcctagcgac ggctcacagg ttttgtaaca 360

agcaatcgaa ggttctggaa tggcgggaaa gggtttagta ccacatgcta tgatgcccac 420

tgtgatctcc agagcaaagt tcgttcgatc gtactgttac tctctctctt tcaaacagaa 480

ttgtccgaat cgtgtgacaa caacagcctg ttctcacaca ctcttttctt ctaaccaagg 540

gggtggttta gtttagtaga acctcgtgaa acttacattt acatatatat aaacttgcat 600

aaattggtca atgcaagaaa tacatatttg gtcttttcta attcgtagtt tttcaagttc 660

ttagatgctt tctttttctc ttttttacag atcatcaagg aagtaattat cta 713

<210> 2

<211> 422

<212> DNA

<213> Saccharomyces cerevisiae

<400> 2

cccacacacc atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg 60

actccgcgca tcgccgtacc acttcaaaac acccaagcac agcatactaa attacccctc 120

tttcttcctc tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga 180

ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt 240

tcttgaaaat tttttttttt gatttttttc tctttcgatg acctcccatt gatatttaag 300

ttaataaatg gtcttcaatt tctcaagttt cagtttcatt tttcttgttc tattacaact 360

ttttttactt cttgctcatt agaaagaaag catagcaatc taatctaagt tttaattaca 420

aa 422

<210> 3

<211> 688

<212> DNA

<213> Saccharomyces cerevisiae

<400> 3

ccatatggag gataagttgg gctgagcttc tgatccaatt tattctatcc attagttgct 60

gatatgtccc accagccaac acttgatagt atctactcgc cattcacttc cagcagcgcc 120

agtagggttg ttgagcttag taaaaatgtg cgcaccacaa gcctacatga ctccacgtca 180

catgaaacca caccgtgggg ccttgttgcg ctaggaatag gatatgcgac gaagacgctt 240

ctgcttagta accacaccac attttcaggg ggtcgatctg cttgcttcct ttactgtcac 300

gagcggccca taatcgcgct ttttttttaa aaggcgcgag acagcaaaca ggaagctcgg 360

gtttcaacct tcggagtggt cgcagatctg gagactggat ctttacaata cagtaaggca 420

agccaccatc tgcttcttag gtgcatgcga cggtatccac gtgcagaaca acatagtctg 480

aagaaggggg ggaggagcat gttcattctc tgtagcagta agagcttggt gataatgacc 540

aaaactggag tctcgaaatc atataaatag acaatatatt ttcacacaat gagatttgta 600

gtacagttct attctctctc ttgcataaat aagaaattca tcaagaactt ggtttgatat 660

ttcaccaaca cacacaaaaa acagtact 688

<210> 4

<211> 232

<212> DNA

<213> Saccharomyces cerevisiae

<400> 4

atcaattttt ttcttttctc tttccccatc ctttacgcta aaataatagt ttattttatt 60

ttttgaatat tttttattta tatacgtata tatagactat tatttatctt ttaatgatta 120

ttaagatttt tattaaaaaa aaattcgctc ctcttttaat gcctttatgc agtttttttt 180

tcccattcga tatttctatg ttcgggttca gcgtatttta agtttaataa ct 232

<210> 5

<211> 399

<212> DNA

<213> Saccharomyces cerevisiae

<400> 5

attaatataa ttatataaaa atattatctt cttttcttta tatctagtgt tatgtaaaat 60

aaattgatga ctacggaaag cttttttata ttgtttcttt ttcattctga gccacttaaa 120

tttcgtgaat gttcttgtaa gggacggtag atttacaagt gatacaacaa aaagcaaggc 180

gctttttcta ataaaaagaa gaaaagcatt taacaattga acacctctat atcaacgaag 240

aatattactt tgtctctaaa tccttgtaaa atgtgtacga tctctatatg ggttactcat 300

aagtgtaccg aagactgcat tgaaagttta tgttttttca ctggaggcgt cattttcgcg 360

ttgagaagat gttcttatcc aaatttcaac tgttatata 399

<210> 6

<211> 221

<212> DNA

<213> Saccharomyces cerevisiae

<400> 6

aaaaagcctt cgagcgtccc aaaaccttct caagcaaggt tttcagtata atgttacatg 60

cgtacacgcg tctgtacaga aaaaaaagaa aaatttgaaa tataaataac gttcttaata 120

ctaacataac tataaaaaaa taaataggga cctagacttc aggttgtcta actccttcct 180

tttcggttag agcggatgtg gggggagggc gtgaatgtaa g 221

<210> 7

<211> 2021

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

taacatctgc ataataggca tttgcaagaa ttactcgtga gtaaggaaag agtgaggaac 60

tatcgcatac ctgcatttaa agatgccgat ttgggcgcga atcctttatt ttggcttcac 120

cctcatacta ttatcagggc cagaaaaagg aagtgtttcc ctccttcttg aattgatgtt 180

accctcataa agcacgtggc ctcttatcga gaaagaaatt accgtcgctc gtgatttgtt 240

tgcaaaaaga acaaaactga aaaaacccag acacgctcga cttcctgtct tcctattgat 300

tgcagcttcc aatttcgtca cacaacaagg tcctagcgac ggctcacagg ttttgtaaca 360

agcaatcgaa ggttctggaa tggcgggaaa gggtttagta ccacatgcta tgatgcccac 420

tgtgatctcc agagcaaagt tcgttcgatc gtactgttac tctctctctt tcaaacagaa 480

ttgtccgaat cgtgtgacaa caacagcctg ttctcacaca ctcttttctt ctaaccaagg 540

gggtggttta gtttagtaga acctcgtgaa acttacattt acatatatat aaacttgcat 600

aaattggtca atgcaagaaa tacatatttg gtcttttcta attcgtagtt tttcaagttc 660

ttagatgctt tctttttctc ttttttacag atcatcaagg aagtaattat ctacttttta 720

caacaaatat aaaacaatgg ctgacatctt cgaaaaccca atgggtttga tgggtttcga 780

atttatcgaa ttggcttctc caactccagg tgttttggaa ccagtttttc aaatattggg 840

cttcactaag gttgcgactc acagatctaa ggacgttcac ttgtaccgtc aaggtggtat 900

caacttgatc ttgaacaacg aaccaaagtc tatcgcttct tacttcgctg ctgaacacgg 960

tccatctgtt tgtggtatgg ctttcagagt tagaaacgct cacgaagcgt acgctagagc 1020

tttggaattg ggtgctcaac cagttgaaat cgaaactggt ccaatggaat tgagattgcc 1080

agctatcaag ggtatcggtg gtgctccatt gtacttgatc gacagattcg aagaaggttc 1140

ttctatctac gacatcgact tcaacttcat cgaaggtgtt gacagaaacc cagttggcgc 1200

gggtctcaag atcatcgacc acttgactca caacgtttac agaggtagaa tggcttactg 1260

ggctggtttc tacgaaaagt tgttcaactt cagagaaatc agatacttcg acatcaaggg 1320

tgaatacact ggtttgactt ctaaggctat gactgctcca gacggtatga tcagaatccc 1380

attgaacgaa gaatcttcta agggtgctgg tcaaatcgaa gaatttttga tgcaattcaa 1440

cggtgaaggt atccaacacg ttgctttctt gactgacgac ttgttgaaga cttgggacgc 1500

tttgaagggt ttgggtatga gattcatgac tgctccacca caaacttact acgaaatgtt 1560

ggaagaaaga ttgccaggtc acggtgaacc agttgaccaa ttgcaagcta gaggtatctt 1620

gttggacggt gcttctcaac caggtgacaa gagattgttg ttgcaaatct tctctgaaac 1680

tttgttgggt ccagttttct tcgaatttat ccaaagaaag ggtgacgacg gtttcggtga 1740

aggtaaattg aattgaattg aaatcgatag atcaattttt ttcttttctc tttccccatc 1800

ctttacgcta aaataatagt ttattttatt ttttgaatat tttttattta tatacgtata 1860

tatagactat tatttatctt ttaatgatta ttaagatttt tattaaaaaa aaattcgctc 1920

ctcttttaat gcctttatgc agtttttttt tcccattcga tatttctatg ttcgggttca 1980

gcgtatttta agtttaataa ctcgaaaatt ctgcgttcgt t 2021

<210> 8

<211> 540

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggatcgtt ggtttgtgtg gtgttgcttc tttggctatc gcttggggtt tgggtttgtg 60

gttgggtttg actgttggta tctctttgat catcggtact gcttactctg ttccaccagt 120

tagattgaag agattctctt tgttggctgc tttgtgtatc ttgactgtta gaggtatcgt 180

tgttaacttg ggtttgttct tgttcttcag aatcggtttg ggttacccac caactttgat 240

cactccaatc tgggttttga ctttgttcat cttggttttc actgttgcta tcgctatctt 300

caaggacgtt ccagacatgg aaggtgacag acaattcaag atccaaactt tgactttgca 360

aatcggtaag caaaacgttt tcagaggtac tttgatcttg ttgactggtt gttacttggc 420

tatggctatc tggggtttgt gggctgctat gccattgaac actgctttct tgatcgtttc 480

tcacttgtgt ttgttggctt tgttgtggtg gagatctcgt gacgttcact tggaatctaa 540

<210> 9

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgtacatgg aaatccgttc gttgatcgta agcatgaacc caaacttgtc ttctttcgaa 60

ttgtctcgtc cagtttctcc attgactaga tctttggttc cattcagatc tactaagttg 120

gttccaagat ctatctctcg tgtttctgct tctatctcta ctccaaactc tgaaactgac 180

aagatctctg ttaagccagt ttacgttcca acttctccaa acagagaatt gagaactcca 240

cactctggtt accacttcga cggtactcca agaaagttct tcgaaggttg gtacttcaga 300

gtttctatcc cagaaaagcg tgagtctttc tgcttcatgt actcggttga aaacccagct 360

ttcagacaat ctttgtctcc attggaagtt gctttgtacg gtccaagatt cactggtgtt 420

ggtgctcaaa tcttgggtgc taacgacaag tacttgtgtc aatacgaaca agactctcac 480

aacttctggg gtgacagaca cgaattggtt ttgggtaaca ctttctctgc tgttccaggt 540

gctaaggctc caaacaagga agttccacca gaagaattta acagaagagt ttcggaaggc 600

ttccaagcta ctccattctg gcaccaaggt cacatctgtg acgacggtag aactgactac 660

gctgaaactg ttaagtctgc tagatgggaa tactctacta gaccagttta cggttggggt 720

gacgttggtg ctaagcaaaa gtctactgcg ggttggccag cggcgttccc agttttcgag 780

ccacactggc aaatctgtat ggcgggcggc ttgtctactg gttggatcga atggggtggt 840

gaaagattcg aatttagaga cgctccatct tactctgaaa agaactgggg tggtggtttc 900

ccaagaaagt ggttctgggt tcaatgtaac gttttcgaag gtgctactgg tgaagttgct 960

ttgactgctg gtggtggttt gagacaattg ccaggtttga ctgaaactta cgaaaacgct 1020

gctttggttt gtgttcacta cgacggtaag atgtacgaat ttgttccatg gaacggtgtt 1080

gttagatggg aaatgtctcc atggggttac tggtacatca ctgctgaaaa cgaaaaccac 1140

gttgttgaat tggaagctag aactaacgaa gctggtactc cattgagagc gccaactact 1200

gaggtaggtc tcgctactgc ttgcagagac tcttgttacg gtgaattgaa gttgcaaatc 1260

tgggaaagat tgtacgacgg ttctaagggt aaggttatct tggaaactaa gtcttctatg 1320

gctgctgttg aaatcggtgg tggtccatgg ttcggtactt ggaagggtga cacttctaac 1380

actccagaat tgttgaagca agctctccaa gttccattgg acttggagtc ggctctcggt 1440

ttggttccat tcttcaagcc accaggtttg taa 1473

<210> 10

<211> 1578

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggaccaat tggttaagac tgaagttact aagaagtctt tcactgctcc agttcaaaag 60

gcttctactc cagttttgac taacaagact gttatctctg gttctaaggt taagtctttg 120

tcttctgctc aatcttcttc ttctggtcca tcttcttctt ctgaagaaga cgactctcgt 180

gacatcgaat ctttggacaa gaagatcaga ccattggaag aattggaagc tctcttgtct 240

tctggtaaca ctaagcaatt gaagaacaag gaagttgctg ctttggttat ccacggtaag 300

ttgccattgt acgctttgga aaagaagttg ggtgacacta ctagagctgt tgctgttaga 360

agaaaggctt tgtctatctt ggctgaagct ccagttttgg cttctgacag attgccatac 420

aagaactacg actacgacag agttttcggt gcttgttgtg aaaacgttat cggttacatg 480

ccattgccag ttggtgttat cggtccattg gttatcgacg gtacttctta ccacatccca 540

atggctacta ctgaaggttg tttggttgct tctgctatga gaggttgtaa ggctatcaac 600

gctggtggtg gcgctactac tgtgctcact aaggacggta tgactagagg tccagttgtt 660

agattcccaa ctttgaagag atctggtgct tgtaagatct ggttggactc tgaagaaggt 720

caaaacgcta tcaagaaggc tttcaactct acttctcgtt tcgctagatt gcaacacatc 780

caaacttgtt tggctggtga cttgttgttc atgagattca gaactactac tggtgacgct 840

atgggtatga acatgatctc taagggtgtt gagtacagct tgaagcaaat ggttgaagaa 900

tacggttggg aagacatgga agttgtttct gtttctggta actactgtac tgacaagaag 960

ccagctgcta tcaactggat cgaaggtaga ggtaagtctg ttgttgctga agctactatc 1020

ccaggtgacg ttgttagaaa ggttttgaag tctgacgttt ctgctttggt tgaattgaac 1080

atcgctaaga acttggttgg ttctgctatg gctggttctg ttggtggttt caacgctcac 1140

gctgctaact tggttactgc tgttttcttg gctttgggtc aagacccagc tcaaaacgtt 1200

gaatcttcta actgtatcac tttgatgaag gaagttgacg gtgacttgag aatctctgtt 1260

tctatgccat ctatcgaagt tggtactatc ggtggtggca ctgtcctcga accacaaggt 1320

gctatgttgg acttgttggg tgttagaggt ccacacgcta ctgctccagg tactaacgct 1380

agacaattgg ctagaatcgt tgcttgtgct gttttggctg gtgaattgtc tttgtgtgct 1440

gctttggctg ctggtcactt ggttcaatct cacatgactc acaacagaaa gccagctgaa 1500

ccaactaagc caaacaactt ggacgctact gacatcaaca gattgaagga cggttctgtt 1560

acttgtatca agtcttaa 1578

<210> 11

<211> 993

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgtcttact tcgacaacta cttcaacgaa atcgttaact ctgttaacga catcatcaag 60

tcttacatct ctggtgacgt tccaaagttg tacgaagcta gttaccactt gttcacttct 120

ggtggtaaga gattgagacc attgatcttg actatctctt ctgacttgtt cggtggtcaa 180

agagaaagag cttactacgc tggtgctgct atcgaagttt tgcacacttt cactttggtt 240

cacgacgaca tcatggacca agacaacatc agaagaggtt tgccaactgt tcacgttaag 300

tacggtttgc cattggctat cttggctggt gacttgttgc acgctaaggc tttccaattg 360

ttgactcaag ctctccgtgg tctcccatct gaaactatca tcaaggcttt cgacatcttc 420

actagatcta tcatcatcat ctctgaaggt caagctgttg acatggaatt tgaagacaga 480

atcgacatca aggaacaaga atacttggac atgatctctc gtaagactgc tgctttgttc 540

tctgcttctt cttctatcgg tgctttgatc gctggtgcta acgacaacga cgttagattg 600

atgtctgact tcggtactaa cttgggtatc gctttccaaa tcgttgacga catcttgggt 660

ttgactgctg acgaaaagga attgggtaag ccagttttct ctgacatcag agaaggtaag 720

aagactatcc tcgttatcaa gactctcgaa ttgtgtaagg aagacgaaaa gaagatcgtt 780

ttgaaggctt tgggtaacaa gtctgcttct aaggaagaat tgatgtcttc tgctgacatc 840

atcaagaagt actctttgga ctacgcttac aacttggctg aaaagtacta caagaacgct 900

atcgactctt tgaaccaagt ttcttctaag tctgacatcc caggtaaggc tttgaagtac 960

ttggctgaat ttactatcag aagaagaaag taa 993

<210> 12

<211> 471

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atcttttata tttaaatctt atctattagt taattttttg taatttatcc ttatatatag 60

tctggttatt ctaaaatatc atttcagtat ctaaaaattc ccctcttttt tcagttatat 120

cttaacaggc gacagtccaa atgttgattt atcccagtcc gattcatcag ggttgtgaag 180

cattttgtca atggtcgaaa tcacatcagt aatagtgcct cttacttgcc tcatagaatt 240

tctttctctt aacgtcaccg tttggtcttt tatagtttcg aaatctatgg tgataccaaa 300

tggtgttccc aattcatcgt tacgggcgta ttttttacca attgaagtat tggaatcgtc 360

aattttaaag tatatctctc ttttacgtaa agcctgcgag atcctcttaa gtatagcggg 420

gaagccatcg ttattcgata ttgtcgtaac aaatactttg atcggcgcta t 471

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tcgctttacc tcataaaact g 21

<210> 14

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaaagccttc gagcgtccca aacttgaaat tgctggcctt 40

<210> 15

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ccaaatttca actgttatat aactccaaag agtatcactc acta 44

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agaccttaac ctactaaata gt 22

<210> 17

<211> 1188

<212> DNA

<213> Saccharomyces cerevisiae

<400> 17

tcgctttacc tcataaaact gatacgagct tctgctatcc tgagggaaac ttcggcagga 60

accagctact agatggttcg attagtcttt cgcccctata cccaaattcg acgatcgatt 120

tgcacgtcag aaccgctacg agcctccacc agagtttcct ctggcttcac cctattcagg 180

catagttcac catctttcgg gtcccaacag ctatgctctt actcaaatcc atccgaagac 240

atcaggatcg gtcgattgtg cacctcttgc gaggccccaa cctacgttca ctttcattac 300

gcgtatgggt tttacaccca aacactcgca tagacgttag actccttggt ccgtgtttca 360

agacgggcgg catataacca ttatgccagc atccttgact tacgtcgcag tcctcagtcc 420

cagctggcag tattcccaca ggctataata cttaccgagg caagctacat tcctatggat 480

ttatcctgcc accaaaactg atgctggccc agtgaaatgc gagattcccc tacccacaag 540

gagcagaggg cacaaaacac catgtctgat caaatgccct tccctttcaa caatttcacg 600

tactttttca ctctcttttc aaagttcttt tcatctttcc atcactgtac ttgttcgcta 660

tcggtctctc gccaatattt agctttagat ggaatttacc acccacttag agctgcattc 720

ccaaacaact cgactcttcg aaggcacttt acaaagaacc gcactcctcg ccacacggga 780

ttctcaccct ctatgacgtc ctgttccaag gaacatagac aaggaacggc cccaaagttg 840

ccctctccaa attacaactc gggcaccgaa ggtaccagat ttcaaatttg agcttttgcc 900

gcttcactcg ccgttactaa ggcaatcccg gttggtttct tttcctccgc ttattgatat 960

gcttaagttc agcgggtact cctacctgat ttgaggtcaa actttaagaa cattgttcgc 1020

ctagacgctc tcttcttatc gataacgttc caatacgctc agtataaaaa aagattagcc 1080

gcagttggta aaacctaaaa cgaccgtact tgcattatac ctcaagcacg cagagaaacc 1140

tctctttgga aaaaaaacat ccaatgaaaa ggccagcaat ttcaagtt 1188

<210> 18

<211> 1036

<212> DNA

<213> Saccharomyces cerevisiae

<400> 18

actccaaaga gtatcactca ctaccaaaca gaatgtttga gaaggaaatg acgctcaaac 60

aggcatgccc cctggaatac caaggggcgc aatgtgcgtt caaagattcg atgattcacg 120

gaattctgca attcacatta cgtatcgcat ttcgctgcgt tcttcatcga tgcgagaacc 180

aagagatccg ttgttgaaag tttttaatat tttaaaattt ccagttacga aaattcttgt 240

ttttgacaaa aatttaatga atagataaaa ttgtttgtgt ttgttacctc tgggccccga 300

ttgctcgaat gcccaaagaa aaagttgcaa agatatgaaa actccacagt gtgttgtatt 360

gaaacggttt taattgtcct ataacaaaag cacagaaatc tctcaccgtt tggaatagca 420

agaaagaaac ttacaagcct agcaagaccg cgcacttaag cgcaggcccg gctggactct 480

ccatctcttg tcttcttgcc cagtaaaagc tctcatgctc ttgccaaaac aaaaaaatcc 540

attttcaaaa ttattaaatt tctttaatga tccttccgca ggttcaccta cggaaacctt 600

gttacgactt ttagttcctc taaatgacca agtttgtcca aattctccgc tctgagatgg 660

agttgccccc ttctctaagc agatcctgag gcctcactaa gccattcaat cggtactagc 720

gacgggcggt gtgtacaaag ggcagggacg taatcaacgc aagctgatga cttgcgctta 780

ctaggaattc ctcgttgaag agcaataatt acaatgctct atccccagca cgacggagtt 840

tcacaagatt accaagacct ctcggccaag gttagactcg ctggctccgt cagtgtagcg 900

cgcgtgcggc ccagaacgtc taagggcatc acagacctgt tattgcctca aacttccatc 960

ggcttgaaac cgatagtccc tctaagaagt ggataaccag caaatgctag caccactatt 1020

tagtaggtta aggtct 1036

<210> 19

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cttcaagcca ccaggtttgt aaaaaaagcc ttcgagcgtc c 41

<210> 20

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

actatttagt aggttaaggt ctacatgtaa ttagttatgt cac 43

<210> 21

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tatcagggcg atggcccact actaacatct gcataatagg catt 44

<210> 22

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cgaacggatt tccatgtaca tgtgttttat atttgttgta a 41

<210> 23

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gtattttaag tttaataact cccacacacc atagcttcaa aatg 44

<210> 24

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gaacgcttgg atagtagcca ttttgtaatt aaaacttaga ttag 44

<210> 25

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cttctctaac actatcttct aatttttcta ataaaaagaa gaa 43

<210> 26

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gtgagtgata ctctttggag ttatataaca gttgaaattt g 41

<210> 27

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ttacaacaaa tataaaacac ttcaattcat catttttttt ttattc 46

<210> 28

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gcctattatg cagatgttag tagtgggcca tcgccctgat aga 43

<210> 29

<211> 1375

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ttcaattcat catttttttt ttattctttt ttttgatttc ggtttctttg aaattttttt 60

gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg agcacagact tagattggta 120

tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg 180

cacagaacaa aaacctgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa 240

cgtgctgcta ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag 300

caaacaaact tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt 360

gaagcattag gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt 420

tccatggagg gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc 480

ttcgaagaca gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt 540

gtatacagaa tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt 600

attgttagcg gtttgaagca ggcggcagaa gaagtaacaa aggaacctag aggccttttg 660

atgttagcag aattgtcatg caagggctcc ctatctactg gagaatatac taagggtact 720

gttgacattg cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg 780

ggtggaagag atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac 840

aagggagacg cattgggtca acagtataga accgtggatg atgtggtctc tacaggatct 900

gacattatta ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt 960

gaacgttaca gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa 1020

aaaactgtat tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa 1080

ttatatcagt tattacccta tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 1140

ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 1200

atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 1260

tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 1320

gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actac 1375

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

tgttggaata gaaatcaact atc 23

<210> 31

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gtattcccac agttaagctt atgtttatat tcattgatcc tat 43

<210> 32

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ccccacacac catagcttca aataaaatga tgataataat attta 45

<210> 33

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ccatgagaaa tgggtgaatg ttgag 25

<210> 34

<211> 167

<212> DNA

<213> Saccharomyces cerevisiae

<400> 34

tgttggaata gaaatcaact atcatctact aactagtatt tacattacta gtatattatc 60

atatacggtg ttagaagatg acgcaaatga tgagaaatag tcatctaaat tagtggaagc 120

tgaaacgcaa ggattgataa tgtaatagga tcaatgaata taaacat 167

<210> 35

<211> 170

<212> DNA

<213> Saccharomyces cerevisiae

<400> 35

ataaaatgat gataataata tttatagaat tgtgtagaat tgcagattcc cttttatgga 60

ttcctaaatc cttgaggaga acttctagta tattctgtat acctaatatt atagccttta 120

tcaacaatgg aatcccaaca attatctcaa cattcaccca tttctcatgg 170

<210> 36

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gtgaatgctg gtcgctatac tgtaacatct gcataatagg c 41

<210> 37

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gtcttaacca attggtccat gtgttttata tttgttgta 39

<210> 38

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gttacttgta tcaagtctta atttttttct tttctctttc cccattat 48

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aaacaacgaa cgcagaattt 20

<210> 40

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

aaattctgcg ttcgttgttt tgtatatgct catttacact c 41

<210> 41

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

gtagttgtcg aagtaagaca tttttgtgtg taaatttagt gaag 44

<210> 42

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ctatcagaag aagaaagtaa ggagattgat aagacttttc ta 42

<210> 43

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

aaatattatt atcatcattt tatatagcgc cgatcaaagt atttg 45

<210> 44

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

taggatcaat gaatataaac ataagcttaa ctgtgggaat ac 42

<210> 45

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gcctattatg cagatgttac agtatagcga ccagcattca cata 44

<210> 46

<211> 1724

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

aagcttaact gtgggaatac tcaggtatcg taagatgcaa gagttcgaat ctcttagcaa 60

ccattatttt tttcctcaac ataacgagaa cacacagggg cgctatcgca cagaatcaaa 120

ttcgatgact ggaaattttt tgttaatttc agaggtcgcc tgacgcatat acctttttca 180

actgaaaaat tgggagaaaa aggaaaggtg agaggccgga accggctttt catatagaat 240

agagaagcgt tcatgactaa atgcttgcat cacaatactt gaagttgaca atattattta 300

aggacctatt gttttttcca ataggtggtt agcaatcgtc ttactttcta acttttctta 360

ccttttacat ttcagcaata tatatatata tttcaaggat ataccattct aatgtctgcc 420

cctatgtctg cccctaagaa gatcgtcgtt ttgccaggtg accacgttgg tcaagaaatc 480

acagccgaag ccattaaggt tcttaaagct atttctgatg ttcgttccaa tgtcaagttc 540

gatttcgaaa atcatttaat tggtggtgct gctatcgatg ctacaggtgt cccacttcca 600

gatgaggcgc tggaagcctc caagaaggtt gatgccgttt tgttaggtgc tgtgggtggt 660

cctaaatggg gtaccggtag tgttagacct gaacaaggtt tactaaaaat ccgtaaagaa 720

cttcaattgt acgccaactt aagaccatgt aactttgcat ccgactctct tttagactta 780

tctccaatca agccacaatt tgctaaaggt actgacttcg ttgttgtcag agaattagtg 840

ggaggtattt actttggtaa gagaaaggaa gacgatggtg atggtgtcgc ttgggatagt 900

gaacaataca ccgttccaga agtgcaaaga atcacaagaa tggccgcttt catggcccta 960

caacatgagc caccattgcc tatttggtcc ttggataaag ctaatgtttt ggcctcttca 1020

agattatgga gaaaaactgt ggaggaaacc atcaagaacg aattccctac attgaaggtt 1080

caacatcaat tgattgattc tgccgccatg atcctagtta agaacccaac ccacctaaat 1140

ggtattataa tcaccagcaa catgtttggt gatatcatct ccgatgaagc ctccgttatc 1200

ccaggttcct tgggtttgtt gccatctgcg tccttggcct ctttgccaga caagaacacc 1260

gcatttggtt tgtacgaacc atgccacggt tctgctccag atttgccaaa gaataaggtt 1320

gaccctatcg ccactatctt gtctgctgca atgatgttga aattgtcatt gaacttgcct 1380

gaagaaggta aggccattga agatgcagtt aaaaaggttt tggatgcagg tatcagaact 1440

ggtgatttag gtggttccaa cagtaccacc gaagtcggtg atgctgtcgc cgaagaagtt 1500

aagaaaatcc ttgcttaata atcagtactg acaataaaaa gattcttgtt ttcaagaact 1560

tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga 1620

tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag 1680

taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actg 1724

Claims (3)

1. The construction method of the recombinant saccharomyces cerevisiae strain for synthesizing the delta-tocotrienol is characterized by comprising the following steps of: integrating exogenous genes hpd, hpt and vte1 into a genome rDNA locus of a saccharomyces cerevisiae BY4742 strain to obtain a recombinant strain VE 01; integrating a thmg1 gene and a ggppssa gene at a delta site of a recombinant strain VE01 genome to obtain a recombinant strain VE 02; the nucleotide sequence of the hpd gene is shown as SEQ ID NO. 7; the nucleotide sequence of the hpt gene is shown as SEQ ID NO. 8; the nucleotide sequence of the vte1 gene is shown as SEQ ID NO. 9; the nucleotide sequence of the thmg1 gene is shown in SEQ ID NO. 10; the nucleotide sequence of the ggppssa gene is shown as SEQ ID NO. 11.

2. A synthetic delta-tocotrienol recombinant saccharomyces cerevisiae strain constructed by the method of claim 1.

3. Use of a recombinant strain of saccharomyces cerevisiae for the synthesis of δ -tocotrienols according to claim 2 for the production of δ -tocotrienols characterized by the steps of: culturing and fermenting the recombinant delta-tocotrienol-synthesizing saccharomyces cerevisiae strain VE02 of claim 2 in a fermentation medium to obtain delta-tocotrienol, wherein the formula of the fermentation medium is 35.66g/L glucose, 24.46g/L peptone, 14.01g/L yeast extract powder and the balance water.

Images