CN113699053B - Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof - Google Patents

Abstract

The invention discloses a recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof. The recombinant saccharomyces cerevisiae disclosed by the invention is used for expressing a beta-carotene ketolase gene CrtW in a constitutive mode, expressing a beta-carotene hydroxylase gene CrtZ in an inducible mode, and synthesizing an intermediate product beta-carotene into astaxanthin. The invention uses the constitutive promoter to regulate the CrtW gene to express firstly, uses the inducible promoter to regulate the CrtZ gene to express later, obviously improves the metabolic flux of the synthesizing path of the saccharomyces cerevisiae astaxanthin, ensures that the astaxanthin yield reaches 464.90mg/L, and has good industrial development prospect.

Description

Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof

Technical Field

The invention belongs to the field of microorganisms, and relates to recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof.

Background

Astaxanthin (3, 3' -dihydroxy-beta, beta ' -carotene-4,4' -dione, C) ₄₀ H ₅₂ O ₄ 596.84) has lipophilicity and hydrophilicity, is one of the most antioxidative antioxidants found in nature so far, and has an antioxidative capacity 800 times that of coenzyme Q10 and 700 times that of anthocyaninMultiple, 550 times vitamin E. Astaxanthin is divided into (3R, 3 'R), (3S, 3' S) and (3R, 3 'S) 3 different configurations according to the difference of two chiral carbons, wherein the (3S, 3' S) configuration has the strongest antioxidant capacity. Astaxanthin has a plurality of conjugated double bonds, can remove free radicals inside and outside cell membranes, reduce oxidation of proteins and lipids and damage of DNA, protect cells and delay aging, is the only carotenoid antioxidant capable of passing through blood brain barriers, has positive treatment and prevention effects on cardiovascular and cerebrovascular diseases, diabetes mellitus, cancers and the like, is known as 'health and soft gold' by European scientists and medical scientists, and has great market value in the fields of medicine, health care products, food, aquaculture and the like. Global astaxanthin production was about 511.8 tons in 2017, with total production values of about dollars 1 million, and proliferated to dollars 1.5 million in 2018. The global astaxanthin production value of 2024 is estimated to be close to 3.4 hundred million dollars, and the astaxanthin production value of China is estimated to be close to 2 hundred million dollars, and the wide market prospect is also pushing the astaxanthin biosynthesis technology to continuously develop.

At present, the species approved for the biosynthesis of astaxanthin are mainly phaffia rhodozyma and rhodococcus pluvialis. The rhodotorula produces astaxanthin with (3R, 3' R) configuration, and the astaxanthin has low antioxidant capacity and poor biological activity and is mainly used as a feed additive. The haematococcus pluvialis can accumulate astaxanthin with a relatively high concentration and a configuration of (3S, 3' S) in vivo, can be used in human foods and cosmetics, but has strict culture conditions, needs to be cultured under the condition of proper illumination throughout the year, has a relatively long period, and therefore, the production economic cost is high for a long time. With the development of synthetic biology in recent years, researchers have turned their eyes to other engineering bacteria with more mature genetic background. Saccharomyces cerevisiae is a recognized safety model microorganism, has clear genetic background and rich molecular operation tools, and can realize high-density fermentation and synthesis of high-quality (3S, 3' S) configuration astaxanthin, so that the realization of high yield of astaxanthin in Saccharomyces cerevisiae has great competitiveness in industrial production. However, a large amount of intermediates can be accumulated in the two-step reticulate metabolic pathway from beta-carotene to astaxanthin synthesis at present, so that the yield of astaxanthin is limited, and therefore, the metabolic flux of the downstream reticulate metabolic pathway is dredged, the accumulation of the intermediates is reduced, and the method has great significance in improving the synthesis of astaxanthin in saccharomyces cerevisiae.

Disclosure of Invention

The invention aims to solve the technical problem of improving the synthesis of astaxanthin in saccharomyces cerevisiae.

In order to solve the technical problems, the invention provides a recombinant saccharomyces cerevisiae which constitutively expresses a beta-carotene ketolase gene CrtW and inducible expresses a beta-carotene hydroxylase gene CrtZ, and an intermediate product beta-carotene is synthesized into astaxanthin.

In some embodiments, in the recombinant Saccharomyces cerevisiae described above, the constitutive promoter used for constitutive expression of the beta-carotene ketolase gene CrtW is TDH3p, and the sequence of TDH3p is shown in SEQ ID NO 9.

In some embodiments, in the recombinant s.cerevisiae described in any of the above, the inducible promoter used for inducible expression of the beta-carotene hydroxylase gene CrtZ is Gal1, and the sequence of Gal1 is shown in SEQ ID No. 6.

In some embodiments, in any of the above recombinant s.cerevisiae, the beta-carotene ketolase gene CrtW is BDC263CrtW derived from Brevibacterium vesiculosum (Brevundimonas vesicularis DC 263), and the sequence of BDC263CrtW is shown in positions 9-734 of SEQ ID NO: 28.

In some embodiments, in the recombinant Saccharomyces cerevisiae of any of the above, the beta-carotene hydroxylase gene CrtZ is AaCrtZ from orange Huang Nong bacillus (Agrobacterium aurantiacum), and the sequence of AaCrtZ is shown in positions 9-497 of SEQ ID NO. 25.

In order to solve the technical problems, the invention also provides a recombinant expression vector of saccharomyces cerevisiae, which comprises a constitutive promoter and a beta-carotene ketolase gene CrtW under the control of the constitutive promoter, and an inducible promoter and a beta-carotene hydroxylase gene CrtZ under the control of the inducible promoter.

In some embodiments, in the recombinant expression vector, the constitutive promoter is TDH3p, and the sequence of TDH3p is shown in SEQ ID NO. 9.

In some embodiments, in any of the above recombinant expression vectors, the beta-carotene ketolase gene CrtW has a terminator downstream, preferably TDH2t, the sequence of TDH2t is shown in SEQ ID No. 12.

In some embodiments, in the recombinant expression vector of any one of the above, the inducible promoter is Gal1, and the sequence of Gal1 is shown in SEQ ID NO. 6.

In some embodiments, in any of the above recombinant expression vectors, the beta-carotene hydroxylase gene CrtZ has a terminator downstream, preferably ADH1t, the sequence of ADH1t being shown in SEQ ID No. 3.

In some embodiments, in any of the above recombinant expression vectors, the beta-carotene ketolase gene CrtW is BDC263CrtW derived from shortwave monad vesicular (Brevundimonas vesicularis DC 263), and the sequence of BDC263CrtW is shown in positions 9-734 of SEQ ID No. 28.

In some embodiments, in any of the above recombinant expression vectors, the beta-carotene hydroxylase gene CrtZ is AaCrtZ derived from bacillus orange Huang Nong (Agrobacterium aurantiacum), and the sequence of AaCrtZ is shown in positions 9-497 in SEQ ID No. 25.

In some embodiments, any one of the above recombinant expression vectors is PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t, the sequence of which is shown in SEQ ID NO: 35.

In order to solve the technical problems, the invention also provides a method for constructing any one of the recombinant saccharomyces cerevisiae, which comprises the step of transferring any one of the recombinant expression vectors into the original saccharomyces cerevisiae to obtain the recombinant saccharomyces cerevisiae;

the Saccharomyces cerevisiae is Saccharomyces cerevisiae capable of synthesizing beta-carotene, and is preferably Saccharomyces cerevisiae Scy10026 (Saccharomyces cerevisiae).

In order to solve the technical problems, the invention also provides a method for producing astaxanthin, which comprises the steps of fermenting and culturing any recombinant saccharomyces cerevisiae to make beta-carotene ketolase gene CrtW be expressed in a constitutive mode and adding D- (+) -galactose to induce beta-carotene hydroxylase gene CrtZ to be expressed in the fermenting and culturing process so as to synthesize astaxanthin.

In some embodiments, the above method of producing astaxanthin, the fermentation culture comprises subjecting the recombinant Saccharomyces cerevisiae described in any of the above to fermentation culture in an initial fermentation medium, at, for example, an initial OD ₆₀₀ Fermenting at 30deg.C, pH5.8-6.2, aeration rate of 3.0vvm, dissolved oxygen of 30% and 300-500rpm for 48 hr, and adding D- (+) -galactose with final concentration of 20g/L for inducing beta-carotene hydroxylase gene CrtZ expression;

before adding D- (+) -galactose, feeding glucose in a feeding way and controlling the feeding speed within a range of 0-1g/L so as to reduce the generation of byproducts as much as possible, feeding the D- (+) -galactose, stopping feeding the glucose in the feeding way, and entering an astaxanthin synthesis stage, wherein a carbon source in a fermentation tank is switched to ethanol, and the carbon source is fed in a feeding way, feeding the ethanol in a feeding way and controlling the feeding speed within a range of 0.5-10g/L, and controlling the temperature within a range of 20-30 ℃;

adding nitrogen source by intermittent feeding with decreasing concentration gradient, adding a certain volume of yeast extract mother liquor into the fermentation tank at intervals, for example, adding 1.5 times of yeast extract every 12 hours (12 h,24h,36h,48h,60 h) from the beginning of fermentation, adding 1.0 times of yeast extract every 12 hours (72 h, 84 h) of fermentation, adding 2 times of yeast extract every 12 hours (96 h, 108 h) of fermentation, and adding 0.5 times of yeast extract every 12 hours (96 h, 108 h) of fermentation.

In some embodiments, in the method of producing astaxanthin of any of the above, the initial fermentation medium is YPD with 3% glucose (10 g/L yeast extract, 20g/L peptone, 30g/L glucose, balance water).

In some embodiments, the method for producing astaxanthin according to any one of the above further comprises, prior to the fermentation culture, a step of subjecting any one of the recombinant s.cerevisiae to a primary seed culture and a secondary seed culture;

the primary seed culture comprises culturing any one of the aboveThe recombinant Saccharomyces cerevisiae is inoculated into a shake flask containing SC-Ura liquid culture medium, and cultured to OD under the conditions of 30 ℃ and 250rpm, for example ₆₀₀ A step of =6-7;

the secondary seed culture comprises transferring the primary seed obtained by primary seed culture into shake flask containing SC-Ura liquid culture medium, and culturing at 30deg.C and 250rpm to OD ₆₀₀ A step of =5-6;

the seed may be inoculated in an amount of 5%.

In order to solve the technical problems, the invention also provides the application of any one of the recombinant saccharomyces cerevisiae and/or any one of the recombinant expression vectors in astaxanthin production.

The recombinant yeast strain for producing astaxanthin disclosed by the invention regulates and controls the expression of beta-carotene ketolase gene CrtW and beta-carotene hydroxylase gene CrtZ on an astaxanthin synthesis path through the combination of constitutive promoters and inducible promoters, the promoters and gene elements with different combinations are cloned on a plasmid expression vector, transformed into Saccharomyces cerevisiae, and the recombinant yeast strain is subjected to fermentation, detection of astaxanthin yield and screening to obtain the high-yield target strain.

The beneficial effects of the invention are as follows: the constitutive promoter is used for regulating and controlling the expression of the CrtW gene, and the inducible promoter is used for regulating and controlling the expression of the Crt Z gene, so that the metabolic flux of the astaxanthin synthesis path of the saccharomyces cerevisiae is obviously improved, the yield and purity of the astaxanthin in the saccharomyces cerevisiae are improved, the yield of the astaxanthin reaches 464.90mg/L, and the method has good industrial development prospect.

Drawings

FIG. 1 is a schematic diagram of two recombinant expression vectors constructed in example 2.

FIG. 2 shows the results of the detection of astaxanthin production by shake flask fermentation in example 4.

FIG. 3 shows the results of fermentation test of the 7L fermenter of recombinant Saccharomyces cerevisiae S1 of example 6.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Saccharomyces cerevisiae S228C (Saccharomyces cerevisiae S228C) is described in the literature "Fisk et al, saccharomyces cerevisiae S288C genome annotation: a working hypothesis. Yeast.2006;23 (12) 857-865", available to the public from Wanhua chemical groups Co., ltd.

The PRS416 carrier is Shanghai-associated biological engineering Co., ltd, and the product number is LM4777.

D- (+) -galactose is an Ala-dine product, with the product number G100367.

AaCrtZ in the examples is CrtZ from orange Huang Nong bacillus (Agrobacterium aurantiacum) and BDC263CrtW is CrtW from Brevibacterium vesiculosus (Brevundimonas vesicularis DC 263).

Example 1: construction of recombinant plasmid PRS416-ADH1t-Gal1-TDH3p-TDH2t

1. PCR amplification is carried out by taking the genome DNA of Saccharomyces cerevisiae S228C (Saccharomyces cerevisiae S228C) as a template and ADH1t_F and ADH1t_R as primers to obtain ADH1t, wherein the sequence of the ADH1t is shown as SEQ ID NO. 3.

ADH1t_F：5’-agctttggacttcttcgccagagg-3’(SEQ ID NO:1)；

ADH1t_R：5’-catgccggtagaggtgtggtcaataag-3’(SEQ ID NO:2)。

2. And performing PCR amplification by taking genomic DNA of Saccharomyces cerevisiae S228C as a template and Gal1_F and Gal1_R as primers to obtain Gal1, wherein the sequence of the Gal1 is shown as SEQ ID NO. 6.

Gal1_F：5’-tatagttttttctccttgacgttaaagtatag-3’(SEQ ID NO:4)；

Gal1_R：5’-ttatattgaattttcaaaaattcttactttttttttggatgg-3’(SEQ ID NO:5)。

3. And performing PCR amplification by taking genomic DNA of saccharomyces cerevisiae S228C as a template and TDH23p_F and TDH23p_R as primers to obtain TDH3p, wherein the sequence of the TDH3p is shown as SEQ ID NO. 9.

TDH3p_F：5’-cagttcgagtttatcattatcaatactgcca-3’(SEQ ID NO:7)；

TDH3p_R：5’-tttgtttgtttatgtgtgtttattcgaaac-3’(SEQ ID NO:8)。

4. And performing PCR amplification by taking genomic DNA of saccharomyces cerevisiae S228C as a template and TDH2t_F and TDH2t_R as primers to obtain TDH2t, wherein the sequence of the TDH2t is shown as SEQ ID NO. 12.

TDH2t_F：5’-atttaactccttaagttactttaatgatttag-3’(SEQ ID NO:10)；

TDH2t_R：5’-gcgaaaagccaattagtgtgatac-3’(SEQ ID NO:11)。

5. Splicing the ADH1t, the Gal1, the TDH3p and the TDH2t obtained in the first step to the fourth step in an OE-PCR mode to obtain the ADH1t-Gal1-TDH3p-TDH2t (SEQ ID NO: 21) with BamHI and SalI enzyme cutting sites at two ends, wherein a BsmBI enzyme cutting site is added between the ADH1t and the Gal1, and a BsaI enzyme cutting site is added between the TDH3p and the TDH2 t.

The OE-PCR system (50. Mu.l reaction system) was as follows:

ADH1t, gal1, TDH3p and TDH2t templates 1. Mu.l each, 1. Mu.l each of the upstream primer, 1. Mu.l each of the downstream primer, 10 XBuffer 5. Mu.l, dNTP 4. Mu.l, pfu DNA Polymerase. Mu.l, and ddH was added ₂ O makes up the volume to 50. Mu.l.

The primers used were as follows:

Primer 1_F：5’-ggatcccatgccggtagaggtgtggtc-3’(SEQ ID NO:13)；

Primer 1_R：5’-tgaaaattcaatataaatgggagacgcgtctcctaaagctttggacttc-3’(SEQ ID NO:14)；

Primer 2_F：5’-aagtccaaagctttaggagacgcgtctcccatttatattgaattttcaaaaattc-3’(SEQ ID NO:15)；

Primer 2_R：5’-tgataaactcgaactgtatagttttttctccttgacgttaaagtatag-3’(SEQ ID NO:16)；

Primer 3_F：5’-gtcaaggagaaaaaactatacagttcgagtttatcattatcaatactg-3’(SEQ ID NO:17)；

Primer 3_R：5’-ggagttaaatatttaggagaccggtctcccattatttgtttgtttatgtg-3’(SEQ ID NO:18)；

Primer 4_F：5’-ataaacaaacaaataatgggagaccggtctcctaaatatttaactccttaag-3’(SEQ ID NO:19)；

Primer 4_R：5’-gtcgacgcgaaaagccaattagtgtgatactaag-3’(SEQ ID NO:20)。

6. cutting ADH1t-Gal1-TDH3p-TDH2t with restriction enzymes SalI and BamHI to obtain gene fragment; cutting a PRS416 vector by using restriction enzymes SalI and BamHI to obtain a vector fragment; the gene segment is connected with the vector segment to obtain a recombinant expression vector PRS416-ADH1t-Gal1-TDH3p-TDH2t, the sequence of which is shown as SEQ ID NO. 22.

Example 2: foreign gene insertion

1. PCR amplification is carried out by taking an AaCrtZ gene as a template and taking a Primer 5_F and a Primer 5_R as primers to obtain BsaI-AaCrtZ-BsaI, the sequence of the BsaI-AaCrtZ-BsaI is shown as SEQ ID NO. 25, and the AaCrtZ gene is shown as the 9 th-497 th position in the SEQ ID NO. 25.

Primer 5_F：5’-ggtctccaatgactaacttcttgatcgttgttg-3’(SEQ ID NO:23)；

Primer 5_R：5’-ggtctccatttaagttctttcttgagcttcag-3’(SEQ ID NO:24)。

2. And performing PCR amplification by using the BDC263CrtW gene as a template and using a Primer 6_F and a Primer 6_R as primers to obtain BsaI-BDC263CrtW-BsaI, wherein the sequence of the BsaI-BDC263CrtW-BsaI is shown as SEQ ID NO. 28, and the BDC263CrtW gene is shown as 9 th-734 th positions in SEQ ID NO. 28.

Primer 6_F：5’-ggtctccaatgtccgctgttactccaatg-3’(SEQ ID NO:26)；

Primer 6_R：5’-ggtctccatttatgaaaataaagaccaccaaggc-3’(SEQ ID NO:27)。

3. PCR amplification is carried out by taking AaCrtZ gene as a template and taking Primer 7_F and Primer 7_R as primers to obtain BsmBI-AaCrtZ-BsmBI, wherein the sequence of the BsmBI-AaCrtZ-BsmBI is shown as SEQ ID NO. 31.

Primer 7_F：5’-cgtctccaatgactaacttcttgatcgttgttg-3’(SEQ ID NO:29)；

Primer 7_R：5’-cgtctcctttaagttctttcttgagcttcagc-3’(SEQ ID NO:30)。

4. And performing PCR amplification by using BDC263CrtW gene as a template and using Primer 8_F and Primer 8_R as primers to obtain BsmBI-BDC263CrtW-BsmBI, wherein the sequence of BsmBI-BDC263CrtW-BsmBI is shown as SEQ ID NO: 34.

Primer 8_F：5’-cgtctccaatgtccgctgttactcc-3’(SEQ ID NO:32)；

Primer 8_R：5’-cgtctcctttatgaaaataaagaccaccaaggc-3’(SEQ ID NO:33)。

5. BsaI-BDC263CrtW-BsaI is digested by BsaI to obtain a gene fragment; PRS416-ADH1t-Gal1-TDH3p-TDH2t obtained in example 1 was digested with BsaI to obtain vector fragment; ligating the gene fragment with the vector fragment to obtain PRS416-ADH1t-Gal1-TDH3p-BDC263CrtW-TDH2t;

cutting BsmBI-AaCrtZ-BsmBI by using BsmBI to obtain a gene fragment; cutting PRS416-ADH1t-Gal1-TDH3p-BDC263CrtW-TDH2t by BsmBI to obtain a vector fragment; the gene segment is connected with the vector segment to obtain a recombinant expression vector PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t, the sequence of which is shown as SEQ ID NO: 35.

6. BsaI-AaCrtZ-BsaI is digested by BsaI to obtain a gene fragment; cutting the PRS416-ADH1t-Gal1-TDH3p-TDH2t vector obtained in example 1 by BsaI to obtain a vector fragment; ligating the gene fragment with the vector fragment to obtain PRS416-ADH1t-Gal1-TDH3p-AaCrtZ-TDH2t;

BsmBI-BDC263CrtW-BsmBI is digested by BsmBI to obtain a gene fragment; cutting PRS416-ADH1t-Gal1-TDH3p-AaCrtZ-TDH2t by BsmBI to obtain vector fragment; connecting the gene segment with the vector segment to obtain a recombinant expression vector PRS416-ADH1t-BDC263CrtW-Gal1-TDH3p-AaCrtZ-TDH2t, wherein the sequence of the recombinant expression vector PRS416-ADH1t-BDC263CrtW-Gal1-TDH3p-AaCrtZ-TDH2t is shown as SEQ ID NO: shown at 36.

A schematic representation of the recombinant expression vectors PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t and PRS416-ADH1t-BDC263CrtW-Gal1-TDH3p-AaCrtZ-TDH2t is shown in FIG. 1.

Example 3: construction of astaxanthin-producing recombinant Yeast Strain

The recombinant expression vectors PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t and PRS416-ADH1t-BDC263CrtW-Gal1-TDH3p-AaCrtZ-TDH2t obtained in the construction of example 2 were transformed into the strain Scy10026 s.cerevisiae (Saccharomyces cerevisiae) producing β -carotene by the lithium acetate method, and were subjected to transformant selection via SC-Ura solid medium (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, uracil-removed mixed amino acid powder 2g/L,2% agar powder) to obtain recombinant s.cerevisiae strains S1 and S2 producing astaxanthin, respectively.

The construction method of the Scy10026 saccharomyces cerevisiae comprises the following steps: transferring CYC1t-BtCrtI-HXT7p-TDH3p-PaCrtB-ADH1t fragment (SEQ ID NO: 37) into Saccharomyces cerevisiae S288C, carrying out homologous recombination and replacing gal1, gal7 and gal10 genes, and integrating into a chromosome; and then the CYC1t-PaCrtY-HXT7p-FBA1p-TmCrtE-TDH2t fragment (SEQ ID NO: 38) is transferred into the yeast, homologous recombination is carried out, the ypl062w gene is replaced, and the yeast is integrated on a chromosome, so that the yeast Scy10026 for producing beta-carotene is obtained.

CYC1t-BtCrtI-HXT7p-TDH3p-PaCrtB-ADH1t (SEQ ID NO: 37):

the 1 st to 40 th homologous sequence is the gal7 gene downstream, the 41 st to 241 st sequence is the terminator CYC1t sequence, the 242 th to 1990 th sequence is the gene BtCrtI sequence, the 1991 st to 2691 th sequence is the promoter HXT7p sequence, the 2692 th to 3371 th sequence is the promoter TDH3p sequence, the 3372 th to 4301 nd sequence is the gene PaCrtB sequence, the 4302 nd to 4629 nd sequence is the terminator ADH1t sequence, and the 4630 th to 4669 th sequence is the gal1 gene downstream homologous sequence.

CYC1t-PaCrtY-HXT7p-FBA1p-TmCrtE-TDH2t (SEQ ID NO: 38):

the 1 st to 40 th homologous sequence is ypl062w gene upstream, the 41 st to 241 st homologous sequence is terminator CYC1t sequence, the 242 st to 1390 th homologous sequence is PaCrtY gene sequence, the 1391 st to 2091 st homologous sequence is promoter HXT7p sequence, the 2092 st to 2911 st homologous sequence is promoter FBA1p sequence, the 2912 nd to 4093 th homologous sequence is gene TmCrtE sequence, the 4094 th to 4493 th homologous sequence is terminator TDH2t sequence, and the 4494 th to 4533 th homologous sequence is ypl062w gene downstream.

Example 4: production of astaxanthin by shaking fermentation

Single colonies of recombinant Saccharomyces cerevisiae strains S1 and S2 were respectively picked and inoculated into 5ml of SC-Ura liquid medium (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, mixed amino acid powder excluding uracil 2g/L, balance water), and cultured overnight at 30℃at 250rpm to obtain primary seeds. The first seed is then inoculated into fresh 5ml SC-Ura liquid medium at an inoculum size of 5% (volume to volume)Culturing the seeds at 30 deg.C and 250rpm to OD ₆₀₀ =5-6, resulting in secondary seeds. Inoculating the secondary seeds into 250mL shake flask containing 50mL YPD culture medium for fermentation culture, and initial OD ₆₀₀ The fermentation was completed for 84 hours after the completion of the fermentation for 48 hours by adding D- (+) -galactose at a final concentration of 2% (2 g/100 ml) to induce astaxanthin production.

1mL of the fermentation broth was centrifuged at 12000rpm for 5min, the supernatant was decanted, 1mL of sterile water was added to resuspend the cells, and the supernatant was decanted. 1mL of 3M HCl was added to resuspend the cells, the mixture was boiled for 5min, ice for 5min, centrifuged, the supernatant was decanted, the boiling water was repeated for 5min, the ice for 5min, and the mixture was repeated 2 times. After washing twice by centrifugation and discarding the supernatant, adding 1mL of acetone to resuspend cells, adding a small amount of quartz sand, shaking for 10min by vortex, centrifuging the acetone extract for 10min at 12000rpm, filtering the supernatant by using a 0.2 mu m organic filter membrane, and adding 200 mu L of the filtered supernatant into a sample bottle for astaxanthin HPLC high performance liquid chromatography detection.

The astaxanthin HPLC high performance liquid chromatography detection conditions are as follows:

the dual-wavelength detection is adopted, the measurement wavelength is set to 450/470nm, and the integral wavelength is 470nm;

column temperature: 25 ℃;

mobile phase: flow rate was 1mL/min, mobile phase B: acetonitrile: water=9:1 (volume ratio), mobile phase C: methanol: isopropanol=3:2 (volume ratio), elution conditions were: 0-15min,0-90% C;15-30min,90% C;30-35min,90-0%C;35-45min,0% C. The astaxanthin concentration was calculated by the external standard method.

Astaxanthin purity was calculated according to the formula:

W＝Ca/C*100％

w is purity, ca is astaxanthin concentration (mg/L), C is carotenoid concentration (mg/L);

the carotenoid concentration is also detected by adopting an HPLC external standard method, and the HPLC detection conditions are consistent with the astaxanthin HPLC high performance liquid chromatography detection conditions.

The astaxanthin yield of recombinant Saccharomyces cerevisiae strain S2 was examined to be 43.72mg/L (purity: 57.51%), the astaxanthin yield of recombinant Saccharomyces cerevisiae strain S1 was 70.67mg/L (purity: 90.65%), and the astaxanthin yields of recombinant Saccharomyces cerevisiae strains S1 and S2 were compared as shown in FIG. 2.

Example 5: fermentation tank fermentation production of astaxanthin

1. Primary seed culture: inoculating S1 into 250mL shake flask containing 50mL SC-Ura liquid culture medium, culturing at 30deg.C and 250rpm to OD ₆₀₀ =6-7, resulting in first order seeds.

2. Secondary seed culture: the first seed is transferred into a 250mL shaking flask filled with 50mL SC-Ura liquid culture medium according to the inoculum size of 5% (volume ratio), and is cultured at 30 ℃ and 250rpm until the OD ₆₀₀ =5-6, resulting in secondary seeds.

3. Inoculating a fermentation tank: transferring the secondary seeds into a 7L fermentation tank with 10% (volume ratio) inoculation amount, starting fermentation, wherein the initial liquid loading amount of the fermentation tank is 3.5L, the initial fermentation medium is YPD (10 g/L yeast extract, 20g/L peptone, 30g/L glucose, and the balance being water) containing 3% glucose, and the initial OD ₆₀₀ =0.5. Fermentation tank parameter setting: the fermentation temperature was 30℃and the pH was controlled at 5.8, the aeration was set at 3.0vvm, the Dissolved Oxygen (DO) was 30%, the rotational speed was in the range of 300-500rpm, and stirring was associated with DO.

The whole fermentation process adopts the D- (+) -galactose inducer to be added at the time point of two stages of boundary points. Glucose was fed in a fed-batch manner before the D- (+) -galactose inducer was added and controlled in the range of 0-1g/L to reduce the production of by-products as much as possible. After 48h of fermentation, adding a D- (+) -galactose inducer with a final concentration of 20g/L, stopping feeding glucose simultaneously, and entering an astaxanthin synthesis stage, wherein a carbon source in the fermentation tank is switched to ethanol, the ethanol is added in a feeding mode, and the concentration of the ethanol in the fermentation tank is controlled to be 0.5-10g/L. In addition, the nitrogen source in the fermentation medium is also an important factor influencing the fermentation. In order to avoid excessive nitrogen source feeding, the invention adopts an intermittent feeding mode with decreasing concentration gradient to feed nitrogen source into the fermentation tank, and adds a certain volume of yeast powder-soaking mother liquor into the fermentation tank at intervals, and the specific feeding process is as follows: 15g yeast extract is added per liter at 12, 24, 36, 48, 60 hours of fermentation; 10g yeast extract powder is added per liter at 72 and 84 hours of fermentation; 5g yeast extract was added per liter at 96 and 108 hours of fermentation.

By the same HPLC detection as in example 4, the final astaxanthin yield in the 7L fermenter was 366.90mg/L, which was 4.19 times higher than the shaking flask fermentation yield (70.67 mg/L).

Example 6

The experiment of example 6 was repeated except that the fermentation pH was controlled to 6.2 and the fermentation temperature after the addition of the D- (+) -galactose inducer was set to 20℃and the fermentation test results were shown in FIG. 3, in which the final 7L fermenter astaxanthin yield was measured to 464.90mg/L by the same HPLC test as in example 4, which was 5.58-fold improvement over the shake flask fermentation yield (70.67 mg/L).

Sequence listing

<110> Wanhua chemical group Co., ltd

Wan Hua Hua Xue (Sichuan) Co., Ltd.

<120> recombinant Saccharomyces cerevisiae for producing astaxanthin and application thereof

<130> DSP1F200403ZX

<160> 38

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

agctttggac ttcttcgcca gagg 24

<210> 2

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

catgccggta gaggtgtggt caataag 27

<210> 3

<211> 328

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

agctttggac ttcttcgcca gaggtttggt caagtctcca atcaaggttg tcggcttgtc 60

taccttgcca gaaatttacg aaaagatgga aaagggtcaa atcgttggta gatacgttgt 120

tgacacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat 180

aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt 240

attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc 300

tcttattgac cacacctcta ccggcatg 328

<210> 4

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tatagttttt tctccttgac gttaaagtat ag 32

<210> 5

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ttatattgaa ttttcaaaaa ttcttacttt ttttttggat gg 42

<210> 6

<211> 668

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60

ttttatgaca tttgaataag aagtaataca aaccgaaaat gttgaaagta ttagttaaag 120

tggttatgca gcttttgcat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180

attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tattatccta tggttgttaa 240

tttgattcgt tgatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 300

accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 360

ctgcgtttca ggaacgcgac cggtgaagac caggacgcac ggaggagagt cttccgtcgg 420

agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 480

gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 540

catttccaca acatataagt aagattagat atggatatgt atatggtggt attgccatgt 600

aatatgatta ttaaacttct ttgcgtccat ccaaaaaaaa agtaagaatt tttgaaaatt 660

caatataa 668

<210> 7

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

cagttcgagt ttatcattat caatactgcc a 31

<210> 8

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tttgtttgtt tatgtgtgtt tattcgaaac 30

<210> 9

<211> 680

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cagttcgagt ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt 60

agtgattttc ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt 120

acatgcccaa aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga 180

acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag 240

aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt 300

ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac 360

ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc 420

atgtatctat ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa 480

agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt 540

atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat 600

tctactttta tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata 660

aacacacata aacaaacaaa 680

<210> 10

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

atttaactcc ttaagttact ttaatgattt ag 32

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gcgaaaagcc aattagtgtg atac 24

<210> 12

<211> 400

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

atttaactcc ttaagttact ttaatgattt agtttttatt attaataatt catgctcatg 60

acatctcata tacacgttta taaaacttaa atagattgaa aatgtattaa agattcctca 120

gggattcgat ttttttggaa gtttttgttt ttttttcctt gagatgctgt agtatttggg 180

aacaattata caatcgaaag atatatgctt acattcgacc gttttagccg tgatcattat 240

cctatagtaa cataacctga agcataactg acactactat catcaatact tgtcacatga 300

gaactctgtg aataattagg ccactgaaat ttgatgcctg aaggaccggc atcacggatt 360

ttcgataaag cacttagtat cacactaatt ggcttttcgc 400

<210> 13

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ggatcccatg ccggtagagg tgtggtc 27

<210> 14

<211> 49

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tgaaaattca atataaatgg gagacgcgtc tcctaaagct ttggacttc 49

<210> 15

<211> 55

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

aagtccaaag ctttaggaga cgcgtctccc atttatattg aattttcaaa aattc 55

<210> 16

<211> 48

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tgataaactc gaactgtata gttttttctc cttgacgtta aagtatag 48

<210> 17

<211> 48

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gtcaaggaga aaaaactata cagttcgagt ttatcattat caatactg 48

<210> 18

<211> 50

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ggagttaaat atttaggaga ccggtctccc attatttgtt tgtttatgtg 50

<210> 19

<211> 52

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

ataaacaaac aaataatggg agaccggtct cctaaatatt taactcctta ag 52

<210> 20

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

gtcgacgcga aaagccaatt agtgtgatac taag 34

<210> 21

<211> 2132

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

ggatcccatg ccggtagagg tgtggtcaat aagagcgacc tcatgctata cctgagaaag 60

caacctgacc tacaggaaag agttactcaa gaataagaat tttcgtttta aaacctaaga 120

gtcactttaa aatttgtata cacttatttt ttttataact tatttaataa taaaaatcat 180

aaatcataag aaattcgctt atttagaagt gtcaacaacg tatctaccaa cgatttgacc 240

cttttccatc ttttcgtaaa tttctggcaa ggtagacaag ccgacaacct tgattggaga 300

cttgaccaaa cctctggcga agaagtccaa agctttagga gacgcgtctc ccatttatat 360

tgaattttca aaaattctta cttttttttt ggatggacgc aaagaagttt aataatcata 420

ttacatggca ataccaccat atacatatcc atatctaatc ttacttatat gttgtggaaa 480

tgtaaagagc cccattatct tagcctaaaa aaaccttctc tttggaactt tcagtaatac 540

gcttaactgc tcattgctat attgaagtac ggattagaag ccgccgagcg ggcgacagcc 600

ctccgacgga agactctcct ccgtgcgtcc tggtcttcac cggtcgcgtt cctgaaacgc 660

agatgtgcct cgcgccgcac tgctccgaac aataaagatt ctacaatact agcttttatg 720

gttatgaaga ggaaaaattg gcagtaacct ggccccacaa accttcaaat caacgaatca 780

aattaacaac cataggataa taatgcgatt agttttttag ccttatttct ggggtaatta 840

atcagcgaag cgatgatttt tgatctatta acagatatat aaatgcaaaa gctgcataac 900

cactttaact aatactttca acattttcgg tttgtattac ttcttattca aatgtcataa 960

aagtatcaac aaaaaattgt taatatacct ctatacttta acgtcaagga gaaaaaacta 1020

tacagttcga gtttatcatt atcaatactg ccatttcaaa gaatacgtaa ataattaata 1080

gtagtgattt tcctaacttt atttagtcaa aaaattagcc ttttaattct gctgtaaccc 1140

gtacatgccc aaaatagggg gcgggttaca cagaatatat aacatcgtag gtgtctgggt 1200

gaacagttta ttcctggcat ccactaaata taatggagcc cgctttttaa gctggcatcc 1260

agaaaaaaaa agaatcccag caccaaaata ttgttttctt caccaaccat cagttcatag 1320

gtccattctc ttagcgcaac tacagagaac aggggcacaa acaggcaaaa aacgggcaca 1380

acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg acacaaggca attgacccac 1440

gcatgtatct atctcatttt cttacacctt ctattacctt ctgctctctc tgatttggaa 1500

aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta ttcccctact tgactaataa 1560

gtatataaag acggtaggta ttgattgtaa ttctgtaaat ctatttctta aacttcttaa 1620

attctacttt tatagttagt ctttttttta gttttaaaac accaagaact tagtttcgaa 1680

taaacacaca taaacaaaca aataatggga gaccggtctc ctaaatattt aactccttaa 1740

gttactttaa tgatttagtt tttattatta ataattcatg ctcatgacat ctcatataca 1800

cgtttataaa acttaaatag attgaaaatg tattaaagat tcctcaggga ttcgattttt 1860

ttggaagttt ttgttttttt ttccttgaga tgctgtagta tttgggaaca attatacaat 1920

cgaaagatat atgcttacat tcgaccgttt tagccgtgat cattatccta tagtaacata 1980

acctgaagca taactgacac tactatcatc aatacttgtc acatgagaac tctgtgaata 2040

attaggccac tgaaatttga tgcctgaagg accggcatca cggattttcg ataaagcact 2100

tagtatcaca ctaattggct tttcgcgtcg ac 2132

<210> 22

<211> 6979

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240

ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300

agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360

cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420

cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480

ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540

aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600

tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660

ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720

aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780

acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840

aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900

gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960

ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020

ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080

atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140

gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200

gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260

aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320

agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380

tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440

tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500

agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560

gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620

aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680

cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740

gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800

gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860

cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920

taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980

acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac gcgaaaagcc 2040

aattagtgtg atactaagtg ctttatcgaa aatccgtgat gccggtcctt caggcatcaa 2100

atttcagtgg cctaattatt cacagagttc tcatgtgaca agtattgatg atagtagtgt 2160

cagttatgct tcaggttatg ttactatagg ataatgatca cggctaaaac ggtcgaatgt 2220

aagcatatat ctttcgattg tataattgtt cccaaatact acagcatctc aaggaaaaaa 2280

aaacaaaaac ttccaaaaaa atcgaatccc tgaggaatct ttaatacatt ttcaatctat 2340

ttaagtttta taaacgtgta tatgagatgt catgagcatg aattattaat aataaaaact 2400

aaatcattaa agtaacttaa ggagttaaat atttaggaga ccggtctccc attatttgtt 2460

tgtttatgtg tgtttattcg aaactaagtt cttggtgttt taaaactaaa aaaaagacta 2520

actataaaag tagaatttaa gaagtttaag aaatagattt acagaattac aatcaatacc 2580

taccgtcttt atatacttat tagtcaagta ggggaataat ttcagggaac tggtttcaac 2640

cttttttttc agctttttcc aaatcagaga gagcagaagg taatagaagg tgtaagaaaa 2700

tgagatagat acatgcgtgg gtcaattgcc ttgtgtcatc atttactcca ggcaggttgc 2760

atcactccat tgaggttgtg cccgtttttt gcctgtttgt gcccctgttc tctgtagttg 2820

cgctaagaga atggacctat gaactgatgg ttggtgaaga aaacaatatt ttggtgctgg 2880

gattcttttt ttttctggat gccagcttaa aaagcgggct ccattatatt tagtggatgc 2940

caggaataaa ctgttcaccc agacacctac gatgttatat attctgtgta acccgccccc 3000

tattttgggc atgtacgggt tacagcagaa ttaaaaggct aattttttga ctaaataaag 3060

ttaggaaaat cactactatt aattatttac gtattctttg aaatggcagt attgataatg 3120

ataaactcga actgtatagt tttttctcct tgacgttaaa gtatagaggt atattaacaa 3180

ttttttgttg atacttttat gacatttgaa taagaagtaa tacaaaccga aaatgttgaa 3240

agtattagtt aaagtggtta tgcagctttt gcatttatat atctgttaat agatcaaaaa 3300

tcatcgcttc gctgattaat taccccagaa ataaggctaa aaaactaatc gcattattat 3360

cctatggttg ttaatttgat tcgttgattt gaaggtttgt ggggccaggt tactgccaat 3420

ttttcctctt cataaccata aaagctagta ttgtagaatc tttattgttc ggagcagtgc 3480

ggcgcgaggc acatctgcgt ttcaggaacg cgaccggtga agaccaggac gcacggagga 3540

gagtcttccg tcggagggct gtcgcccgct cggcggcttc taatccgtac ttcaatatag 3600

caatgagcag ttaagcgtat tactgaaagt tccaaagaga aggttttttt aggctaagat 3660

aatggggctc tttacatttc cacaacatat aagtaagatt agatatggat atgtatatgg 3720

tggtattgcc atgtaatatg attattaaac ttctttgcgt ccatccaaaa aaaaagtaag 3780

aatttttgaa aattcaatat aaatgggaga cgcgtctcct aaagctttgg acttcttcgc 3840

cagaggtttg gtcaagtctc caatcaaggt tgtcggcttg tctaccttgc cagaaattta 3900

cgaaaagatg gaaaagggtc aaatcgttgg tagatacgtt gttgacactt ctaaataagc 3960

gaatttctta tgatttatga tttttattat taaataagtt ataaaaaaaa taagtgtata 4020

caaattttaa agtgactctt aggttttaaa acgaaaattc ttattcttga gtaactcttt 4080

cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc gctcttattg accacacctc 4140

taccggcatg ggatccacta gttctagagc ggccgccacc gcggtggagc tccagctttt 4200

gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg 4260

tgtgaaattg ttatccgctc acaattccac acaacatagg agccggaagc ataaagtgta 4320

aagcctgggg tgcctaatga gtgaggtaac tcacattaat tgcgttgcgc tcactgcccg 4380

ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 4440

gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 4500

tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 4560

aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4620

gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4680

aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4740

ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4800

tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 4860

tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4920

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4980

tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 5040

ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 5100

tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 5160

aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 5220

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 5280

aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 5340

ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 5400

acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 5460

ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 5520

gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 5580

taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 5640

tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5700

gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5760

cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 5820

aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 5880

cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 5940

tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 6000

gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 6060

tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 6120

gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 6180

ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 6240

cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 6300

agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 6360

gggttccgcg cacatttccc cgaaaagtgc cacctgggtc cttttcatca cgtgctataa 6420

aaataattat aatttaaatt ttttaatata aatatataaa ttaaaaatag aaagtaaaaa 6480

aagaaattaa agaaaaaata gtttttgttt tccgaagatg taaaagactc tagggggatc 6540

gccaacaaat actacctttt atcttgctct tcctgctctc aggtattaat gccgaattgt 6600

ttcatcttgt ctgtgtagaa gaccacacac gaaaatcctg tgattttaca ttttacttat 6660

cgttaatcga atgtatatct atttaatctg cttttcttgt ctaataaata tatatgtaaa 6720

gtacgctttt tgttgaaatt ttttaaacct ttgtttattt ttttttcttc attccgtaac 6780

tcttctacct tctttattta ctttctaaaa tccaaataca aaacataaaa ataaataaac 6840

acagagtaaa ttcccaaatt attccatcat taaaagatac gaggcgcgtg taagttacag 6900

gcaagcgatc cgtcctaaga aaccattatt atcatgacat taacctataa aaataggcgt 6960

atcacgaggc cctttcgtc 6979

<210> 23

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

ggtctccaat gactaacttc ttgatcgttg ttg 33

<210> 24

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

ggtctccatt taagttcttt cttgagcttc ag 32

<210> 25

<211> 506

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

ggtctccaat gactaacttc ttgatcgttg ttgctactgt tttggttatg gaattgactg 60

cttactctgt tcacagatgg atcatgcacg gtccattggg ttggggttgg cacaagtctc 120

accacgaaga acacgaccac gctttggaaa agaacgactt gtacggtttg gttttcgctg 180

ttatcgctac tgttttgttc actgttggtt ggatctgggc tccagttttg tggtggatcg 240

ctttgggtat gactgtttac ggtttgatct acttcgtttt gcacgacggt ttggttcacc 300

aaagatggcc attcagatac atcccaagaa agggttacgc tagaagattg taccaagctc 360

acagattgca ccacgctgtt gaaggtagag accactgtgt ttctttcggt ttcatctacg 420

ctccaccagt tgacaagttg aagcaagact tgaagatgtc tggtgttttg agagctgaag 480

ctcaagaaag aacttaaatg gagacc 506

<210> 26

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

ggtctccaat gtccgctgtt actccaatg 29

<210> 27

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

ggtctccatt tatgaaaata aagaccacca aggc 34

<210> 28

<211> 743

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

ggtctccaat gtccgctgtt actccaatga gtagagttgt ccctaatcaa gcattgattg 60

gtttgacttt agcaggtttg attgctgctg cttggttgac attgcatata tacggtgtct 120

acttccacag atggacaatc tggtctgttt tgaccgtccc attaattgta gctggtcaaa 180

cttggttgtc cgtaggttta ttcatagttg ctcatgatgc aatgcacggt agtttggcac 240

cagccagacc tagattaaac acagcaatcg gttctttggc tttagcattg tatgccggtt 300

ttagattcac cccattgaaa actgcccatc acgctcatca cgctgcacca ggtacagcag 360

atgaccctga ttttcatgcc gacgctccaa gagcattttt gccttggttc tatggtttct 420

ttagaaccta cttcggttgg agagaattag cagttttgac tgtattagtt gcagtcgccg 480

tattaatatt gggtgctaga atgccaaatt tgttagtctt ttgggccgct cctgcattgt 540

tgtctgcctt acaattgttc actttcggta catggttgcc acatagacac accgatgacg 600

cctttcctga taatcataac gctagaacat ctccattcgg tcctgtttta tcattgttga 660

cctgtttcca tttcggtaga catcacgaac atcacttgac accttggaag ccttggtggt 720

ctttattttc ataaatggag acc 743

<210> 29

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

cgtctccaat gactaacttc ttgatcgttg ttg 33

<210> 30

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

cgtctccttt aagttctttc ttgagcttca gc 32

<210> 31

<211> 505

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

cgtctccaat gactaacttc ttgatcgttg ttgctactgt tttggttatg gaattgactg 60

cttactctgt tcacagatgg atcatgcacg gtccattggg ttggggttgg cacaagtctc 120

accacgaaga acacgaccac gctttggaaa agaacgactt gtacggtttg gttttcgctg 180

ttatcgctac tgttttgttc actgttggtt ggatctgggc tccagttttg tggtggatcg 240

ctttgggtat gactgtttac ggtttgatct acttcgtttt gcacgacggt ttggttcacc 300

aaagatggcc attcagatac atcccaagaa agggttacgc tagaagattg taccaagctc 360

acagattgca ccacgctgtt gaaggtagag accactgtgt ttctttcggt ttcatctacg 420

ctccaccagt tgacaagttg aagcaagact tgaagatgtc tggtgttttg agagctgaag 480

ctcaagaaag aacttaaagg agacg 505

<210> 32

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

cgtctccaat gtccgctgtt actcc 25

<210> 33

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

cgtctccttt atgaaaataa agaccaccaa ggc 33

<210> 34

<211> 742

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

cgtctccaat gtccgctgtt actccaatga gtagagttgt ccctaatcaa gcattgattg 60

gtttgacttt agcaggtttg attgctgctg cttggttgac attgcatata tacggtgtct 120

acttccacag atggacaatc tggtctgttt tgaccgtccc attaattgta gctggtcaaa 180

cttggttgtc cgtaggttta ttcatagttg ctcatgatgc aatgcacggt agtttggcac 240

cagccagacc tagattaaac acagcaatcg gttctttggc tttagcattg tatgccggtt 300

ttagattcac cccattgaaa actgcccatc acgctcatca cgctgcacca ggtacagcag 360

atgaccctga ttttcatgcc gacgctccaa gagcattttt gccttggttc tatggtttct 420

ttagaaccta cttcggttgg agagaattag cagttttgac tgtattagtt gcagtcgccg 480

tattaatatt gggtgctaga atgccaaatt tgttagtctt ttgggccgct cctgcattgt 540

tgtctgcctt acaattgttc actttcggta catggttgcc acatagacac accgatgacg 600

cctttcctga taatcataac gctagaacat ctccattcgg tcctgtttta tcattgttga 660

cctgtttcca tttcggtaga catcacgaac atcacttgac accttggaag ccttggtggt 720

ctttattttc ataaaggaga cg 742

<210> 35

<211> 8154

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240

ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300

agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360

cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420

cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480

ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540

aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600

tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660

ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720

aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780

acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840

aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900

gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960

ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020

ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080

atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140

gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200

gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260

aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320

agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380

tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440

tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500

agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560

gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620

aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680

cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740

gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800

gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860

cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920

taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980

acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac gcgaaaagcc 2040

aattagtgtg atactaagtg ctttatcgaa aatccgtgat gccggtcctt caggcatcaa 2100

atttcagtgg cctaattatt cacagagttc tcatgtgaca agtattgatg atagtagtgt 2160

cagttatgct tcaggttatg ttactatagg ataatgatca cggctaaaac ggtcgaatgt 2220

aagcatatat ctttcgattg tataattgtt cccaaatact acagcatctc aaggaaaaaa 2280

aaacaaaaac ttccaaaaaa atcgaatccc tgaggaatct ttaatacatt ttcaatctat 2340

ttaagtttta taaacgtgta tatgagatgt catgagcatg aattattaat aataaaaact 2400

aaatcattaa agtaacttaa ggagttaaat aatttatgaa aataaagacc accaaggctt 2460

ccaaggtgtc aagtgatgtt cgtgatgtct accgaaatgg aaacaggtca acaatgataa 2520

aacaggaccg aatggagatg ttctagcgtt atgattatca ggaaaggcgt catcggtgtg 2580

tctatgtggc aaccatgtac cgaaagtgaa caattgtaag gcagacaaca atgcaggagc 2640

ggcccaaaag actaacaaat ttggcattct agcacccaat attaatacgg cgactgcaac 2700

taatacagtc aaaactgcta attctctcca accgaagtag gttctaaaga aaccatagaa 2760

ccaaggcaaa aatgctcttg gagcgtcggc atgaaaatca gggtcatctg ctgtacctgg 2820

tgcagcgtga tgagcgtgat gggcagtttt caatggggtg aatctaaaac cggcatacaa 2880

tgctaaagcc aaagaaccga ttgctgtgtt taatctaggt ctggctggtg ccaaactacc 2940

gtgcattgca tcatgagcaa ctatgaataa acctacggac aaccaagttt gaccagctac 3000

aattaatggg acggtcaaaa cagaccagat tgtccatctg tggaagtaga caccgtatat 3060

atgcaatgtc aaccaagcag cagcaatcaa acctgctaaa gtcaaaccaa tcaatgcttg 3120

attagggaca actctactca ttggagtaac agcggacatt tttgtttgtt tatgtgtgtt 3180

tattcgaaac taagttcttg gtgttttaaa actaaaaaaa agactaacta taaaagtaga 3240

atttaagaag tttaagaaat agatttacag aattacaatc aatacctacc gtctttatat 3300

acttattagt caagtagggg aataatttca gggaactggt ttcaaccttt tttttcagct 3360

ttttccaaat cagagagagc agaaggtaat agaaggtgta agaaaatgag atagatacat 3420

gcgtgggtca attgccttgt gtcatcattt actccaggca ggttgcatca ctccattgag 3480

gttgtgcccg ttttttgcct gtttgtgccc ctgttctctg tagttgcgct aagagaatgg 3540

acctatgaac tgatggttgg tgaagaaaac aatattttgg tgctgggatt cttttttttt 3600

ctggatgcca gcttaaaaag cgggctccat tatatttagt ggatgccagg aataaactgt 3660

tcacccagac acctacgatg ttatatattc tgtgtaaccc gccccctatt ttgggcatgt 3720

acgggttaca gcagaattaa aaggctaatt ttttgactaa ataaagttag gaaaatcact 3780

actattaatt atttacgtat tctttgaaat ggcagtattg ataatgataa actcgaactg 3840

tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 3900

ttttatgaca tttgaataag aagtaataca aaccgaaaat gttgaaagta ttagttaaag 3960

tggttatgca gcttttgcat ttatatatct gttaatagat caaaaatcat cgcttcgctg 4020

attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tattatccta tggttgttaa 4080

tttgattcgt tgatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 4140

accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 4200

ctgcgtttca ggaacgcgac cggtgaagac caggacgcac ggaggagagt cttccgtcgg 4260

agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 4320

gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 4380

catttccaca acatataagt aagattagat atggatatgt atatggtggt attgccatgt 4440

aatatgatta ttaaacttct ttgcgtccat ccaaaaaaaa agtaagaatt tttgaaaatt 4500

caatataaat gactaacttc ttgatcgttg ttgctactgt tttggttatg gaattgactg 4560

cttactctgt tcacagatgg atcatgcacg gtccattggg ttggggttgg cacaagtctc 4620

accacgaaga acacgaccac gctttggaaa agaacgactt gtacggtttg gttttcgctg 4680

ttatcgctac tgttttgttc actgttggtt ggatctgggc tccagttttg tggtggatcg 4740

ctttgggtat gactgtttac ggtttgatct acttcgtttt gcacgacggt ttggttcacc 4800

aaagatggcc attcagatac atcccaagaa agggttacgc tagaagattg taccaagctc 4860

acagattgca ccacgctgtt gaaggtagag accactgtgt ttctttcggt ttcatctacg 4920

ctccaccagt tgacaagttg aagcaagact tgaagatgtc tggtgttttg agagctgaag 4980

ctcaagaaag aacttaaagc tttggacttc ttcgccagag gtttggtcaa gtctccaatc 5040

aaggttgtcg gcttgtctac cttgccagaa atttacgaaa agatggaaaa gggtcaaatc 5100

gttggtagat acgttgttga cacttctaaa taagcgaatt tcttatgatt tatgattttt 5160

attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt 5220

ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag 5280

gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgggatc cactagttct 5340

agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc 5400

gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 5460

tccacacaac ataggagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 5520

gtaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 5580

ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 5640

ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 5700

agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 5760

catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 5820

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 5880

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 5940

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 6000

cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 6060

caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 6120

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 6180

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 6240

taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 6300

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 6360

tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 6420

gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 6480

catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 6540

atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 6600

ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 6660

gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 6720

agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 6780

gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 6840

agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 6900

catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 6960

aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 7020

gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 7080

taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 7140

caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 7200

ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 7260

ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 7320

tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 7380

aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 7440

actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 7500

catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 7560

agtgccacct gggtcctttt catcacgtgc tataaaaata attataattt aaatttttta 7620

atataaatat ataaattaaa aatagaaagt aaaaaaagaa attaaagaaa aaatagtttt 7680

tgttttccga agatgtaaaa gactctaggg ggatcgccaa caaatactac cttttatctt 7740

gctcttcctg ctctcaggta ttaatgccga attgtttcat cttgtctgtg tagaagacca 7800

cacacgaaaa tcctgtgatt ttacatttta cttatcgtta atcgaatgta tatctattta 7860

atctgctttt cttgtctaat aaatatatat gtaaagtacg ctttttgttg aaatttttta 7920

aacctttgtt tatttttttt tcttcattcc gtaactcttc taccttcttt atttactttc 7980

taaaatccaa atacaaaaca taaaaataaa taaacacaga gtaaattccc aaattattcc 8040

atcattaaaa gatacgaggc gcgtgtaagt tacaggcaag cgatccgtcc taagaaacca 8100

ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc 8154

<210> 36

<211> 8154

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240

ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300

agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360

cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420

cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480

ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540

aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600

tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660

ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720

aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780

acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840

aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900

gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960

ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020

ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080

atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140

gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200

gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260

aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320

agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380

tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440

tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500

agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560

gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620

aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680

cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740

gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800

gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860

cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920

taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980

acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac gcgaaaagcc 2040

aattagtgtg atactaagtg ctttatcgaa aatccgtgat gccggtcctt caggcatcaa 2100

atttcagtgg cctaattatt cacagagttc tcatgtgaca agtattgatg atagtagtgt 2160

cagttatgct tcaggttatg ttactatagg ataatgatca cggctaaaac ggtcgaatgt 2220

aagcatatat ctttcgattg tataattgtt cccaaatact acagcatctc aaggaaaaaa 2280

aaacaaaaac ttccaaaaaa atcgaatccc tgaggaatct ttaatacatt ttcaatctat 2340

ttaagtttta taaacgtgta tatgagatgt catgagcatg aattattaat aataaaaact 2400

aaatcattaa agtaacttaa ggagttaaat aatttaagtt ctttcttgag cttcagctct 2460

caaaacacca gacatcttca agtcttgctt caacttgtca actggtggag cgtagatgaa 2520

accgaaagaa acacagtggt ctctaccttc aacagcgtgg tgcaatctgt gagcttggta 2580

caatcttcta gcgtaaccct ttcttgggat gtatctgaat ggccatcttt ggtgaaccaa 2640

accgtcgtgc aaaacgaagt agatcaaacc gtaaacagtc atacccaaag cgatccacca 2700

caaaactgga gcccagatcc aaccaacagt gaacaaaaca gtagcgataa cagcgaaaac 2760

caaaccgtac aagtcgttct tttccaaagc gtggtcgtgt tcttcgtggt gagacttgtg 2820

ccaaccccaa cccaatggac cgtgcatgat ccatctgtga acagagtaag cagtcaattc 2880

cataaccaaa acagtagcaa caacgatcaa gaagttagtc atttttgttt gtttatgtgt 2940

gtttattcga aactaagttc ttggtgtttt aaaactaaaa aaaagactaa ctataaaagt 3000

agaatttaag aagtttaaga aatagattta cagaattaca atcaatacct accgtcttta 3060

tatacttatt agtcaagtag gggaataatt tcagggaact ggtttcaacc ttttttttca 3120

gctttttcca aatcagagag agcagaaggt aatagaaggt gtaagaaaat gagatagata 3180

catgcgtggg tcaattgcct tgtgtcatca tttactccag gcaggttgca tcactccatt 3240

gaggttgtgc ccgttttttg cctgtttgtg cccctgttct ctgtagttgc gctaagagaa 3300

tggacctatg aactgatggt tggtgaagaa aacaatattt tggtgctggg attctttttt 3360

tttctggatg ccagcttaaa aagcgggctc cattatattt agtggatgcc aggaataaac 3420

tgttcaccca gacacctacg atgttatata ttctgtgtaa cccgccccct attttgggca 3480

tgtacgggtt acagcagaat taaaaggcta attttttgac taaataaagt taggaaaatc 3540

actactatta attatttacg tattctttga aatggcagta ttgataatga taaactcgaa 3600

ctgtatagtt ttttctcctt gacgttaaag tatagaggta tattaacaat tttttgttga 3660

tacttttatg acatttgaat aagaagtaat acaaaccgaa aatgttgaaa gtattagtta 3720

aagtggttat gcagcttttg catttatata tctgttaata gatcaaaaat catcgcttcg 3780

ctgattaatt accccagaaa taaggctaaa aaactaatcg cattattatc ctatggttgt 3840

taatttgatt cgttgatttg aaggtttgtg gggccaggtt actgccaatt tttcctcttc 3900

ataaccataa aagctagtat tgtagaatct ttattgttcg gagcagtgcg gcgcgaggca 3960

catctgcgtt tcaggaacgc gaccggtgaa gaccaggacg cacggaggag agtcttccgt 4020

cggagggctg tcgcccgctc ggcggcttct aatccgtact tcaatatagc aatgagcagt 4080

taagcgtatt actgaaagtt ccaaagagaa ggttttttta ggctaagata atggggctct 4140

ttacatttcc acaacatata agtaagatta gatatggata tgtatatggt ggtattgcca 4200

tgtaatatga ttattaaact tctttgcgtc catccaaaaa aaaagtaaga atttttgaaa 4260

attcaatata aatgtccgct gttactccaa tgagtagagt tgtccctaat caagcattga 4320

ttggtttgac tttagcaggt ttgattgctg ctgcttggtt gacattgcat atatacggtg 4380

tctacttcca cagatggaca atctggtctg ttttgaccgt cccattaatt gtagctggtc 4440

aaacttggtt gtccgtaggt ttattcatag ttgctcatga tgcaatgcac ggtagtttgg 4500

caccagccag acctagatta aacacagcaa tcggttcttt ggctttagca ttgtatgccg 4560

gttttagatt caccccattg aaaactgccc atcacgctca tcacgctgca ccaggtacag 4620

cagatgaccc tgattttcat gccgacgctc caagagcatt tttgccttgg ttctatggtt 4680

tctttagaac ctacttcggt tggagagaat tagcagtttt gactgtatta gttgcagtcg 4740

ccgtattaat attgggtgct agaatgccaa atttgttagt cttttgggcc gctcctgcat 4800

tgttgtctgc cttacaattg ttcactttcg gtacatggtt gccacataga cacaccgatg 4860

acgcctttcc tgataatcat aacgctagaa catctccatt cggtcctgtt ttatcattgt 4920

tgacctgttt ccatttcggt agacatcacg aacatcactt gacaccttgg aagccttggt 4980

ggtctttatt ttcataaagc tttggacttc ttcgccagag gtttggtcaa gtctccaatc 5040

aaggttgtcg gcttgtctac cttgccagaa atttacgaaa agatggaaaa gggtcaaatc 5100

gttggtagat acgttgttga cacttctaaa taagcgaatt tcttatgatt tatgattttt 5160

attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt 5220

ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag 5280

gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgggatc cactagttct 5340

agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc 5400

gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 5460

tccacacaac ataggagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 5520

gtaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 5580

ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 5640

ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 5700

agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 5760

catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 5820

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 5880

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 5940

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 6000

cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 6060

caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 6120

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 6180

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 6240

taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 6300

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 6360

tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 6420

gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 6480

catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 6540

atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 6600

ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 6660

gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 6720

agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 6780

gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 6840

agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 6900

catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 6960

aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 7020

gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 7080

taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 7140

caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 7200

ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 7260

ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 7320

tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 7380

aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 7440

actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 7500

catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 7560

agtgccacct gggtcctttt catcacgtgc tataaaaata attataattt aaatttttta 7620

atataaatat ataaattaaa aatagaaagt aaaaaaagaa attaaagaaa aaatagtttt 7680

tgttttccga agatgtaaaa gactctaggg ggatcgccaa caaatactac cttttatctt 7740

gctcttcctg ctctcaggta ttaatgccga attgtttcat cttgtctgtg tagaagacca 7800

cacacgaaaa tcctgtgatt ttacatttta cttatcgtta atcgaatgta tatctattta 7860

atctgctttt cttgtctaat aaatatatat gtaaagtacg ctttttgttg aaatttttta 7920

aacctttgtt tatttttttt tcttcattcc gtaactcttc taccttcttt atttactttc 7980

taaaatccaa atacaaaaca taaaaataaa taaacacaga gtaaattccc aaattattcc 8040

atcattaaaa gatacgaggc gcgtgtaagt tacaggcaag cgatccgtcc taagaaacca 8100

ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc 8154

<210> 37

<211> 4669

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

ttacagtctt tgtagataat gaatctgacc atctaaattt tttgtacaga aaaaaaagaa 60

aaatttgaaa tataaataac gttcttaata ctaacataac tattaaaaaa aataaatagg 120

gacctagact tcaggttgtc taactccttc cttttcggtt agagcggatg tgggaggagg 180

gcgtgaatgt aagcgtgaca taactaatta catgatatcg acaaaggaaa aggggcctgt 240

tttatatcct aatatcgtta gagttctgtc cttggaagac gtttggcaac aactgattaa 300

taaaagatgc tggagtagta gtgtcgtccc taggaaagaa atagaagaac aagaaagtga 360

caaagtagca tgctaagcag taataaatcc agtgtgactc agtcttcctt gtttgctcag 420

gtgcatattt cttctgagag tcctgtaact tcctaggcaa agggttttgt ccgaatgact 480

tacagacctg gtcagaagtt aatttagatc ctgccaagac tattgggact cctgtaccag 540

ggtgagttga tgcaccgacg aaaaataaat tgtcatatct gttagtagag tcctttgttg 600

aaggtctgaa ccacaaaacc tgaaagacat catgtgataa acctaatatt gatcccctcc 660

acaagttgaa cttagattgc cagactgatg ggtcgttgac ctcctcgtgt tctatcaagt 720

tagcgaagtt gttgactccc aacctcctct ctatgacctc caagaccatc tttctagccc 780

tgttgaccaa ctctgggtag ttctcctctg ctgagttacc tgtcttagac ttcatgtgac 840

caattggaac taaaactata attgagtcct tatttggagg tgctgcagac tcgtctatcc 900

ttgaaggaac gttgacgtag aaagatgctt cagaaggcaa accgaaatcg ttaaaaatct 960

cgtcgaatga ctccttgtaa gcctcagcca agaaaatgtt gtggacgtcc aattgaggga 1020

ccttagtaga cattgaccag taaaaagaaa ttgatgaaga tgttaatttc ttagatgcca 1080

aagtcttctt tgtccagttg caaggtggca acaagtggtg gtaagcgtag accaagtcag 1140

cgttgcagac gacagcgtct gcctcaatga cttctccaga ctccaaagtg acaccagtga 1200

ccctcttgtc tttatcgaca gtgttaatct tagcgacagg agattggtac ctgaactcag 1260

ctccgtactt cttagaagct atagactcca acttttggac gaccatgttg aaaccacccc 1320

ttgggtacca aattccctct gcgaactctg tatattgcaa caatgagtag actgcaggtg 1380

cgtcgtaagg tgacataccc atgtacattg tttgaaaagt aaaagccatc ctcatctttt 1440

ttgtttggaa gtattttgat gctctgtcgt atatcttacc gaataagtgc aacctaaaaa 1500

tctctgggac gtactgtaac cttattaagt cccatatagt ttcgaagttt ctctttatag 1560

caatgaatgt accctgctcg tagtggacgt gtgtctcttt catgaaatct aagaacctac 1620

cgaatcctaa aggtccctca atcctgtcca actcaccctt catctttgtt aagtctgatg 1680

acaattggac agcgtcaccg tcgtcgaaat ggactttgta attgttgtca catcttaata 1740

agtccaagtg gtctcctatc ctctcgtcta agtcagcgaa agcgtcctca aacaacttag 1800

gcatcaagta caatgaaggt ccctggtcga acctgtgacc gtcgtggtga atgaaagagc 1860

accttcctcc agagaagtcg ttcttctcga cgacagtgac tctgaaaccc tcccttgcta 1920

accttgctgc tgttgcagta cctcctattc cagcacctat gacgacaatg tgcttcttct 1980

gatcagacat ttttttgatt aaaattaaaa aaactttttg tttttgtgtt tattctttgt 2040

tcttagaaaa gacaagttga gcttgtttgt tcttgatgtt ttattatttt acaatagctg 2100

caaatgaaga atagattcga acattgtgaa gtattggcat atatcgtctc tatttatact 2160

tttttttttt cagttctagt atattttgta ttttcctcct tttcattctt tcagttgcca 2220

ataagttaca ggggatctcg aaagatggtg gggatttttc cttgaaagac gactttttgc 2280

catctaattt ttccttgttg cctctgaaaa ttatccagca gaagcaaatg taaaagatga 2340

acctcagaag aacacgcagg ggcccgaaat tgttcctacg agaagtagtg ggtcataaaa 2400

agtttattcc ctggaaaaaa aattttgcgt tgcctttctg gagaattttt tcgaattagc 2460

gtgctgccac tgcatgcatt tctgagaagt gtgggcattc ttccaccagt tgttcctcct 2520

aaaaaaaaaa aagatttcct accccgcaca aattcctgca tacccctcat ttccacgggg 2580

taaaaaaaaa acatcccgta gcagcctctg acggagccgt atggaacaat cctggataaa 2640

tatggcggaa aaacggctct ggctgcggct ttgtttaagg cagaaactag acagttcgag 2700

tttatcatta tcaatactgc catttcaaag aatacgtaaa taattaatag tagtgatttt 2760

cctaacttta tttagtcaaa aaattagcct tttaattctg ctgtaacccg tacatgccca 2820

aaataggggg cgggttacac agaatatata acatcgtagg tgtctgggtg aacagtttat 2880

tcctggcatc cactaaatat aatggagccc gctttttaag ctggcatcca gaaaaaaaaa 2940

gaatcccagc accaaaatat tgttttcttc accaaccatc agttcatagg tccattctct 3000

tagcgcaact acagagaaca ggggcacaaa caggcaaaaa acgggcacaa cctcaatgga 3060

gtgatgcaac ctgcctggag taaatgatga cacaaggcaa ttgacccacg catgtatcta 3120

tctcattttc ttacaccttc tattaccttc tgctctctct gatttggaaa aagctgaaaa 3180

aaaaggttga aaccagttcc ctgaaattat tcccctactt gactaataag tatataaaga 3240

cggtaggtat tgattgtaat tctgtaaatc tatttcttaa acttcttaaa ttctactttt 3300

atagttagtc ttttttttag ttttaaaaca ccaagaactt agtttcgaat aaacacacat 3360

aaacaaacaa aatgtcacaa ccaccattat tggaccacgc tacacaaact atggcaaacg 3420

gttctaaatc tttcgctact gctgctaaat tattcgaccc agcaacaaga agatctgtat 3480

tgatgttgta cacctggtgt agacattgcg atgacgttat agatgaccaa actcacggtt 3540

ttgcttcaga agctgcagcc gaagaagaag ctacacaaag attggcaaga ttaagaactt 3600

tgacattagc tgcattcgaa ggtgccgaaa tgcaagatcc agcttttgcc gctttccaag 3660

aagttgcatt aacccatggt attactccta gaatggcttt ggatcactta gacggttttg 3720

caatggatgt cgcccaaaca agatacgtaa ccttcgaaga cactttaaga tattgttacc 3780

atgtcgccgg tgttgtcggt ttgatgatgg ctagagtaat gggtgttaga gatgaaagag 3840

ttttagatag agcatgtgac ttgggtttag ccttccaatt gacaaacata gctagagata 3900

taatagatga cgcagccata gacagatgct atttgccagc tgaatggtta caagatgcag 3960

gtttgactcc tgaaaattac gctgcaagag aaaacagagc cgctttagcc agagttgctg 4020

aaagattgat agatgcagcc gaaccatatt acatctcttc acaagctggt ttgcatgatt 4080

tgccacctag atgcgcatgg gccattgcta ccgcaagatc tgtttacaga gaaatcggta 4140

ttaaagtcaa ggctgcaggt ggttccgcat gggatagaag acaacacact tctaaaggtg 4200

aaaagatcgc tatgttgatg gccgctcctg gtcaagttat tagagcaaag accaccagag 4260

tcaccccaag accagccggt ttatggcaaa gacctgttta aagctttgga cttcttcgcc 4320

agaggtttgg tcaagtctcc aatcaaggtt gtcggcttgt ctaccttgcc agaaatttac 4380

gaaaagatgg aaaagggtca aatcgttggt agatacgttg ttgacacttc taaataagcg 4440

aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac 4500

aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc 4560

ctgtaggtca ggttgctttc tcaggtatag catgaggtcg ctcttattga ccacacctct 4620

accggcatgc tctaaaccag cattgggcag ctgtctatat gaattataa 4669

<210> 38

<211> 4533

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

atagaattgg attatgtaaa aggtgaagat accattgtag tttgtacaga aaaaaaagaa 60

aaatttgaaa tataaataac gttcttaata ctaacataac tattaaaaaa aataaatagg 120

gacctagact tcaggttgtc taactccttc cttttcggtt agagcggatg tgggaggagg 180

gcgtgaatgt aagcgtgaca taactaatta catgatatcg acaaaggaaa aggggcctgt 240

tttaacgatg agtcgtcata atggcttgca atgctgctaa taccggaaca ggcggcttgc 300

cgctcagaat acgtagccga tcggtcagcg tgagttttcc cgcataaaaa cgggcaatta 360

aatcttcagg taaaccataa aaacgctgca taacccgcca gcgtgaatcg gcgggtccgg 420

ctaaaaacag catgcgattc agcatgcgga aaaagcgctg ctgctgccag cgctcgcggg 480

caaaatgcgt aatggcctgg tgaattgagg ccgacgtaaa gacatcaagt gcgctcaggc 540

ggtcggccac ggcaaccgcc agcggcagtg aatagccggt ggtaggatgg aacagaccgg 600

cacgtaatcc actacaggcc agggggcgct gctgccagaa tgcgtcggca ttgcccgaca 660

gggtgatggg taaggcgccc tgttcttcac gcagcaatgt ctgaagctgc caaccctgtt 720

gcgcggcata gtcgcaaata ttttgccgcg cgcgttcagg atctaatgtc gcattatcga 780

tatagtgcgt gtcttcaatt aacaatctgg tcggcgagag cggcaggctg tacacgaagc 840

gataaccatt ttgctgatcg accgtggcat ccatgataat gggagacgat aaaccatgcg 900

gttggctcaa tcgccattcc tggccaataa acgcctggaa gcccacgctc agtgctgagt 960

ttgccgcata accccgcccg tcaatcaccg cgcgtgcacc gataacctga ccctttttca 1020

accgaacaga ttccgcatta acctctgcga ccgcggtatc catccacaag tgcgggccaa 1080

actgtcgctg taaaacctca gcgaaacgct gagaagtaat acagaagtag ccgctgttca 1140

gcttacgacg gcgtgtggga aagcgcacct gatagtcggg ccagtgatga accaccagcg 1200

gagctatcca gcgatgttgg ctctcagtca aatcatcgtg gtgaaatgac cacgtatgat 1260

tcccgcccgc ctggggtgcg gcgtcgataa gcaaaatacg catatcaggt tgctgctgct 1320

gaagacgcag ggcgataagg ccattcgcga gtccagcccc cacgagaatc agatcataat 1380

gcggttgcat ttttttgatt aaaattaaaa aaactttttg tttttgtgtt tattctttgt 1440

tcttagaaaa gacaagttga gcttgtttgt tcttgatgtt ttattatttt acaatagctg 1500

caaatgaaga atagattcga acattgtgaa gtattggcat atatcgtctc tatttatact 1560

tttttttttt cagttctagt atattttgta ttttcctcct tttcattctt tcagttgcca 1620

ataagttaca ggggatctcg aaagatggtg gggatttttc cttgaaagac gactttttgc 1680

catctaattt ttccttgttg cctctgaaaa ttatccagca gaagcaaatg taaaagatga 1740

acctcagaag aacacgcagg ggcccgaaat tgttcctacg agaagtagtg ggtcataaaa 1800

agtttattcc ctggaaaaaa aattttgcgt tgcctttctg gagaattttt tcgaattagc 1860

gtgctgccac tgcatgcatt tctgagaagt gtgggcattc ttccaccagt tgttcctcct 1920

aaaaaaaaaa aagatttcct accccgcaca aattcctgca tacccctcat ttccacgggg 1980

taaaaaaaaa acatcccgta gcagcctctg acggagccgt atggaacaat cctggataaa 2040

tatggcggaa aaacggctct ggctgcggct ttgtttaagg cagaaactag atccaactgg 2100

caccgctggc ttgaacaaca ataccagcct tccaacttct gtaaataacg gcggtacgcc 2160

agtgccacca gtaccgttac ctttcggtat acctcctttc cccatgtttc caatgccctt 2220

catgcctcca acggctacta tcacaaatcc tcatcaagct gacgcaagcc ctaagaaatg 2280

aataacaata ctgacagtac taaataattg cctacttggc ttcacatacg ttgcatacgt 2340

cgatatagat aataatgata atgacagcag gattatcgta atacgtaata gttgaaaatc 2400

tcaaaaatgt gtgggtcatt acgtaaataa tgataggaat gggattcttc tatttttcct 2460

ttttccattc tagcagccgt cgggaaaacg tggcatcctc tctttcgggc tcaattggag 2520

tcacgctgcc gtgagcatcc tctctttcca tatctaacaa ctgagcacgt aaccaatgga 2580

aaagcatgag cttagcgttg ctccaaaaaa gtattggatg gttaatacca tttgtctgtt 2640

ctcttctgac tttgactcct caaaaaaaaa aaatctacaa tcaacagatc gcttcaatta 2700

cgccctcaca aaaacttttt tccttcttct tcgcccacgt taaattttat ccctcatgtt 2760

gtctaacgga tttctgcact tgatttatta taaaaagaca aagacataat acttctctat 2820

caatttcagt tattgttctt ccttgcgtta ttcttctgtt cttctttttc ttttgtcata 2880

tataaccata accaagtaat acatattcaa aatggcttat accgcaatgg cagcaggaac 2940

tcagtcattg cagttgagga cagtcgcctc ttaccaggag tgcaactcaa tgaggtcttg 3000

cttcaagttg accccattca agtcattcca cggtgtcaac ttcaacgttc cttctttagg 3060

tgccgccaac tgcgaaatca tgggtcactt gaaattgggt tctttgccat acaaacagtg 3120

ttcagtatca tctaagtcaa ctaagactat ggcccagttg gtagatttgg cagagaccga 3180

gaaagccgag ggaaaggata tcgagttcga ttttaacgag tatatgaagt ctaaggctgt 3240

cgctgttgat gcagccttgg ataaggccat ccctttggag tatccagaga agatccatga 3300

gtctatgagg tactcattgt tggccggagg aaaaagggtc agacctgcat tatgcatcgc 3360

tgcttgcgag ttagtaggtg gttctcagga cttggccatg ccaaccgcat gtgccatgga 3420

aatgattcat accatgtcat tgattcacga tgatttgcct tgcatggaca acgacgactt 3480

cagaagggga aagcctacca atcacaaggt tttcggagag gacactgctg ttttagccgg 3540

tgacgcattg ttatctttcg cttttgaaca catcgccgtt gccacatcaa aaactgtccc 3600

atctgacagg accttgagag tcatttctga gttgggtaaa accatcggtt cacagggatt 3660

ggtcggaggt caggtagtcg acatcacttc tgagggagac gccaacgtcg acttaaagac 3720

attggagtgg attcacattc acaagactgc cgtcttgttg gaatgctctg ttgtttctgg 3780

aggaatcttg ggtggagcta ccgaggatga gattgctaga ataagaagat acgccaggtg 3840

cgtcggtttg ttgttccagg ttgtcgacga cattttggat gtcaccaagt cttcagagga 3900

attgggaaag accgccggta aagacttatt gaccgacaag gctacctacc ctaagttgat 3960

gggtttggag aaggccaaag agtttgcagc agaattagct accagggcaa aggaagagtt 4020

gtcatcattc gaccagatca aggcagcccc tttgttagga ttggccgatt acatcgcttt 4080

caggcaaaac taaatttaac tccttaagtt actttaatga tttagttttt attattaata 4140

attcatgctc atgacatctc atatacacgt ttataaaact taaatagatt gaaaatgtat 4200

taaagattcc tcagggattc gatttttttg gaagtttttg tttttttttc cttgagatgc 4260

tgtagtattt gggaacaatt atacaatcga aagatatatg cttacattcg accgttttag 4320

ccgtgatcat tatcctatag taacataacc tgaagcataa ctgacactac tatcatcaat 4380

acttgtcaca tgagaactct gtgaataatt aggccactga aatttgatgc ctgaaggacc 4440

ggcatcacgg attttcgata aagcacttag tatcacacta attggctttt cgcattgatc 4500

tcctcttggg aacggtgagt gcaacgaatg cga 4533

Claims (2)

1. A method for producing astaxanthin, comprising the steps of subjecting recombinant saccharomyces cerevisiae to fermentation culture such that the beta-carotene ketolase gene CrtW is constitutively expressed and adding D- (+) -galactose during the fermentation culture to induce the expression of the beta-carotene hydroxylase gene CrtZ to synthesize astaxanthin;

the fermentation culture comprises the steps of carrying out fermentation culture on the recombinant saccharomyces cerevisiae in an initial fermentation culture medium, fermenting the saccharomyces cerevisiae with pH of 5.8-6.2, and then adding D- (+) -galactose to induce beta-carotene hydroxylase gene CrtZ to express;

before D- (+) -galactose is added, glucose is fed in and controlled within the range of 0-1 g/L; d- (+) -galactose is added, meanwhile, glucose is stopped being fed, ethanol is fed and controlled within the range of 0.5-10g/L, and the temperature is controlled within 20-30 ℃;

the nitrogen source is supplemented in a concentration gradient decreasing intermittent feeding mode during fermentation culture;

the recombinant saccharomyces cerevisiae is obtained by transferring a recombinant expression vector PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t shown in SEQ ID NO. 35 into the saccharomyces cerevisiae capable of synthesizing beta-carotene.

2. The method according to claim 1, characterized in that: the method also comprises the step of carrying out primary seed culture and secondary seed culture on the recombinant saccharomyces cerevisiae before fermentation culture;

the primary seed culture comprises inoculating the recombinant Saccharomyces cerevisiae into a shake flask filled with SC-Ura liquid culture medium, and culturing to OD ₆₀₀ A step of =6-7;

the secondary seed culture comprises transferring the primary seed obtained by primary seed culture into shake flask containing SC-Ura liquid culture medium, and culturing to OD ₆₀₀ Step=5-6.