CN113699053A - 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 constitutively expresses beta-carotene ketolase gene CrtW, and inductively expresses beta-carotene hydroxylase gene CrtZ, so that an intermediate product beta-carotene is synthesized into astaxanthin. According to the invention, the constitutive promoter is used for regulating the expression of the CrtW gene first, and the inducible promoter is used for regulating the expression of the CrtZ gene later, so that the metabolic flux of the synthetic path of the saccharomyces cerevisiae astaxanthin is obviously improved, the astaxanthin yield reaches 464.90mg/L, and the industrial development prospect is good.

Description

Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof

Technical Field

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

Background

Astaxanthin (3,3 ' -dihydroxy-beta, beta ' -carotene-4,4 ' -dione, C)₄₀H₅₂O₄596.84) has lipophilicity and hydrophilicity, and is one of the antioxidants with the strongest oxidation resistance found in nature so far, and the antioxidant capacity of the antioxidant is 800 times that of coenzyme Q10, 700 times that of anthocyanin and 550 times that of vitamin E. The astaxanthin has two asymmetric carbons which are positioned at the 3 and 3 ' positions of a beta-ionone ring, and is divided into 3 different configurations of (3R,3 ' R), (3S,3 ' S) and (3R,3 ' S) according to the difference of the 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 the oxidation of protein and lipid and the damage of DNA, protect cells and delay senility, is the only carotenoid antioxidant which can pass through a blood brain barrier, has positive treatment and prevention effects on cardiovascular and cerebrovascular diseases, diabetes, cancers and the like, is known as 'healthy soft gold' by European and American scientists and medical scientists, and has great market value in the fields of medicines, health products, foods, aquaculture and the like. The global astaxanthin production in 2017 was about 511.8 tons, the total yield was about $ 1 billion, and the yield in 2018 proliferated to $ 1.5 billion. The global astaxanthin production value is estimated to approach 3.4 billion dollars in 2024, while the astaxanthin production value in China will approach 2 billion dollars, and the wide market prospect also pushes the astaxanthin biosynthesis technology to develop continuously.

Currently, the species approved for the biosynthesis of astaxanthin are primarily phaffia rhodozyma and haematococcus pluvialis. The phaffia rhodozyma produces astaxanthin with (3R, 3' R) configuration, has low oxidation resistance and poor biological activity, and is mainly used for feed additives. The haematococcus pluvialis can accumulate astaxanthin with higher concentration configuration (3S, 3' S) in vivo, can be used in human food and cosmetics, but has rigorous culture conditions, needs to be cultured under the condition of proper illumination throughout the year, has longer period, and has long-term high production economic cost. With the development of synthetic biology in recent years, researchers have turned their eyes to other engineering bacteria with mature genetic backgrounds. The saccharomyces cerevisiae is a well-known safe mode microorganism, has clear genetic background and abundant molecular operation tools, can realize high-density fermentation and synthesize high-quality astaxanthin with a (3S, 3' S) configuration, and therefore, the realization of high yield of the astaxanthin in the saccharomyces cerevisiae has great competitiveness in industrial production. However, at present, a large number of intermediates are accumulated in the two-step reticular metabolic pathway from beta-carotene to astaxanthin synthesis, and the yield of astaxanthin is limited, so that the metabolic flux of the downstream reticular metabolic pathway is dredged, the accumulation of the intermediates is reduced, and the method has great significance for 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, inducibly expresses a beta-carotene hydroxylase gene CrtZ, and synthesizes an intermediate product beta-carotene into astaxanthin.

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

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

In some embodiments, in the recombinant Saccharomyces cerevisiae of any of the above embodiments, the β -carotene ketolase gene CrtW is BDC263CrtW derived from Brevundimonas vesiculosus (Brevundimonas vesicularis DC263), and the sequence of BDC263CrtW is shown in SEQ ID NO:28 at positions 9-734.

In some embodiments, in any of the above recombinant Saccharomyces cerevisiae, the β -carotene hydroxylase gene CrtZ is AaCrtZ derived from Agrobacterium aurantium (Agrobacterium aurantiacaum), and the sequence of AaCrtZ is shown as positions 9-497 in SEQ ID NO. 25.

In order to solve the technical problems, the invention also provides a recombinant expression vector of the 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 above 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 recombinant expression vectors described above, the β -carotene ketolase gene CrtW has a terminator downstream, preferably TDH2t, and TDH2t has the sequence shown in SEQ ID NO. 12.

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

In some embodiments, in any one of the recombinant expression vectors described above, a terminator is located downstream of the β -carotene hydroxylase gene CrtZ, preferably the terminator is ADH1t, and the sequence of ADH1t is shown in SEQ ID NO. 3.

In some embodiments, in any of the recombinant expression vectors described above, the β -carotene ketolase gene CrtW is BDC263CrtW derived from Brevundimonas vesicularis (Brevundimonas vesicularis DC263), and the sequence of BDC263CrtW is shown in SEQ ID NO:28 at positions 9-734.

In some embodiments, in any of the recombinant expression vectors described above, the β -carotene hydroxylase gene CrtZ is AaCrtZ derived from Agrobacterium aurantiacus (Agrobacterium aurantiacaum), and the sequence of AaCrtZ is shown as positions 9-497 in SEQ ID No. 25.

In some embodiments, the recombinant expression vector of any one of the above, wherein the recombinant expression vector is PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t, and the sequence 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 starting saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae;

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

In order to solve the technical problems, the invention also provides a method for producing astaxanthin, which comprises the steps of performing fermentation culture on any one of the recombinant saccharomyces cerevisiae so that the beta-carotene ketolase gene CrtW is constitutively expressed, and adding D- (+) -galactose to induce the expression of the beta-carotene hydroxylase gene CrtZ during the fermentation culture so as to synthesize astaxanthin.

In some embodiments, the above method for producing astaxanthin, wherein said fermentative culture comprises subjecting the recombinant Saccharomyces cerevisiae of any of the above to fermentative culture in an initial fermentation medium, e.g., at the initial OD₆₀₀Fermenting at 30 deg.C and pH5.8-6.2 with ventilation of 3.0vvm for 48h under the conditions of dissolved oxygen 30% and 300-500rpm, and adding D- (+) -galactose with final concentration of 20g/L to induce the expression of beta-carotene hydroxylase gene CrtZ;

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

supplementing a nitrogen source by adopting an intermittent feeding mode with decreasing concentration gradient, adding a certain volume of yeast extract powder mother liquor into a fermentation tank at intervals, for example, adding 1.5 times of yeast extract powder every 12 hours (12 th hour, 24 th hour, 36 th hour, 48 th hour and 60 th hour) from the beginning of fermentation by taking the concentration of yeast extract in an initial fermentation culture medium as 1.0 times, supplementing 5 times, then adding 1.0 times of yeast extract powder every 12 hours (72 th and 84 th hour of fermentation), supplementing 2 times, then adding 0.5 times of yeast extract powder every 12 hours (96 th and 108 th hour of fermentation), and supplementing 2 times.

In some embodiments, the method of producing astaxanthin according to any one of the above, wherein the initial fermentation medium is YPD containing 3% glucose (10g/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 the step of subjecting the recombinant Saccharomyces cerevisiae according to any one of the above to primary seed culture and secondary seed culture before fermentation culture;

the primary seed culture comprises inoculating any one of the recombinant Saccharomyces cerevisiae strains into a shake flask containing SC-Ura liquid medium, and culturing at 30 deg.C and 250rpm to OD₆₀₀A step of 6-7;

the secondary seed culture comprises transferring the primary seed obtained from the primary seed culture to a shake flask containing SC-Ura liquid medium, and culturing at 30 deg.C and 250rpm to OD₆₀₀A step of 5-6;

the first seed may be inoculated at 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 vector in the production of astaxanthin.

The recombinant yeast strain for producing astaxanthin regulates the expression of beta-carotene ketolase gene CrtW and beta-carotene hydroxylase gene CrtZ on an astaxanthin synthesis path through the combination of a constitutive promoter and an inducible promoter, clones promoters and gene elements with different combinations on a plasmid expression vector, transfers the plasmid expression vector into saccharomyces cerevisiae, and obtains a high-yield target strain through fermentation, astaxanthin yield detection and screening.

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

Drawings

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

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

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

Detailed Description

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

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

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

Saccharomyces cerevisiae S228C (Saccharomyces cerevisiae S228C) in the literature "Fisk et al. Saccharomyces cerevisiae S288C genome identification: a working hypothesis. Yeast.2006; 23(12) 857-.

The PRS416 vector is a product of Shanghai Lianmai bioengineering, Inc., and has a cargo number LM 4777.

The D- (+) -galactose is an aradin product with the product number of G100367.

In the examples, AaCrtZ is Agrobacterium aurantiacaum (Agrobacterium aurantiacaum) derived CrtZ, and BDC263CrtW is Brevundimonas vesicularis (Brevundimonas vesicularis DC263) derived CrtW.

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

Firstly, PCR amplification is carried out by taking the genomic 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 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)。

Secondly, PCR amplification is carried out by taking the genome DNA of the 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)。

Thirdly, taking the genome DNA of the saccharomyces cerevisiae S228C as a template and TDH3p _ F and TDH3p _ R as primers to carry out PCR amplification 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)。

And fourthly, carrying out PCR amplification by using the genome DNA of the 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)。

Fifthly, splicing ADH1t, Gal1, TDH3p and TDH2t obtained in the first step to the fourth step in an OE-PCR mode to obtain ADH1t-Gal1-TDH3p-TDH2t (SEQ ID NO:21) with digestion sites of BamHI and SalI at two ends, wherein BsmBI digestion sites are added between ADH1t and Gal1, and BsaI digestion sites are added between TDH3p and TDH2 t.

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

1. mu.l each of ADH1t, Gal1, TDH3p and TDH2t templates, 1. mu.l each of upstream primer, 1. mu.l each of downstream primer, 5. mu.l of 10 XBuffer, 4. mu.l of dNTP, 1. mu.l of Pfu DNA Polymerase were added to the mixture, and ddH was added thereto₂O make 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)。

sixthly, double enzyme digestion is carried out on ADH1t-Gal1-TDH3p-TDH2t by using restriction endonucleases SalI and BamHI to obtain a gene fragment; carrying out double digestion on the PRS416 vector by using restriction enzymes SalI and BamHI to obtain a vector fragment; the gene fragment is connected with the vector fragment to obtain a recombinant expression vector PRS416-ADH1t-Gal1-TDH3p-TDH2t, and the sequence is shown as SEQ ID NO. 22.

Example 2: insertion of foreign genes

Firstly, using AaCrtZ gene as a template, and using Primer 5_ F and Primer 5_ R as primers to carry out PCR amplification to obtain BsaI-AaCrtZ-BsaI, wherein the sequence of the BsaI-AaCrtZ-BsaI is shown as SEQ ID NO. 25, and the AaCrtZ gene is shown as 9-497 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)。

Secondly, BsaI-BDC263CrtW-BsaI is obtained by PCR amplification by taking the BDC263CrtW gene as a template and taking Primer 6_ F and Primer 6_ R as primers, the sequence of the BsaI-BDC263CrtW-BsaI is shown as SEQ ID NO:28, and the BDC263CrtW gene is shown as the 9 th to 734 th sites in the SEQ ID NO: 28.

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

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

And thirdly, performing PCR amplification by using the AaCrtZ gene as a template and using 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)。

And fourthly, performing PCR amplification by using the 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 the 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)。

Fifthly, digesting BsaI-BDC263CrtW-BsaI by BsaI enzyme to obtain a gene segment; the PRS416-ADH1t-Gal1-TDH3p-TDH2t obtained in example 1 was digested with BsaI to obtain a vector fragment; connecting the gene fragment with the vector fragment to obtain PRS416-ADH1t-Gal1-TDH3p-BDC263CrtW-TDH2 t;

using BsmBI to perform enzyme digestion on BsmBI-AaCrtZ-BsmBI to obtain a gene fragment; cutting PRS416-ADH1t-Gal1-TDH3p-BDC263CrtW-TDH2t by BsmBI enzyme to obtain a vector fragment; the gene fragment is connected with the vector fragment to obtain a recombinant expression vector PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t, and the sequence is shown as SEQ ID NO. 35.

Sixthly, carrying out enzyme digestion on BsaI-AaCrtZ-BsaI by BsaI to obtain a gene fragment; the PRS416-ADH1t-Gal1-TDH3p-TDH2t vector obtained in example 1 was digested with BsaI to obtain a vector fragment; connecting the gene fragment with the vector fragment to obtain PRS416-ADH1t-Gal1-TDH3p-AaCrtZ-TDH2 t;

using BsmBI to perform enzyme digestion on BsmBI-BDC263CrtW-BsmBI to obtain a gene fragment; cutting PRS416-ADH1t-Gal1-TDH3p-AaCrtZ-TDH2t by BsmBI enzyme to obtain a vector fragment; the gene fragment is connected with the vector fragment to obtain a recombinant expression vector PRS416-ADH1t-BDC263CrtW-Gal1-TDH3p-AaCrtZ-TDH2t, the sequence of which is shown in SEQ ID NO: shown at 36.

The recombinant expression vectors PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t and PRS416-ADH1t-BDC263CrtW-Gal1-TDH3p-AaCrtZ-TDH2t are schematically shown in FIG. 1.

Example 3: construction of recombinant Yeast strains for the production of astaxanthin

The recombinant expression vectors PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t and PRS416-ADH1t-BDC263CrtW-Gal1-TDH3p-AaCrtZ-TDH2t obtained in example 2 were transformed into β -carotene-producing strain Scy10026 Saccharomyces cerevisiae (Saccharomyces cerevisiae) by a lithium acetate method, and transformants were selected by 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 astaxanthin-producing recombinant yeast strains S1 and S2, respectively.

The construction method of the Scy10026 Saccharomyces cerevisiae is as follows: transferring the CYC1t-BtCrtI-HXT7p-TDH3p-PaCrtB-ADH1t fragment (SEQ ID NO:37) into Saccharomyces cerevisiae S288C, carrying out homologous recombination, replacing gal1, gal7 and gal10 genes, and integrating the genes into a chromosome; then, the CYC1t-PaCrtY-HXT7p-FBA1p-TmCrtE-TDH2t fragment (SEQ ID NO:38) was transferred into it, homologous recombination was carried out, the ypl062w gene was replaced, and the gene was integrated into the chromosome, thereby obtaining the beta-carotene-producing yeast Scy 10026.

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

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

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

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

Example 4: astaxanthin production by shake flask fermentation

Single colonies of recombinant Saccharomyces cerevisiae strains S1 and S2 were picked and inoculated into 5ml of SC-Ura liquid medium (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of mixed amino acid powder without uracil, and the balance water), and cultured overnight at 30 ℃ and 250rpm to obtain primary seeds. Then inoculating the primary seed with 5% (volume ratio) of inoculum size to fresh 5ml SC-Ura liquid medium for secondary seed culture, culturing at 30 deg.C and 250rpm to OD₆₀₀Secondary seeds were obtained 5-6. Inoculating the secondary seed into 250mL shake flask containing 50mL YPD medium, fermenting and culturing to obtain initial OD₆₀₀After fermentation for 48 hours, D- (+) -galactose was added at a final concentration of 2% (2g/100ml) to induce astaxanthin production, and the co-fermentation was terminated for 84 hours to obtain a fermentation broth.

Taking 1mL fermentation liquor, centrifuging at 12000rpm for 5min, pouring off the supernatant, adding 1mL sterile water to resuspend cells, centrifuging, and pouring off the supernatant. Add 1mL 3M HCl heavy suspension cells, boiling water bath 5min, ice bath 5min, centrifugation, pour off the supernatant, repeat boiling water bath 5min, ice bath 5min, repeat 2 times. Centrifuging and washing twice, discarding the supernatant, adding 1mL of acetone to resuspend cells, adding a small amount of quartz sand, performing vortex oscillation for 10min, centrifuging the acetone extracting solution at 12000rpm for 10min, filtering the supernatant by using a 0.2 mu m organic filter membrane, and adding 200 mu L of the supernatant into a sample bottle for astaxanthin HPLC detection.

The astaxanthin HPLC detection conditions are as follows:

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

column temperature: 25 ℃;

mobile phase: flow rate 1mL/min, mobile phase B: acetonitrile to water 9:1 (volume ratio), mobile phase C: methanol to 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. And calculating the concentration of the astaxanthin by an external standard method.

The astaxanthin purity was calculated according to the following formula:

W＝Ca/C*100％

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

wherein the carotenoid concentration is detected by adopting an HPLC external standard method, and the HPLC detection condition is consistent with the astaxanthin HPLC detection condition.

The astaxanthin production of recombinant s.cerevisiae strain S2 was found to be 43.72mg/L (purity of 57.51%), the astaxanthin production of recombinant s.cerevisiae strain S1 was found to be 70.67mg/L (purity of 90.65%), and a comparison of the astaxanthin production of recombinant s.cerevisiae strains S1 and S2 is shown in fig. 2.

Example 5: fermentation tank for producing astaxanthin

First-stage seed culture: inoculating S1 into 250mL shake flask containing 50mL SC-Ura liquid culture medium, culturing at 30 deg.C and 250rpm to OD₆₀₀First-order seeds were obtained 6-7.

II, secondary seed culture: inoculating the first seed at 5% (volume ratio) into 250mL shake flask containing 50mL SC-Ura liquid culture medium, culturing at 30 deg.C and 250rpm to OD₆₀₀Secondary seeds were obtained 5-6.

Thirdly, inoculating in a fermentation tank: transferring the second seed to 7L fermenter with 10% (volume ratio) for fermentation, wherein the initial liquid loading of the fermenter is 3.5L, the initial fermentation medium is YPD (10g/L yeast extract, 20g/L peptone, 30g/L glucose, and water as the rest) containing 3% glucose, and the initial OD is₆₀₀0.5. Setting fermentation tank parameters: the fermentation temperature was 30 ℃, pH was controlled at 5.8, aeration was 3.0vvm, Dissolved Oxygen (DO) was 30%, rotation speed was 300-500rpm, and stirring was related to DO.

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

Through HPLC detection same as that in example 4, the astaxanthin yield of the final 7L fermentation tank reaches 366.90mg/L, which is improved by 4.19 times compared with the shake 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 D- (+) -galactose inducer was set to 20 deg.C, and the fermentation test results are shown in FIG. 3, in which the astaxanthin production in the final 7L fermentor reached 464.90mg/L, which was measured by the same HPLC test as in example 4, and was improved by 5.58 times as compared with the shake flask fermentation yield (70.67 mg/L).

Sequence listing

<110> Wanhua chemical group, 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 (10)

1. A recombinant saccharomyces cerevisiae constitutively expresses a beta-carotene ketolase gene CrtW, inducibly expresses a beta-carotene hydroxylase gene CrtZ, and synthesizes an intermediate product beta-carotene into astaxanthin.

2. The recombinant saccharomyces cerevisiae of claim 1, wherein: the constitutive promoter for constitutive expression of the beta-carotene ketolase gene CrtW is TDH3 p; and/or

The inducible promoter used for inducible expression of the beta-carotene hydroxylase gene CrtZ is Gal 1.

3. A recombinant expression vector of Saccharomyces cerevisiae 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.

4. The recombinant expression vector of claim 3, wherein: the constitutive promoter is TDH3 p; and/or

The inducible promoter is Gal 1.

5. The recombinant expression vector according to claim 3 or 4, wherein: the recombinant expression vector is PRS416-ADH1t-AaCrtZ-Gal1-TDH3p-BDC263CrtW-TDH2t, and the sequence is shown in SEQ ID NO. 35.

6. A method for constructing the recombinant Saccharomyces cerevisiae of claim 1 or 2, comprising the steps of transferring the recombinant expression vector of any one of claims 3-5 into a starting Saccharomyces cerevisiae to obtain a recombinant Saccharomyces cerevisiae;

the starting saccharomyces cerevisiae is saccharomyces cerevisiae capable of synthesizing beta-carotene.

7. A method for producing astaxanthin comprising the steps of subjecting the recombinant Saccharomyces cerevisiae as set forth in claim 1 or 2 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.

8. The method of claim 7, wherein: the fermentation culture comprises the steps of carrying out fermentation culture on the recombinant saccharomyces cerevisiae in an initial fermentation culture medium, wherein the fermentation pH is 5.8-6.2, and adding D- (+) -galactose to induce the expression of a beta-carotene hydroxylase gene CrtZ;

before adding D- (+) -galactose, feeding glucose and controlling the glucose to be in a range of 0-1 g/L; stopping adding glucose while adding D- (+) -galactose, adding ethanol while controlling at 0.5-10g/L, and controlling at 20-30 deg.C;

and supplementing a nitrogen source in an intermittent feeding mode with decreasing concentration gradient during the fermentation culture.

9. The method according to claim 7 or 8, characterized in that: before fermentation culture, the method also comprises the step of performing primary seed culture and secondary seed culture on the recombinant saccharomyces cerevisiae of claim 1 or 2;

the primary seed culture comprises inoculating the recombinant Saccharomyces cerevisiae of claim 1 or 2 into a shake flask containing SC-Ura liquid medium, and culturing to OD₆₀₀A step of 6-7;

the second-stage seed culture comprises transferring the first-stage seed obtained by the first-stage seed culture to a shake flask containing SC-Ura liquid culture medium, and culturing to OD₆₀₀5-6.

10. Use of a recombinant s.cerevisiae as claimed in claim 1 or 2 and/or a recombinant expression vector as claimed in any one of claims 3 to 5 for the production of astaxanthin.

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