Methods to improve carotenoid production by Pharf red yeast

Abstract

This invention discloses a method for improving carotenoid production in *Phaefflera rubrum* yeast. The invention obtains recombinant *Phaefflera rubrum* yeast by upregulating the expression level of glutathione S-transferase (GST1) in *Phaefflera rubrum*. Fermentation experiments on YPD and YM media showed that the biomass, carotenoid yield, and astaxanthin yield of the recombinant *Phaefflera rubrum* yeast were all higher than those of the recipient *Phaefflera rubrum* yeast. This recombinant *Phaefflera rubrum* yeast has significant industrial value in the preparation of carotenoids and / or astaxanthin.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a method for improving the production of carotenoids by Pharfovia rubescens. Background Technology

[0002] Carotenoids are unsaturated compounds with terpene groups. More than 700 different structures of carotenoids have been identified, each containing multiple conjugated double bonds. Carotenoids can be derived from the basic structure of lycopene, which contains 11 conjugated double bonds, through reactions such as oxidation, hydrogenation, dehydrogenation, cyclization, carbon skeleton rearrangement, and degradation. Carotenoid molecules are divided into two categories based on the presence or absence of oxygen groups: those without oxygen atoms are called carotenes, and those containing oxygen functional groups (such as hydroxyl, epoxy, methoxy, ketone, and hydroxy acid groups) are called xanthophylls. Carotenoids mainly include lycopene, α-carotene, β-carotene, and γ-carotene; xanthophylls mainly include zeaxanthin, astaxanthin, and lutein. Carotenoids have antioxidant, immunomodulatory, anticancer, and anti-aging effects. Lutein, for example, has antioxidant and light-filtering properties, which can protect eyesight to some extent, prevent vision decline, and prevent eye diseases such as cataracts. Natural astaxanthin is currently the most potent antioxidant, playing a crucial role in aquaculture by reducing pigmentation in salmon and crustaceans. Astaxanthin can prevent lipid oxidation, improve metabolism, combat aging, and enhance animal reproductive and growth performance. The market prospects for astaxanthin are vast.

[0003] Because of the low fermentation temperature of Pharfogel's red yeast, which results in high energy consumption during production; the low pigment yield of wild strains; and the long culture cycle of strains, industrial production of Pharfogel's red yeast is limited. Therefore, research on the genetic improvement of the physiological and biochemical properties of Pharfogel's red yeast is very important. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to increase the content of carotenoids and / or astaxanthin in Pharfia redis.

[0005] To solve the above-mentioned technical problems, the present invention provides a method for constructing recombinant red Paffa yeast, the method comprising upregulating (increasing or raising) the expression level of the GST1 gene encoding glutathione S-transferase in red Paffa yeast to obtain recombinant red Paffa yeast.

[0006] The recombinant Pharfogel's carotenoid and / or astaxanthin content is higher than that of Pharfogel's red yeast.

[0007] Furthermore, in the above method, the glutathione S-transferase is selected from any one of the following proteins (A1)-A3):

[0008] A1) A protein with the amino acid sequence shown in SEQ ID No. 3;

[0009] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein shown in A1) and having more than 80% identity with the protein shown in A1) and having glutathione S-transferase activity.

[0010] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0011] In this invention, the protein described in A1) consists of 247 amino acids.

[0012] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0014] Table 1: Sequence of Labels

[0015] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0016] Furthermore, in the above method, the preparation method of the recombinant bacteria includes introducing the GST1 gene into Pharfogel's red yeast rice, which serves as the recipient bacteria.

[0017] Furthermore, in the above method, the GST1 gene is introduced into the recipient bacteria through an expression cassette containing the GST1 gene.

[0018] Furthermore, in the above method, the GST1 gene is selected from any one of B1-B3:

[0019] B1) The nucleotide sequence of the coding strand is the DNA molecule shown in positions 473-1216 of SEQ ID No. 10;

[0020] B2) The coding sequence of the coding strand is the DNA molecule shown in positions 473-1216 of SEQ ID No. 10;

[0021] B3) DNA molecules that share more than 80% identity with DNA molecules defined by B1) or B2) and encode proteins that have glutathione S-transferase function.

[0022] Furthermore, in the above method, the expression cassette is introduced into the recipient bacteria via a recombinant expression vector with the nucleotide sequence SEQ ID No. 10.

[0023] In the above methods, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0024] In the above method, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0025] In one embodiment of the present invention, the expression cassette containing the glutathione S-transferase gene is a DNA molecule with the nucleotide sequence shown in positions 7-1410 of SEQ ID No. 10, and the expression cassette contains the promoter TEFIp and ADH1t. The nucleotide sequence of the promoter TEFIp is shown in positions 7-466 of SEQ ID No. 10; the nucleotide sequence of the gene encoding GST1 is shown in positions 473-1216 of SEQ ID No. 10; and the nucleotide sequence of the terminator ADH1t is shown in positions 1223-1410 of SEQ ID No. 10.

[0026] In a second aspect, the present invention provides recombinant red Pharf yeast constructed using any of the methods described above.

[0027] Thirdly, the invention provides the use of the aforementioned recombinant red Pharf yeast in the production of carotenoids and / or astaxanthin.

[0028] In a fourth aspect, the present invention provides a method for preparing carotenoids and / or astaxanthin, the method comprising preparing carotenoids and / or astaxanthin using the above-described recombinant Pharfogel's yeast.

[0029] Fifthly, the present invention provides the application of the above-mentioned glutathione S-transferase or related biological materials in the preparation of recombinant red Paffir yeast producing carotenoids and / or astaxanthin; said biological material is any one of C1)-C3):

[0030] C1) is the nucleic acid molecule that encodes the above-mentioned glutathione S-transferase;

[0031] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0032] C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette described in C2).

[0033] In one embodiment of the present invention, the Phaffia rhodozyma as the recipient bacterium may be Phaffia rhodozyma AS2.1557.

[0034] This invention obtains a strain with high carotenoid production through functional evolution. Through genome resequencing and transcriptome sequencing analysis, a key influencing factor affecting carotenoid synthesis in Pharfogel's red yeast was identified, which can then be used as a method to improve carotenoid production in Pharfogel's red yeast.

[0035] The recombinant Pharfovia erythrosporum provided by this invention has higher biomass, carotenoid content, and astaxanthin content than the initial strain. This recombinant Pharfovia erythrosporum has significant industrial value in the preparation of carotenoids and / or astaxanthin. Attached Figure Description

[0036] Figure 1 Physical map of recombinant expression plasmid YCp-TA-GST1.

[0037] Figure 2 This is a calculation method for determining carotenoid content using ultraviolet spectrophotometry. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Saccharomyces cerevisiae YS58 is disclosed in the literature “Teunissen AWRH, Holub E, van den Hucht J, van der Berg JA, Stensma HY. (1993). Sequence of the openreading frame of the FLO1 gene from Saccharomyces cerevisiae. Yeast, 9:423-427.” The public can obtain the above biological material from the applicant. The obtained biological material is only used for repeating the experiments of this invention and cannot be used for other purposes.

[0041] Phaffia rhodozyma AS2.1557 is disclosed in the literature “Phaff HJ, Miller MW, Yoneyama M, et al. A comparative study of the yeast florae associated with trees on the Japanese islands and on the west coast of North America[J]. Fermentation Technology Today, 1972, 1(1):759-774.” The public can obtain the above-mentioned biological material from the applicant. The above-mentioned biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0042] YPD medium: Dissolve 20g glucose, 20g peptone, and 10g yeast powder in distilled water, bring the volume to 1L, natural pH; sterilize by moist heat at 8 psi for 30 min.

[0043] YM medium: Dissolve 20g glucose, 5g peptone, 3g yeast extract, and 3g malt extract in distilled water, bring the volume to 1L, and set to natural pH; sterilize by moist heat at 8 psi for 30 min.

[0044] The fermentation culture temperature in the following examples is 20°C.

[0045] In the following examples, the analysis of carotenoid content was performed using ultraviolet spectrophotometry. In the analysis of astaxanthin content, astaxanthin (Aladdin Reagent (Shanghai) Co., Ltd., product catalog number A141428) was used as the standard, and a standard curve was plotted by high performance liquid chromatography to quantitatively analyze astaxanthin.

[0046] Example 1: Construction of a carotenoid-producing Pharfovia rubescens strain

[0047] 1.1 Obtaining the GST1 gene

[0048] 1.1.1 PCR amplification of the GST1 gene

[0049] Based on the reported GST1 gene sequence of *Phaeopsyrum rubrum* (GenBank accession number KX384908.1, 2016-09-26), the following primers were designed:

[0050] Upstream primer P1:

[0051] 5'-caatctaatctaagttttaattacaaa tctaga ATGAGCACCGAACTGAAGAAAC-3' (SEQ ID No. 1, lowercase letters are homologous arms, and the underlined part is the XbaI restriction site)

[0052] Downstream primer P2:

[0053] 5'-taataataaaaatcataaatcataagaaattcgc ggatcc TCACTTCGTGCTCGGAGG-3' (SEQ ID No. 2, lowercase letters indicate homologous arms, and underlined areas indicate BamHI restriction enzyme recognition sites)

[0054] 1.1.2. PCR amplification to obtain the GST1 gene

[0055] Using genomic DNA of Pharfia redis as a template, PCR amplification was performed with primers P1 and P2 to obtain PCR amplification products containing the gene GST1.

[0056] The GST1 gene sequence is shown in positions 473-1216 of SEQ ID No. 10.

[0057] The PCR reaction system described above is as follows: 50 ng genomic DNA, primer P1 final concentration 0.3 μmol / L, primer P2 final concentration 0.3 μmol / L, KOD-Plus-Neo DNA polymerase 1 μL, 10×KOD buffers 5 μL, 2 mM dNTPs 5 μL, 25 mM MgSO4 2+ Add 2 μL of deionized water to bring the system to 50 μL, and mix well.

[0058] The PCR reaction conditions described above are: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 59℃ annealing for 30 seconds, 68℃ extension for 1 minute, for a total of 30 cycles; and 68℃ extension for 10 minutes to ensure complete product extension.

[0059] 1.2 Construction of YCp-TA

[0060] 1.2.1 Design the following primers:

[0061] Upstream primer P3:

[0062] 5'-gtatcacgaggccctttcgtcttcaa gaattc GAATCCTTACATCACACCCAAT-3' (SEQ ID No. 4, lowercase letters indicate homologous arms, and underlined areas indicate EcoRI restriction site)

[0063] Downstream primer P4:

[0064] 5'-c ggatcc aagtgggtaagcaagtt tctaga TTTGTAATTAAAACTTAGATTAGAT-3' (SEQ ID No. 5, lowercase letters are homologous arms, and the underlined sequences are the BamHI and XbaI restriction enzyme recognition sites, respectively)

[0065] Upstream primer P5:

[0066] 5'-caaa tctaga aacttgcttacccactt ggatcc GCGAATTTCTTATGATTTATGAT-3' (SEQ ID No. 6, lowercase letters are homologous arms, and the underlined sequences are the XbaI and BamHI restriction enzyme recognition sites, respectively)

[0067] Downstream primer P6:

[0068] 5'-ggttgaaggctctcaagggcatcg gtcgac CCGGTAGAGATGTGG-3' (SEQ ID No. 7, lowercase letters indicate homologous arms, and underlined areas indicate SalI restriction enzyme recognition sites)

[0069] Using genomic DNA of Saccharomyces cerevisiae YS58 as a template, PCR amplification was performed using primers P3 and P4, and primers P5 and P6 to obtain PCR amplification products, which were named TEFIp and ADH1t, respectively.

[0070] The PCR reaction system described above is as follows: template 50 ng, primer final concentration 0.3 μmol / L, KOD-Plus-Neo DNA polymerase 1 uL, 10×KOD buffers 5 uL, 2 mM dNTPs 5 uL, 25 mM Mg2+ 2 uL, and deionized water is added to bring the system to 50 uL and mixed well.

[0071] The PCR reaction conditions were as follows: denaturation at 94℃ for 5 minutes; denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 68℃ for 30 seconds, for a total of 30 cycles; and extension at 68℃ for 10 minutes to ensure complete product extension.

[0072] 1.2.2 Construction of recombinant plasmids YCp-TA and YCp-TA-GST1

[0073] YCp50 was double-digested with EcoRI and SalI to obtain a large vector fragment. The gene fragments TEFIp and ADH1t, along with the large vector fragment obtained from the double digestion, were electroporated into Saccharomyces cerevisiae YS58. The recombinant plasmid was obtained by ligation using homologous arms and named YCp-TA. YCp-TA was sequenced, and the sequencing results showed that the nucleotide sequence of YCp-TA is shown in SEQ ID No. 9.

[0074] The nucleotide sequence of YCp50 is shown in SEQ ID No. 8.

[0075] YCp-TA was double-digested with XbaI and BamHI to obtain a large vector fragment. The PCR amplification product containing the GST1 gene obtained in section 1.1.2 was electroporated into *Saccharomyces cerevisiae* YS58, and ligated using homologous arms to obtain the recombinant expression plasmid, named YCp-TA-GST1. YCp-TA-GST1 was sequenced, and the sequencing results showed that the nucleotide sequence of YCp-TA-GST1 is shown in SEQ ID No. 10. The physical map of the recombinant expression plasmid YCp-TA-GST1 is shown below. Figure 1 As shown.

[0076] The recombinant plasmid YCp-TA-GST1 contains an expression cassette with nucleotide sequences as shown in positions 7-1410 of SEQ ID No. 10, the expression cassette containing the promoters TEFIp and ADH1t. The nucleotide sequence of the promoter TEFIp is shown in positions 7-466 of SEQ ID No. 10; the nucleotide sequence of the gene encoding GST1 is shown in positions 473-1216 of SEQ ID No. 10; and the nucleotide sequence of the terminator ADH1t is shown in positions 1223-1410 of SEQ ID No. 10.

[0077] Example 2: Construction of recombinant bacteria PR / YCp-TA and PR / YCp-TA-GST1

[0078] 2.1 Construction of recombinant bacteria PR / YCp-TA and PR / YCp-TA-GST1

[0079] Plasmids YCp-TA and YCp-TA-GST1 were transformed into *Phaefflera rubescens* AS2.1557 (hereinafter referred to as host strain PR). Transformants were screened on YPD plates containing 50 μg / mL LG418. Because the plasmids contained the G418 gene, the transformants containing plasmids YCp-TA and YCp-TA-GST1 grew on YPD plates containing 50 μg / mL LG418, while the host strain PR did not grow. The transformant plasmids were extracted and sequenced to confirm their correctness, proving that plasmids YCp-TA and YCp-TA-GST1 were successfully introduced into the host strain PR. The transformant strain containing plasmid YCp-TA-GST1 was named recombinant strain PR / YCp-TA-GST1, and the transformant strain containing the empty vector plasmid YCp-TA was named PR / YCp-TA.

[0080] Total RNA was extracted from the control strain PR / YCp-TA and the recombinant strain PR / YCp-TA-GST1. Reverse transcription was performed using a qRT-PCR kit (SYBR Green I) to obtain the corresponding cDNA. Using this cDNA as a template, the expression level of the GST1 gene was relatively quantified using the Quant two-step real-time qRT-PCR method (SYBR Green I) with primer pair GST1QPF-2 / GST1QPR-2, using Actin as an internal control. Experimental data were collected using LightCycler96 software SW 1.1 (Roche). Differences in gene expression levels were analyzed using the ΔΔCT method. The ACT1 gene in *Phaefflera rubrum* was used as a control. Each experiment was performed in triplicate. The results showed that the expression level of GST1 in strain PR / YCp-TA-GST1 was 2.40 times that of the control strain PR / YCp-TA, indicating enhanced GST1 expression in the recombinant strain PR / YCp-TA-GST1.

[0081] Primer GST1QPF-2: GGGTGTTTTG GTCACCGTTC (SEQ ID No. 11);

[0082] Primer GST1QPR-2: CTCATCGTCG GCTCTCTCTC (SEQ ID No. 12);

[0083] Primer ACTINQPF-1: CCCCAAGGCT AACAGAGAGA (SEQ ID No. 13);

[0084] Primer ACTINQPR-1: CAGAGGCGTA CAAAGAAAGC A (SEQ ID No. 14).

[0085] 2.2 Production of carotenoids and astaxanthin by recombinant bacteria in YPD medium

[0086] PR / YCp-TA and PR / YCp-TA-GST1 were inoculated into 2.5 mL of YPD medium and activated at 20°C and 200 rpm for 48 h. Then, they were transferred at a 10% inoculum and cultured for 24 h. Finally, they were inoculated into 20 mL of YPD liquid medium (250 mL Erlenmeyer flasks) at a 10% inoculum, with three flasks containing each recombinant strain. All flasks were then incubated at 20°C for 84 h.

[0087] After cultivation, the culture media of PR / YCp-TA and PR / YCp-TA-GST1 were taken separately. The bacterial cells were disrupted by acid-heat method, extracted with acetone, and the carotenoid content in each sample was determined by ultraviolet spectrophotometry. The astaxanthin content in each sample was determined by high performance liquid chromatography. The experiment was repeated three times.

[0088] Cell wall disruption method: Take 5 mL of fermentation broth into a 7 mL centrifuge tube, centrifuge at 8000 rpm for 3 min, and rinse the cells three times with deionized water. Then add 1.8 mL of HCl solution (3 mol / L), shake thoroughly and soak for 30 min, then heat in a boiling water bath for 8-9 min. After observing that the cells become flocculent, immediately cool in an ice bath.

[0089] After cooling to room temperature, centrifuge at 8000 rpm for 5 min and discard the supernatant. Wash twice with deionized water and discard the supernatant. Then add 5 mL of acetone, extract by shaking in the dark for 15 min until the bacterial cells are colorless. Centrifuge at 8000 rpm for 5 min and collect the supernatant.

[0090] Determination of carotenoid content by ultraviolet spectrophotometry: The absorbance (A value) is measured at 474 nm using an ultraviolet spectrophotometer. The calculation of carotenoid content is as follows... Figure 2 As shown.

[0091] Determination of astaxanthin content by high performance liquid chromatography:

[0092] The samples were analyzed by HPLC (Agilent 1260TCC DEACN13785, DAD detector). The ODS column specifications were Eclipse Plus C18, 4.6 x 250 mm, 5 μm. The mobile phase was methanol:acetonitrile = 9:1, the flow rate was 1 mL / min, the column temperature was 30℃, the injection volume was 10 μL, and the detection wavelength was 478 nm. 500 μL of the extract was filtered into a chromatographic vial, and then analyzed by HPLC. The peak area was substituted into the standard curve formula to calculate the astaxanthin content in the fermentation broth.

[0093] Table 2: Effects of GST1 overexpression in PR on the content and yield of carotenoids and astaxanthin (YPD liquid medium)

[0094]

[0095] Note: DCW refers to stem cells.

[0096] Table 2 shows that the biomass, carotenoid production, and astaxanthin production of the GST1 overexpressing strain PR (YCp-TA-GST1) were increased by 7%, 26%, and 38%, respectively, during fermentation in YPD medium compared with the original strain PR, demonstrating that overexpression of GST1 in Pharfia rubra PR increased biomass and carotenoid production.

[0097] 2.3 Production of carotenoids by recombinant bacteria in YM medium

[0098] PR / YCp-TA and PR / YCp-TA-GST1 were inoculated into 2.5 mL of YPD medium and activated at 20°C and 200 rpm for 48 h. Then, they were transferred at a 10% inoculum and cultured for 24 h. Finally, they were inoculated into 20 mL of LYM liquid medium (250 mL Erlenmeyer flasks) at a 10% inoculum, with three flasks containing each recombinant strain. All flasks were then incubated at 20°C for 84 h.

[0099] After the culture was completed, the culture media of PR / YCp-TA and PR / YCp-TA-GST1 were taken and subjected to cell disruption and extraction treatment using the method described in 2.2 of Example 2. The content of carotenoids in each sample was determined by ultraviolet spectrophotometry, and the content of astaxanthin in each sample was determined by high performance liquid chromatography.

[0100] Table 3: Effects of GST1 overexpression in PR on the content and yield of carotenoids and astaxanthin (YM liquid medium)

[0101]

[0102] Note: DCW refers to stem cells.

[0103] Table 3 shows that the biomass, carotenoid production, and astaxanthin production of the GST1-expressing strain PR / YCp-TA-GST1 were increased by 12%, 44%, and 65%, respectively, during fermentation in YM medium compared to the starting strain PR, demonstrating that overexpression of GST1 in Pharfia przewalskii increased biomass and carotenoid production.

[0104] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. sequence list <110> Institute of Microbiology, Chinese Academy of Sciences <120> Methods to improve carotenoid production by Pharf red yeast <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 55 <212> DNA <213> Artificial Sequence <400> 1 caatctaatc taagttttaa ttacaaatct agaatgagca ccgaactgaa gaaac 55 <210> 2 <211> 58 <212> DNA <213> Artificial Sequence <400> 2 taataataaa aatcataaat cataagaaat tcgcggatcc tcacttcgtg ctcggagg 58 <210> 3 <211> 247 <212> PRT <213> Phaffia rhodozyma <400> 3 Met Ser Thr Glu Leu Lys Lys Pro Leu Glu Leu Tyr Ser Ala Ser Thr 1 5 10 15 Pro Asn Gly Gln Lys Ala Thr Val Phe Leu Glu Glu Leu Lys Gln Ile 20 25 30 Tyr Pro Ala Phe Asp Tyr His Val Asn Glu Ile Asp Ile Gln Lys Asn 35 40 45 Glu Gln Lys Glu Glu Trp Phe Leu Lys Ile Asn Pro Asn Gly Arg Ile 50 55 60 Pro Ala Leu Val Asp Pro Asn Arg Gly Asp His Ala Val Trp Glu Ser 65 70 75 80 Ala Ala Ile Leu Leu Tyr Leu Glu Lys His Tyr Asp Pro Asp His Lys 85 90 95 Phe Ser Phe Gly Asp Ser Ala Asp Glu Asp Asn Phe Arg Ser Glu Ile 100 105 110 Leu Gln Trp Met Phe Phe Val His Gly Gly Ile Gly Pro Met Gln Gly 115 120 125 Gln Ala Val Phe Phe Ser Lys Tyr Ala Pro Glu Gln Ile Pro Tyr Ala 130 135 140 Ile Lys Arg Tyr Gln Asp Glu Thr Lys Arg Leu Tyr Ser Val Leu Asp 145 150 155 160 Thr Arg Leu Arg Asn Arg Asp Tyr Leu Val Gly Pro Gly Arg Gly Lys 165 170 175 Tyr Ser Ile Ala Asp Ala Asn Ala Leu Pro Trp Val Phe Trp Ser Pro 180 185 190 Phe Ala Gly Ile Asp His Val Glu Ile Pro Thr Gly Val Lys Ala Trp 195 200 205 Leu Asp Arg Asn Leu Glu Arg Glu Pro Thr Met Arg Gly Leu Lys Val 210 215 220 Gly Pro Asn Thr Ser Ser Asp Met Ile Glu Gln Ile Lys Lys Tyr Gly 225 230 235 240 Ala Leu Pro Pro Ser Thr Lys 245 <210> 4 <211> 54 <212> DNA <213> Artificial Sequence <400> 4 gtatcacgag gccctttcgt cttcaagaat tcgaatcctt acatcacacc caat 54 <210> 5 <211> 55 <212> DNA <213> Artificial Sequence <400> 5 cggatccaag tgggtaagca agtttctaga tttgtaatta aaacttagat tagat 55 <210> 6 <211> 56 <212> DNA <213> Artificial Sequence <400> 6 caaatctaga aacttgctta cccacttgga tccgcgaatt tcttatgatt tatgat 56 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <400> 7 ggttgaaggc tctcaagggc atcggtcgac ccggtagaga tgtgg 45 <210> 8 <211> 7700 <212> DNA <213> Artificial Sequence <400> 8 gaattcgaca tggaggccca gaataccctc cttgacagtc ttgacgtgcg cagctcaggg 60 gcatgatgtg actgtcgccc gtacatttag cccatacatc cccatgtata atcatttgca 120 tccatacatt ttgatggccg cacggcgcga agcaaaaatt acggctcctc gctgcagacc 180 tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt gtccccacgc cgcgcccctg 240 tagagaaata taaaaggtta ggatttgcca ctgaggttct tctttcatat acttcctttt 300 aaaatcttgc taggatacag ttctcacatc acatccgaac ataaacaacc gatatctcta 360 gaggatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc 420 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat 480 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttggggcgc 540 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact600 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgaccgatgc ccttgagagc 660 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat 720 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt 780 cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg 840 aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cgcagtatag cgaccagcat tcacatacga ttgacgcatg atattacttt ctgcgcactt aacttcgcat ctgggcagat gatgtcgagg cgaaaaaaaa tataaatcac gctaacattt gataaata gacaactac aatataaaa aactatacaa atgacaagtt cttgaaaaca agaatctttt tattgtcagt actgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 1200 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 1260 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 1320 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 1380 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 1440 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 1500 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 1560 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 1620 ctaatacctg gaatgctgtt ttgccgggga tcgcagtggt gagtaaccat gcatcatcag 1680 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 1740 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 1800 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 1860 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg 1920 aaacgtgagt cttttcctta cccatggttg tttatgttcg gatgtgatgt gagaactgta 1980 tcctagcaag attttaaaag gaagtatatg aaagaagaac ctcagtggca aatcctaacc 2040 ttttatattt ctctacaggg gcgcggcgtg gggacaattc aacgcgtctg tgaggggagc 2100 gtttccctgc tcgcaggtct gcagcgagga gccgtaattt ttgcttcgcg ccgtgcggcc 2160 atcaaaatgt atggatgcaa atgattatac atggggatgt atgggctaaa tgtacgggcg 2220 acagtcacat catgcccctg agctgcgcac gtcaagactg tcaaggaggg tattctgggc 2280 ctccatgtcc ccgggaatct cggtcgtaat gatttttata atgacgaaaa aaaaaaaatt 2340 ggaaagaaaa cccccccccc gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct 2400 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc 2460 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac 2520 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc 2580 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc 2640 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag aaattccccc ttacacggag gcatcaagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagg tgggccattc tcatgaagaa tatcttgaat ttattgtcat attactagtt ggtgtggaag tccatatatc ggtgatcaat atagtggttg acatgctggc tagtcaacat tgagcctttt gatcatgcaa fatherattacg gtattttaca atcaaatatc aaacttaact attgacttta 3240. 3240. 3240. 3240. 3240. 3240. 3240. 3240. 3240. 3240. 3240. 3240 agcttttaac ttgtatccta ggttatctat gctgtctcac catagagaat attack tcagaatgta tgtccatgat tcgccgggta aatacatata atacacaaat ctggcttaat 3360 aaagtctata atatatctca taaagaagtg ctaaattggc tagtgctata tatttttaag 3420 aaaatttctt ttgactaagt ccatatcgac tttgtaaaag ttcactttag catacatata 3480 ttacacgagc cagaaattgt aacttttgcc taaaatcaca aattgcaaaa tttaattgct 3540 tgcaaaaggt cacatgctta taatcaactt ttttaaaaat ttaaaatact ttttatttt 3600 ttattttaa acataaatga aataatttat ttattgttta tgattaccga aacataaaac 3660 ctgctcaaga aaaagaaact gttttgtcct tggaaaaaaa gcactaccta ggagcggcca 3720 aaatgccgag gctttcatag cttaaactct ttacagaaaa taggcattat agatcagttc 3780 gagttttctt attcttcctt ccggttttat cgtcacagtt ttacagtaaa tagtatcac 3840 ctcttagagt taactatgag ataagcaagt atcatctcat ttcatttacc tgaagtcgag 3900 taaacagaaa atccaattgt tgatgaacct caatgactta gaactatcta tcggcagatc 3960 atataaagag gatttaggta cctagaggac tgtacctgga gtatatatat atatatatat 4020 atattatctc aactatagtc catagaggtt tctttcttga ggccttaaac tgctaaagaa 4080 tgatattggt ggaatgcaag caccaatctc tcttctttcg taactgttca tatacttcaa 4140 accaagaatg taacgggcat tgacccatcc aaaaccttca gtagctgccc ctttaaagtc 4200 agcaccttga ttaccgtatt ctgcttcaac acgatgagga tctgttcctc ttgtgacatc 4260 atatttttca accacaatac cattataatc gacaaaagcc tttgtcatca tgaaaagcca 4320 tctataagct agcctattcg ttacagttaa ataaccataa gaacggaggc cttcccaagc 4380 aagaatttga tggggtgccc aaccaaatgg atagtcccat tgtctaattg gtctcgaaat 4440 agaaattggg cctcgagaac gctccgtaca tgcagctaaa cctccaagca tctctaactt 4500 gggtagtgct ttctccacca ttttctgtgc ttgctccttc gtggcaagtc cagcccataa 4560 tgcccagaat gtagttgcgg attcgtatga cgttctgtgc ttgatttttg tgttgtagtc 4620 aaagaaaaac cccgactcgt catcccacat atatttggta attgatgagg caacgctaat 4680 tatcaacata tagattgtta tctatctgca tgaacacgaa atctttactt gacgacttga 4740 ggctgatggt gtttatgcaa agaaaccact gtgtttaata tgtgtcactg tttgatatta 4800 ctgtcagcgt agaagataat agtaaaagcg gttaataagt gtatttgaga taagtgtgat 4860 aaagttttta cagcgaaaag acgataaata caagaaaatg attacgagga tacggagaga 4920 ggtatgtaca tgtgtattta tatactaagc tgccggcggt tgtttgcaag accgagaaaa 4980 ggctagcaag aatcgggtca ttgtagcgta tgcgcctgtg aacattctct tcaacaagtt 5040 tgattccatt gcggtgaaat ggtaaaagtc aaccccctgc gatgtatatt ttcctgtaca 5100 atcaatcaaa aagccaaatg atttagcatt atctttacat cttgttattt tacagatttt 5160 atgtttagat cttttatgct tgcttttcaa aaggcctgca ggcaagtgca caaacaatac 5220 ttaaataaat actactcagt aataacctat ttcttagcat ttttgacgaa atttgctatt 5280 ttgttagagt cttttacacc atttgtctcc acacctccgc ttacatcaac accaataacg 5340 ccatttaatc taagcgcatc accaacattt tctggcgtca gtccaccagc taacataaaa 5400 tgtaagctct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc 5460 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc 5520 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc 5580 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata 5640 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg 5700 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 5760 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 5820 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 5880 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 5940 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 6000 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 6060 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 6120 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 6180 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 6240 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 6300 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 6360 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 6420 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 6480 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 6540 gattatcaaa aggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 6600 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 6660 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6720 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6780 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6840 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 6900 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg 6960 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 7020 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 7080 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 7140 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 7200 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata 7260 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 7320 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 7380 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 7440 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 7500 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 7560 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 7620 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca7680 cgaggccctt tcgtcttcaa 7700 <210> 9 <211> 7745 <212> DNA <213> Artificial Sequence <400> 9 gaattcgaat ccttacatca cacccaatcc cccacaagtg atcccccaca caccatagct 60 tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg 120 taccacttca aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt 180 gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt 240 tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt 300 ttttgatttt tttctctttc gatgacctcc cattgatatt taagttaata aacggtcttc 360 aatttctcaa gtttcagttt catttttctt gttctattac aacttttttt acttcttgct 420 cattagaaag aaagcatagc aatctaatct aagttttaat tacaaatcta gaaacttgct 480 tacccacttg gatccgcgaa tttcttatga tttatgattt ttattattaa ataagttata 540 aaaaaaataa gtgtatacaa atttaaagt gactcttagg ttttaaaacg aaaattctta 600 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca tgaggtcgct 660 cttattgacc acatctctac cgggtcgacc gatgcccttg agagccttca acccagtcag 720 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttcttat 780 catgcaactc gtaggacagg tgccggcagc gctctgggtc atttcggcg aggaccgctt 840 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct 900 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat 960 cgccggcatg gcggccgcag tatagcgacc agcattcaca tacgattgac gcatgatatt 1020 actttctgcg cacttaactt cgcatctggg cagatgatgt cgaggcgaaa aaaataataa 1080 atcacgctaa catttgatta aatagaaca actacaatat aaaaaaacta tacaaatgac 1140 aagttcttga aaacaagaat ctttttattg tcagtactga ttagaaaaac tcatcgagca 1200 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc 1260 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaattc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga cattacaaa caggatcga atgcaaccgg cgcaggaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttgcc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attcgcga gcccatttat 1920. acccatata atcagcatcc atgttggaat ttaatcgcgg cctcgaaacg tgagtctttt ccttacccat ggttgttat gttcggatgt gatgtgagaa ctgtatccta gcaagatttt 2040 aaaaggaagt atatgaaaga agaacctcag tggcaaatcc taacctttta tatttctcta 2100 caggggcgcg gcgtggggac aattcaacgc gtctgtgagg ggagcgtttc cctgctcgca 2160 ggtctgcagc gaggagccgt aatttttgct tcgcgccgtg cggccatcaa aatgtatgga 2220 tgcaaatgat tatacatggg gatgtatggg ctaaatgtac gggcgacagt cacatcatgc 2280 ccctgagctg cgcacgtcaa gactgtcaag gagggtattc tgggcctcca tgtccccggg 2340 aatctcggtc gtaatgattt ttataatgac gaaaaaaaa aaattggaaa gaaaaccccc 2400 cccccgcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac 2460 ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg 2520 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt 2580 cggttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt 2640 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac 2700 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag 2760 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt 2820 gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatt cccccttaca 2880 cggaggcatc aagtgaccaa acaggaaaaaa accgccctta acatggcccg ctttatcaga 2940 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac 3000 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcaggtgggc cattctcatg 3060 aagaatatct tgaatttatt gtcatattac tagttggtgt ggaagtccat atatcggtga 3120 tcaatatagt ggttgacatg ctggctagtc aacattgagc cttttgatca tgcaaaatata 3180 ttacggtatt ttacaatcaa atatcaaact taactattga ctttataact tatttaggtg 3240 gtaacattct tataaaaaag aaaaaaaatta ctgcaaaaca gtactagctt ttaacttgta 3300 tcctaggtta tctatgctgt ctcaccatag agaatattac ctatttcaga atgtatgtcc 3360 atgattcgcc gggtaaatac atataataca caaatctggc ttaataaagt ctataatata 3420 tctcataaag aagtgctaaa ttggctagtg ctatatattt ttaagaaaat ttcttttgac 3480 taagtccata tcgactttgt aaaagttcac tttagcatac atatattaca cgagccagaa 3540 attgtaactt ttgcctaaaa tcacaaattg caaaatttaa ttgcttgcaa aaggtcacat 3600 gcttataatc aactttttta aaaatttaaa atactttttt attttttat tttaaacata 3660 aatgaaataa tttatttat gtttatgatt accgaaacat aaaacctgct caagaaaaag 3720 aaactgtttt gtccttggaa aaaaagcact acctaggagc ggccaaaatg ccgaggcttt 3780 catagcttaa actctttaca gaaaataggc attatagatc agttcgagtt ttcttattct 3840 tccttccggt tttatcgtca cagttttaca gtaataataagt atcacctctt agagttaact 3900 atgagataag caagtatcat ctcatttcat ttacctgaag tcgagtaaac agaaaatcca 3960 attgttgatg aacctcaatg acttagaact atctatcggc agatcatata aagaggattt 4020 aggtacctag aggactgtac ctggagtata tatatatata tatatatatt atctcaacta 4080 tagtccatag aggtttcttt cttgaggcct taaactgcta aagaatgata ttggtggaat 4140 gcaagcacca atctctcttc tttcgtaact gttcatatac ttcaaaccaa gaatgtaacg 4200 ggcattgacc catccaaaac cttcagtagc tgccccttta aagtcagcac cttgattacc 4260 gtattctgct tcaacacgat gaggatctgt tcctcttg acatcatatt tttcaaccac 4320 aataccatta taatcgacaa aagccttttgt catcatgaaa agccatctat aagctagcct 4380 attcgttaca gttaaataac cataagaacg gaggccttcc caagcaagaa tttgatgggg 4440 tgcccaacca aatggatagt cccattgtct aattggtctc gaaatagaaa ttgggcctcg 4500 agaacgctcc gtacatgcag ctaaacctcc aagcatctct aacttgggta gtgctttctc 4560 caccattttc tgtgcttgct cttcgtggc aagtccagcc cataatgccc agaatgtagt 4620 tgcggattcg tatgacgttc tgtgcttgat ttttgtgttg tagtcaaaga aaaaccccga 4680 ctcgtcatcc cacatatatt tggtaattga tgaggcaacg ctaattatca acatatagat 4740 tgttatctat ctgcatgaac acgaaatctt tacttgacga cttgaggctg atggtgtta 4800 tgcaaagaaa ccactgtgtt tatatgtgt cactgtttga tattactgtc agcgtagaag 4860 ataatagtaa aagcggttaa taagtgtatt tgagataagt gtgataaagt ttttacagcg 4920 aaaagacgat aaatacaaga aaatgattac gaggatacgg agagaggtat gtacatgtgt 4980 atttatatac taagctgccg gcggttgttt gcaagaccga gaaaaggcta gcaagaatcg 5040 ggtcattgta gcgtatgcgc ctgtgaacat tctcttcaac aagtttgatt ccattgcggt 5100 gaaatggtaa aagtcaaccc cctgcgatgt atattttcct gtacaatcaa tcaaaaagcc 5160 aaatgattta gcattatctt tacatcttgt tattttacag attttatgtt tagatctttt 5220 atgcttgctt ttcaaaaggc ctgcaggcaa gtgcacaaac aatacttaaa taaatactac 5280 tcagtaataa cctatttctt agcatttttg acgaaatttg ctattttgtt agagtctttt 5340 acaccatttg tctccacacc tccgcttaca tcaacaccaa taacgccatt taatctaagc 5400 gcatcaccaa cattttctgg cgtcagtcca ccagctaaca taaaatgtaa gctctgcctc 5460 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 5520 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 5580 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 5640 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac 5700 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg 5760 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5820 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5880 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5940 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 6000 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 6060 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 6120 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 6180 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6240 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 6300 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactaga ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa aggttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6480 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatctttct acggggtctg 6540. acgctcagtg gacgaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 6780 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 6840 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 6960. cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 7080 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 7140 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 7200 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 7260 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata 7320 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 7380 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 7440 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 7500 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 7560 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 7620 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 7680 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 7740 ttcaa 7745 <210> 10 <211> 8472 <212> DNA <213> Artificial Sequence <400> 10 gaattcgaat ccttacatca cacccaatcc cccacaagtg atcccccaca caccatagct 60 tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg 120 taccacttca aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt 180 gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt 240 tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt 300 ttttgatttt tttctctttc gatgacctcc cattgatatt taagttaata aacggtcttc 360 aatttctcaa gtttcagttt catttttctt gttctattac aacttttttt acttcttgct 420 cattagaaag aaagcatagc aatctaatct aagttttaat tacaaatcta gaatgagcac 480 cgaactgaag aaacctcttg agctctactc ggcgagcaca ccgaacggcc agaaggccac 540 tgtgttcttg gaggaactga agcagatcta cccagctttc gactatcatg tgaacgagat 600 cgatatccag aagaatgaac agaaggagga gtggttcctg aagatcaacc ccaacgggcg 660 gatccccgct ttggtcgatc ctaacagggg ggaccatgct gtgtgggagt ccgccgccat 720 actgctgtat ctagagaagc actatgaccc agaccacaag ttctcattcg gcgactcggc 780 tgatgaggat aactttagga gtgagatcct ccagtggatg tttttcgtcc acggagggat 840 cggtccgatg caaggccagg cggttttctt ctcgaaatat gcgcccgagc agatcccata 900 tgccatcaag aggtaccaag atgagaccaa acgactgtac tcggtgctcg ataccaggct 960 ccggaacaga gactatctgg tcggtccggg aagaggtaag tactcgatcg cagacgcaaa 1020 cgcgcttccc tgggtgtttt ggtcaccgtt cgcgggggatc gatcatgtgg agattccgac 1080 gggagtgaag gcatggctgg acaggaacct ggagagagag ccgacgatga gggggctcaa 1140 ggtcggaccg aacacgtcct cagatatgat cgaacagatc aagaagtatg gggcgcttcc 1200 tccgagcacg aagtgaggat ccgcgaattt cttatgattt atgattttta ttattaaata 1260 agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa 1320 attcttattc ttgagtact ctttcctgta ggtcaggttg ctttctcagg tagcatga 1380 ggtcgctctt attgaccaca tctctaccgg gtcgaccgat gcccttgaga gccttcacc 1440 cagtcagctc cttccggtgg gcgcggggca tgactcgt cgccgcactt atgactgtct 1500 tctttatcat gcaaccgta ggacaggtgc cggcagcgct ctggtcatt tcggcgagg 1560 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc 1620 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgttcggc gagaagcagg 1680 ccattatcgc cggcatggcg gccgcagtat agcgaccagc atcacatac gattgacgca 1740 tgatattact ttctgcgcac ttaacttcgc atctggggcag atgatgtcga ggcgaaaaaa 1800 ataataatc acgctacat ttgattaaa tegacaact aacataaa aaactatac 1860 aaatgacaag ttctgaaa caagaatctt tttattgtca gtactgatta gaaaaactca 1920 tcgagcatca atgaaactg catttattc attcaggat tatcaatacc atattttga 1980 aaaagccgtt tctgtaatga aggaaaac tcaccgaggc agttccatag gatggcaaga 2040 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 2100 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 2160 aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg 2220 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga 2280 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc 2340 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc 2400 tggaatgctg tttgccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg 2460 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc 2520 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca 2580 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc 2640 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct cgaaacgtga 2700 gtctttctct tacccatggt tgttatgtt cggatgtgat gtgagaactg tatcctagca 2760 agattttaaa aggaagtata tgaaagaaga acctcagtgg caaatcctaa ccttttatat 2820 ttctctacag gggcgcggcg tggggacaat tcaacgcgtc tgtgagggga gcgtttccct 2880 gctcgcaggt ctgcagcgag gagccgtaat ttttgcttcg cgccgtgcgg ccatcaaaat 2940 gtatggatgc aaatgattat acatggggat gtatgggcta aatgtacggg cgacagtcac 3000 atcatgcccc tgagctgcgc acgtcaagac tgtcaaggag ggtattctgg gcctccatgt 3060 ccccgggaat ctcggtcgta atgattttta taatgacgaa aaaaaaaaaa ttggaaagaa 3120 aacccccccc ccgcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt 3180 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac 3240 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa 3300 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca 3360 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg 3420 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata 3480 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc 3540 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaattccc 3600 ccttacacgg aggcatcaag tgaccaaaca ggaaaaacc gccttaaca tggcccgctt 3660 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca 3720 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag cttaccgca ggtgggccat 3780 tctcatgaag aatatcttga atttattgtc atattactag ttggtgtgga agtccatata 3840 tcggtgatca atatagtggt tgacatgctg gctagtcaac attgagcctt ttgatcatgc 3900 aaatatatta cggtatttta caatcaaata tcaaacttaa ctattgactt tataacttat 3960 ttaggtggta acattcttat aaaaaagaaa aaaattactg caaaacagta ctagctttta 4020 acttgtatcc taggttatct atgctgtctc accatagaga atattaccta tttcagaatg 4080 tatgtccatg attcgccggg taatacata tatacacaa atctggctta ataaagtcta 4140 tatatatct cataaagaag tgctaaattg gctagtgcta tatattttta agaaaatttc 4200 ttttgactaa gtccatatcg actttgtaaa agttcacttt agcatacata tattacacga 4260 gccagaaatt gtaacttttg cctaaatca caaattgcaa aatttaattg cttgcaaaag 4320 gtcacatgct tataatcaac ttttttaaaa attaaaata cttttttatt ttttattttt 4380 aaacataaat gaaataattt atttattgtt tatgattacc gaaacataaa acctgctcaa 4440 gaaaaagaaa ctgttttgtc cttggaaaaa aagcactacc taggagcggc caaaatgccg 4500 aggctttcat agcttaaact cttacagaa aataggcatt atagatcagt tcgagttttc 4560 ttattcttcc ttccggtttt atcgtcacag ttttacagta aataagtatc acctcttaga 4620 gttaactatg agataagcaa gtatcatctc atttcattta cctgaagtcg agtaaacaga 4680 aaatccaatt gttgatgaac ctcaatgact tagaactatc tatcggcaga tcatataaag 4740 aggatttagg tacctagagg actgtacctg gagtatatat atatatatat atatattatc 4800 tcaactatag tccatagagg tttctttctt gaggccttaa actgctaaag aatgatattg 4860 gtggaatgca agcaccaatc tctcttcttt cgtaactgtt catatacttc aaaccaagaa 4920 tgtaacgggc attgacccat ccaaaacctt footgctgc ccctttaaag tcagcacctt 4980 gattaccgta ttctgcttca acacgatgag gatctgttcc tcttgtgaca tcatattttt 5040 caaccacaat accattataa tcgacaaaag cctttgtcat catgaaaagc catctataag 5100 ctagcctatt cgttacagtt aaataaccat aagaacggag gccttcccaa gcaagaattt 5160 gatggggtgc ccaaccaaat ggatagtccc attgtctaat tggtctcgaa atagaaattg 5220 ggcctcgaga acgctccgta catgcagcta aacctccaag catctctaac ttgggtagtg 5280 ctttctccac cattttctgt gcttgctcct tcgtggcaag tccagcccat aatgcccaga 5340 atgtagttgc ggattcgtat gacgttctgt gcttgatttt tgtgttgtag tcaaagaaaa 5400 accccgactc gtcatcccac atatatttgg taattgatga ggcaacgcta attatcaaca 5460 tatagattgt tatctatctg catgaacacg aaatctttac ttgacgactt gaggctgatg 5520 gtgtttatgc aaagaaacca ctgtgtttaa tatgtgtcac tgtttgatat tactgtcagc 5580 gtagagagata atagtaaaag cggttaataa gtgtatttga gataagtgtg ataaagtttt 5640 tacagcgaaa agacgataaa tacaagaaaa tgattacgag gatacggaga gaggtatgta 5700 catgtgtatt tatatactaa gctgccggcg gttgtttgca agaccgagaa aaggctagca 5760 agaatcgggt cattgtagcg tatgcgcctg tgaacattct cttcaacaag tttgattcca 5820 ttgcggtgaa atggtaaaag tcaacccct gcgatgtata ttttcctgta caatcaatca 5880 aaaagccaaa tgatttagca ttatctttac atcttgttat tttacagatt ttatgtttag 5940 atcttttatg cttgcttttc aaaaggcctg caggcaagtg cacaaacaat acttaaataa 6000 atactactca gtaataacct atttcttagc atttttgacg aaatttgcta ttttgttaga 6060 gtcttttaca ccatttgtct ccacacctcc gcttacatca acaccaataa cgccatttaa 6120 tctaagcgca tcaccaacat tttctggcgt cagtccacca gctaacataa aatgtaagct 6180 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 6240 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 6300 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta 6360 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt 6420 gaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg 6480 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 6540 gcggtaatac ggttatccac agaatcaggg gataacgcag gaagaacat gtgagcaaaa 6600 ggccagcaaa aggcaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6660 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6720 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6780 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6840 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6900 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6960 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 7020 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 7080 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaga 7140 gttggtagct cttgatccgg caacaacc accgctgta gcggtggtttt tttgtttgc 7200 aagcagcaga tcgcgcag aaaaaagga tccagaag atccttgat ctttctacg 7260 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttgtcat gagatttaca 7320 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 7380 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctactca 7440 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 7500 attack gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 7560 ccggctccag atttatcagc aaaaaccag ccagccggaa gggccgagcg cagaagtggt 7620 cctgcaactt tatccgcctc catccagtct atttattgtt gccgggaagc tagtagt 7680 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca 7740 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 7800 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7860 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7920 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7980 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacgggga tataccgcg 8040 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 8100 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 8160 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 8220 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 8280 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 8340 attagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 8400 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 8460 ttcgtcttc aa 8472 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 gggtgttttg gtcaccgttc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 ctcatcgtcg gctctctctc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 ccccaaggct aacagagaga 20 <210> 14 <211> twenty one <212> DNA <213> Artificial Sequence <400> 14 cagaggcgta caaagaaagc a 21

Claims

1. A method for constructing recombinant Pharfia redis yeast, characterized in that: The method includes upregulating the encoding of glutathione S-transferase in Rhodophora oryzae. GST1 Gene expression levels were measured to obtain recombinant red Pharf yeast.

2. The method as described in claim 1, characterized in that: The glutathione S-transferase is selected from any one of the proteins listed in A1) or A2): A1) A protein with the amino acid sequence shown in SEQ ID No. 3; A2) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1).

3. The method as described in claim 1 or 2, characterized in that: The method for preparing the recombinant red Paffo yeast includes: GST1 Genes were introduced into Pharfia rubra, the recipient strain.

4. The method as described in claim 3, characterized in that: The GST1 Genes contain the aforementioned GST1 The gene expression cassette was introduced into the recipient bacteria.

5. The method according to any one of claims 1-4, characterized in that: The GST1 The gene is selected from either B1 or B2: B1) The nucleotide sequence of the coding strand is the DNA molecule shown in positions 473-1216 of SEQ ID No. 10; B2) The coding sequence of the coding strand is the DNA molecule shown in positions 473-1216 of SEQ ID No. 10; B3) is more than 80% identical to the DNA molecule defined in B1) or B2) and encodes the DNA molecule of the glutathione S-transferase as described in claim 1 or 2.

6. The method according to any one of claims 1-5, characterized in that: The expression cassette was introduced into the recipient bacteria via a recombinant expression vector with the nucleotide sequence SEQ ID No.

10.

7. Recombinant red Paffo yeast constructed by the method of any one of claims 1-6.

8. The use of the recombinant red Pharf yeast of claim 7 in the production of carotenoids and / or astaxanthin.

9. A method for preparing carotenoids and / or astaxanthin, characterized in that: The method includes preparing carotenoids and / or astaxanthin using the recombinant red Paffo yeast of claim 7.

10. The use of the glutathione S-transferase or related biological material as described in claim 1 or 2 in the preparation of recombinant Pharf yeast producing carotenoids and / or astaxanthin; wherein the biological material is any one of C1)-C3). C1) A nucleic acid molecule encoding the glutathione S-transferase as described in claim 1 or 2; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette described in C2).

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