High-yield sclareol recombinant strain as well as construction method and application thereof

Abstract

The invention discloses a high-yield sclareol recombinant strain as well as a construction method and application thereof, and belongs to the technical field of microbial metabolism engineering. The invention provides recombinant yarrowia lipolytica SC37 with high yield of sclareol, which is characterized in that a sclareol biosynthesis path is firstly constructed, then acetyl coenzyme A in peroxidase is guided to a mevalonic acid path, and then a precursor acetyl coenzyme A and a cofactor NADPH are introduced by optimizing an intracellular mevalonic acid metabolism path to obtain the recombinant strain SC37. The invention provides a recombinant strain SC37 which takes waste oil as a substrate and is high in sclareol yield, and by introducing and optimizing the expression of a sclareol synthesis path, the sclareol yields in shake flask batch fed-batch fermentation and bioreactor batch fed-batch fermentation respectively reach 2.5 g / L and 20.18 g / L. The recombinant strain SC37 has important values for reducing environmental pollution and increasing waste oil economy, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial metabolic engineering, and particularly relates to a recombinant strain for high-yield sclareol and a construction method and application thereof. BACKGROUND

[0002] Waste cooking oil (WCO) is a waste product in frying oil, containing triglycerides, polar compounds and non-volatile compounds. Most of the WCO is poured into the sewage pipe, causing water pollution. Therefore, recycling waste oil as a raw material for producing value-added chemicals is of great significance for preventing environmental pollution, and can also produce additional economic value of WCO.

[0003] As an oleaginous yeast, Yarrowia lipolytica has the characteristics of salt tolerance, low temperature tolerance and growth in low pH environment. In terms of metabolism, it has the advantages of utilizing glucose, waste cooking oil, ethanol, glycerol and other substrates for growth, and is an excellent chassis cell for synthesizing natural products. Secondly, Yarrowia lipolytica has a high flux of tricarboxylic acid (TCA) cycle, which provides a rich source of acetyl-CoA precursors for the synthesis of terpenoids. The abundant lipid droplets and subcellular structures in its cytoplasm also provide a place for the storage of hydrophobic products. In addition, Yarrowia lipolytica also has a high flux of pentose phosphate pathway (PPP) metabolism, which can provide the required cofactors for product synthesis. The above advantages of Yarrowia lipolytica make it an excellent metabolic engineering chassis strain.

[0004] Sclareol (also known as hard tail butterfly) is mainly derived from the plant sclareol, and is a plant secondary metabolite. It is commonly seen as white crystalline powder, easily soluble in organic solvents such as ethanol and acetone, and difficult to dissolve in water. Its chemical molecule C 20 H 36 O2, molecular weight is 308.5, and the chemical structural formula is shown below. Sclareol is mainly used for the synthesis of sclareolide and ambrox, etc. natural amber, and can also be used for the preparation of perfumes, and has important application value in the pharmaceutical and cosmetic industries.

[0005]

[0006] Chinese patent (CN 117264792A) discloses a high-sclareol-producing recombinant Yarrowia lipolytica, which is constructed by overexpressing pyrophosphate lysadalenediol synthase mutant gene CaLPPSR227Q, sclarol synthase gene SsTPS and sclarol synthase gene SaGGPP in Yarrowia lipolytica po1f, and expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMGR and isoprene pyrophosphate isomerase gene idi. The highest sclarol yield of the strain in a 5L fermenter is only 8510mg / L, and the yield still needs to be improved. Therefore, it is urgent to develop a high-sclareol-producing recombinant strain. SUMMARY

[0007] To solve the above problems existing in the prior art, the purpose of the present application is to provide a high-sclareol-producing recombinant strain and a construction method and use thereof.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0009] The present application provides a construction method of a high-sclareol-producing recombinant strain, which comprises the following steps:

[0010] (1) introducing a sclarol biosynthesis pathway into a host strain to obtain a recombinant strain A, wherein the host strain is Yarrowia lipolytica;

[0011] Alternatively, the construction method further comprises the following step: (2) using peroxisomal acetyl-CoA for the mevalonate pathway in the recombinant strain A to obtain a recombinant strain B;

[0012] Alternatively, the construction method further comprises the following step: (3) strengthening the mevalonate pathway and weakening at least one of the competing pathways leading to squalene and lipid synthesis in the recombinant strain B to obtain a recombinant strain C;

[0013] Alternatively, the construction method further comprises the following step: (4) overexpressing acetyl-CoA and co-factor NADPH in the recombinant strain C to obtain a high-sclareol-producing recombinant strain D.

[0014] Further, the introduction of the sclarol biosynthesis pathway in step (1) is overexpressing pyrophosphate lysadalenediol synthase gene tPvLPPS and sclarol synthase gene PvSCS at E1 and B1 sites of the genome of the host strain; the nucleotide sequence of the pyrophosphate lysadalenediol synthase gene tPvLPPS is shown in SEQ ID NO: 1, and the nucleotide sequence of the sclarol synthase gene PvSCS is shown in SEQ ID NO: 2.

[0015] Further, a pair of polypeptide interaction tags RIAD and RIDD are connected to the C-terminus of the tPvLPPS and PvTPS genes, and the nucleotide sequence of the RIAD and RIDD is shown as SEQ ID NO: 6.

[0016] Further, the way of using peroxisomal acetyl-CoA for the mevalonate pathway in step (2) is as follows: sequentially integrate the mvaE, ERG13 and tHMGR genes with the C-terminus of a targeting tag ePTS1 into different sites of the genome of the host strain, and the different sites are as follows:

[0017] 1) integrate the DNA fragment P TEFin -mvaE-ePTS1-T XPR2 into the A1 site, and the nucleotide sequence of the mvaE gene is shown as SEQ ID NO: 3;

[0018] 2) integrate the DNA fragment P TEFin -ERG13-ePTS1-T XPR2 into the A3 site, and the nucleotide sequence of the ERG13 gene is shown as SEQ ID NO: 4;

[0019] 3) integrate the DNA fragment P TEFin -tHMGR-ePTS1-T XPR2 into the A08 site, and the nucleotide sequence of the tHMGR is shown as SEQ ID NO: 5.

[0020] Further, the way of strengthening the mevalonate pathway in step (3) is selected from at least one of 1) to 8):

[0021] 1) integrate the DNA fragment P TEFin -ERG20-tHMGR-T XPR2 into the host D17 site, and the nucleotide sequence of ERG20 is shown as SEQ ID NO: 7;

[0022] 2) integrate the DNA fragment P TEFin -SsGGPPS-T XPR2 into the host MFE1 site, and the nucleotide sequence of SsGGPPS is shown as SEQ ID NO: 8;

[0023] 3) integrate the DNA fragment P TEFin -tPvLPPS-RIDD-T XPR2 into the host XPR2 site, and the nucleotide sequence of tPvLPPS is shown as SEQ ID NO: 1;

[0024] 4) integrate the DNA fragment P TEFin-PvSCS-RIAD-T XPR2 The nucleotide sequence of PvSCS is shown as SEQ ID NO: 2.

[0025] 5) replace the DNA fragment P TEFin -tHMGR-T XPR2 integrated into the host F17 site;

[0026] 6) replace the DNA fragment P TEFin -tHMGR-T XPR2 -P EXP -PaGGPPS-T LIP integrated into the host F17 site, and the nucleotide sequence of PaGGPPS is shown as SEQ ID NO: 9.

[0027] 7) replace the DNA fragment P ERG11 in the host with the DNA fragment P ERG9 , and the nucleotide sequence of P ERG11 is shown as SEQ ID NO: 10.

[0028] 8) replace the DNA fragment P TEFin -mvaE-T LIP integrated into the host C4 site, and the nucleotide sequence of mvaE is shown as SEQ ID NO: 3.

[0029] Further, in step (3), the way to weaken the competitive pathway to squalene synthesis is to replace the promoter P ERG9 of squalene synthase with the promoter P ERG11 .

[0030] Further, in step (4), the overexpression of acetyl-CoA is the overexpression of at least one of acetyl-CoA synthase ACS, pyruvate dehydrogenase PDH, and knockout of citrate synthase CIT1.

[0031] The nucleotide sequence of ACS is shown as SEQ ID NO: 11.

[0032] The nucleotide sequence of PDH is shown as SEQ ID NO: 12.

[0033] The nucleotide sequence of CIT1 is shown as SEQ ID NO: 13.

[0034] Further, in step (4), the overexpression of co-factor NADPH is the overexpression of at least one of zwf, gnd, malate enzyme mael, isocitrate dehydrogenase IDH, and NAD kinase DADK in the pentose phosphate pathway.

[0035] The nucleotide sequence of the zwf is shown as SEQ ID NO: 14;

[0036] The nucleotide sequence of the gnd is shown as SEQ ID NO: 15;

[0037] The nucleotide sequence of the mae1 is shown as SEQ ID NO: 16;

[0038] The nucleotide sequence of the IDH is shown as SEQ ID NO: 17;

[0039] The nucleotide sequence of the DADK is shown as SEQ ID NO: 18.

[0040] The application further provides a Yarrowia lipolytica recombinant strain obtained by the construction method.

[0041] The application further provides a use of the Yarrowia lipolytica recombinant strain in producing sclerol.

[0042] Further, the substrate for producing sclerol is waste oil.

[0043] Further, the waste oil is discarded edible oil.

[0044] The application has the following beneficial effects:

[0045] The application provides a recombinant Yarrowia lipolytica strain SC37 with high sclerol yield, which is constructed by first constructing a sclerol biosynthesis path, then guiding acetyl coenzyme A in the peroxisome to the mevalonate path, and finally introducing a precursor acetyl coenzyme A and a cofactor NADPH to obtain the recombinant strain SC37. The application provides a recombinant strain SC37 with high sclerol yield and using waste oil as a substrate, which is constructed by introducing and optimizing the expression of the sclerol synthesis path, and the sclerol yield reaches 2.5 g / L and 20.18 g / L in a shake flask batch and a bioreactor batch fed-batch fermentation, respectively. The recombinant strain SC37 of the application has important values in reducing environmental pollution and increasing waste oil economy, and has a good application prospect.

[0046] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above technical idea of the application.

[0047] The above content of the application will be further described in detail through the following embodiment form. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Yonsei alcohol production of recombinant strain SC01 with waste cooking oil and glucose as carbon source, respectively.

[0049] Figure 2 Yonsei alcohol production of recombinant strain SC05, recombinant strain SC15, and recombinant strain SC16.

[0050] Figure 3 Yonsei alcohol production of recombinant strain SC20, recombinant strain SC25, and recombinant strain SC37.

[0051] Figure 4 Yonsei alcohol production of recombinant strain SC38 in bioreactor batch fed fermentation. DETAILED DESCRIPTION

[0052] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0053] The waste oil used in the specific embodiments of the present application is waste cooking oil.

[0054] In the following experiments, if the temperature is not specified, it means that the reaction is carried out under normal temperature conditions, and the normal temperature is room temperature, which is 25±5℃.

[0055] The initial strain sampled in the following examples is Yarrowia lipolytica po1f (genotype ATCC MYA-2613, ura3-302, leu2-270, xpr2-322, axp-2, leu2-270) purchased from a strain collection company by the company in the early stage.

[0056] Example 1: Construction of high-yield recombinant strain of yonsei alcohol

[0057] 1) Overexpression of pyrophosphoryl-lycopene diolsynthase gene (tPvLPPS) and yonsei alcohol synthase gene (PvSCS) from Prunella vulgaris

[0058] First, the donor DNA expression cassette (P TEFin -tPvLPPS-T XPR2 , PvSCS-T) overexpressing tPvLPPS and PvSCS in the Yarrowia lipolytica genome was constructed. TEFin -PvSCS-T XPR2 ); the donor DNA fragment includes 1000bp homologous arms upstream and downstream of E1 and B1 sites amplified from Yarrowia lipolytica po1f as a template (gene integration site reference Wheeldon et al., ACS Synth. Biol. 2017, 6, 402-409).

[0059] The donor DNA and gRNA were transformed into the starting strain po1f by chemical transformation method with the aid of CRISPR-Cas9 technology (the transformation process was referred to Wheeldon et al., ACS Synth. Biol. 2017, 6, 402-409), coated on YNB plate, static culture, screening, verified by colony PCR, and then subcultured on YPD+5-FOA solid medium for plasmid loss to obtain the recombinant strain SC01.

[0060] Specifically, the Yarrowia lipolytica po1f was used as a template to amplify the 1000 bp homologous arm fragments E1-LH, E1-RH, B1-LH and B1-RH on the upstream and downstream of the neutral sites E1 and B1 on the chromosome, as well as the promoters P TEFin and terminators T XPR2 . The genes tPvLPPS (SEQ ID NO: 1) and PvSCS (SEQ ID NO: 2) were obtained after codon optimization, and a synthase single expression donor DNA was constructed: E1-LH, P TEFin -tPvLPPS-T XPR2 , E1-RH and B1-LH, P TEFin -PvSCS-T XPR2 , B1-RH were fused by PCR and then ligated into the plasmid pCRISPRyl to obtain the plasmids pCRISPRyl-E1-tPvLPPS and pCRISPRyl-B1-PvSCS containing the donor DNA. A pair of simple polypeptide interaction tags RIAD and RIDD were connected to the C-terminus of the PvLPPS and PvTPS genes, and this structure realized the effective assembly of the two enzymes in vivo. The nucleotide sequences of the RIAD and RIDD are shown in SEQ ID NO: 6. The plasmids pCASyl-E1 and pCASyl-B1 containing the integration site PAM sequence were obtained by amplifying the PAM sequence containing E1 and B1 sites from the pCASyl template using the primer pairs E01-sg-F / E01-sg-R and B01-sg-F / B01-sg-R, respectively, and then ligating. 500 ng of the plasmids pCRISPRyl-E1-tPvLPPS and pCASyl-E1 (or pCRISPRyl-B1-PvSCS and pCASyl-B1) containing the donor DNA were transformed into the Yarrowia lipolytica po1f strain by chemical transformation method, coated on YNB screening plate, and incubated at 30°C for 3-4 days. The transformants were incubated overnight in liquid YPD medium, verified by colony PCR, inoculated into YPD liquid without antibiotics, and then streaked on YPD plates containing 5-FOA (5-fluoroorotic acid) for plasmid loss. The strain after plasmid loss was named as the recombinant strain SC01.

[0061] The recombinant strain SC01 was activated in YPD medium and cultured at 220 rpm for 16-20 h, then transferred to 25 mL YPD medium / 100 mL flask with glucose and waste oil as carbon source, respectively, at an initial OD 600 = 0.2, 30°C, 220 rpm for 120 h, and the yield of sclareol was determined. The experimental results are shown in Table 1. Figure 1 As shown in Table 1, the yield of sclareol with glucose as carbon source was significantly higher than that with waste oil as carbon source.

[0062] 2) Increase the utilization rate of waste oil and increase the yield of sclareol

[0063] In order to further increase the utilization rate of waste oil, on the basis of the recombinant strain A, the mvaE (SEQ ID NO: 3) containing peroxisome localization peptide, ERG13 (SEQ ID NO: 4), tHMGR (SEQ ID NO: 5) were overexpressed to obtain the recombinant strain SC05, and the three genes can synthesize methylmalonyl acid from acetyl-CoA after β-oxidation of long-chain fatty acids in peroxisomes, and then the synthesized methylmalonyl acid passes through the peroxisome to the cytoplasm for the synthesis of sclareol. The yield of sclareol of the recombinant strain SC05 with waste oil as carbon source was significantly increased, reaching 150 mg / L( Figure 2 ).

[0064] Specifically: first, the 1000 bp homologous arm sequences upstream and downstream of the integration site, the promoter, the terminator and the structural gene were amplified respectively using Yarrowia lipolytica po1f genome as template, and then ligated into pCRISPRyl plasmid to obtain a plasmid containing donor DNA; then, the pCASyl plasmid containing the integration site PAM sequence was obtained by amplifying the ligated plasmid with the corresponding primer pair, and the pCRISPRyl plasmid and the pCASyl plasmid (each 500 ng) were transformed into the recombinant strain SC01 by chemical transformation method, and then coated on YNB selection plate and incubated at 30°C for 3-4 days, and then verified by colony PCR, and then subcultured on YPD+5-FOA plate to lose plasmid, and then the strain after losing plasmid was preserved and used for the next round of gene editing. The following genomic editing work follows a similar procedure.

[0065] Through fermentation extraction detection, the yield of sclareol of the recombinant strain SC05 with waste oil as carbon source was significantly increased, reaching 150 mg / L( Figure 2 ), which can significantly increase the yield of sclareol by guiding acetyl-CoA in mitochondria to methylmalonyl acid.

[0066] 3) Strengthen the MVA pathway to increase the yield of sclareol

[0067] On the basis of the recombinant strain SC05, the mevalonate (MVA) pathway was strengthened, and the squalene synthesis pathway was weakened; by means of the CRISPR / Cas9 system, the key genes in the MVA pathway were placed behind the P TEFin promoter and integrated into different sites of the genome to obtain the recombinant strain SC15; subsequently, with SC15 as the starting strain, the squalene synthase promoter P ERG9 was replaced with the weaker promoter P ERG11 to obtain the recombinant strain SC16.

[0068] The strengthened mevalonate pathway includes one or more of 1) to 8), and the following seven are adopted in this embodiment: 1) to 4), 6) to 8).

[0069] 1) The DNA fragment P TEFin -ERG20-tHMGR-T XPR2 is integrated into the host D17 site, and the nucleotide sequence of ERG20 is shown in SEQ ID NO: 7;

[0070] 2) The DNA fragment P TEFin -SsGGPPS-T XPR2 is integrated into the host MFE1 site, and the nucleotide sequence of SsGGPPS is shown in SEQ ID NO: 8;

[0071] 3) The DNA fragment P TEFin -tPvLPPS-RIDD-T XPR2 is integrated into the host XPR2 site, and the nucleotide sequence of tPvLPPS is shown in SEQ ID NO: 1;

[0072] 4) The DNA fragment P TEFin -PvSCS-RIAD-T XPR2 is integrated into the host A2 site, and the nucleotide sequence of PvSCS is shown in SEQ ID NO: 2;

[0073] 5) The DNA fragment P TEFin -tHMGR-T XPR2 is integrated into the host F17 site;

[0074] 6) The DNA fragment P TEFin -tHMGR-T XPR2 -P EXP -PaGGPPS-T LIP is integrated into the host F17 site, and the nucleotide sequence of PaGGPPS is shown in SEQ ID NO: 9;

[0075] 7) The DNA fragment P ERG11 is replaced with PERG9 , P ERG11 The nucleotide sequence of P is shown as SEQ ID NO: 10.

[0076] 8) The DNA fragment P TEFin -mvaE-T LIP is integrated into the host C4 site, and the nucleotide sequence of mvaE is shown as SEQ ID NO: 3.

[0077] After fermentation extraction detection, the results are shown as Figure 2 The yield of sclareol of the recombinant strain SC15 reached 800 mg / L. The yield of sclareol of the recombinant strain SC16 was further increased to 1.2 g / L.

[0078] On the basis of the recombinant strain SC16, the copy number of the PvLPPS gene and the PvTPS gene was further increased, that is, after overexpression of one PvLPPS gene and one PvTPS gene on the genome, the recombinant strain SC20 was obtained. After fermentation extraction detection, the results are shown as Figure 3 After increasing the copy number of the PvLPPS gene and the PvTPS gene, the yield of sclareol was increased to 2.1 g / L.

[0079] 4) Strengthening the supply of precursors acetyl-CoA and reducing power NADPH to improve the product synthesis ability of the chassis cell

[0080] On the basis of the recombinant strain SC20, the supply of the precursor acetyl-CoA in the strain was strengthened. With the help of the CRISPR / Cas9 system, the donor DNA expression cassette of acetyl-CoA synthetase ACS (SEQ ID NO: 11), pyruvate dehydrogenase PDH (SEQ ID NO: 12), and citrate synthase CIT1 (SEQ ID NO: 13) was constructed, and was integrated into different sites of the genome by chemical transformation. After fermentation extraction and detection, the results are shown as Figure 3 It is concluded that the integration and expression of ACS and the knockout of CIT1 are most beneficial to the accumulation of sclareol, and the recombinant strain is named SC25, with a yield of 2.3 g / L.

[0081] On the basis of the recombinant strain SC25, the supply of reducing power NADPH was enhanced, and with the help of the CRISPR / Cas9 system, the donor DNA expression cassette of zwf (SEQ ID NO: 14), gnd (SEQ ID NO: 15), mae1 (SEQ ID NO: 16), IDH (SEQ ID NO: 17), and DADK (SEQ ID NO: 18) genes was constructed, and was integrated into different sites on the genome. After fermentation detection and extraction, the results are shown as Figure 3As shown, after the integration of zwf, gnd and IDH genes, the yield of sclareol reached 2.5 g / L, and the recombinant strain was named as SC37 (i.e., the recombinant strain D).

[0082] Example 2: Batch fed-batch fermentation of the recombinant strain

[0083] In order to test the high-density fermentation capacity of the recombinant strain, a shake flask batch fed-batch experiment was performed, and the SC37 was back-supplemented with the screening marker ura and leu genes (only used for marking), and was named as the recombinant strain SC38.

[0084] (1) Batch fed-batch fermentation of the recombinant strain in a shake flask

[0085] The batch fed-batch fermentation of the recombinant strain SC38 was performed in a shake flask, and YPD medium was used, with 50 mL / 250 mL shake flask, and the initial OD 600 of the inoculation was 0.2. The fermentation was performed at 30℃ and 220 rpm for 5 days, and the yield of sclareol reached 2.5 g / L.

[0086] (2) Batch fed-batch fermentation of the recombinant strain in a parallel bioreactor

[0087] The batch fed-batch fermentation was performed by using a 5L bioreactor system, and the volume of the batch fed-batch fermentation was 2.5L. YPD medium was used in the batch fed-batch fermentation, with a volume of 2.5L, and the OD 600 of the inoculation was 0.4, and the pH was 5.5. The feeding medium was 10x YPD medium and the feeding concentration of glucose was 800 g / L. In the batch fed-batch fermentation, when the glucose was consumed, the exponential feeding was started, and the enzymes were sampled every 4 hours, and the OD 600 and the residual sugar in the medium were detected, and the yield of sclareol was detected every 12 hours. The highest yield of the recombinant strain SC38 was 20.18 g / L Figure 4 ).

[0088] In summary, the present application provides a recombinant Yarrowia lipolytica strain SC37 with high yield of sclareol, which is constructed by first constructing a sclareol biosynthesis pathway, then guiding acetyl coenzyme A in the peroxisome to the mevalonate pathway, and then introducing the precursor acetyl coenzyme A and the cofactor NADPH to obtain the recombinant strain SC37. The present application provides a recombinant strain SC37 with high yield of sclareol and using waste oil as a substrate, and by introducing and optimizing the expression of the sclareol synthesis pathway, the yield of sclareol in the batch fed-batch fermentation in a shake flask and a bioreactor reaches 2.5 g / L and 20.18 g / L, respectively. The recombinant strain SC37 of the present application has important value for reducing environmental pollution and increasing waste oil economy, and has good application prospect.

[0089] The nucleotide sequence involved in the present application is as follows:

[0090] SEQ ID NO: 1

[0091] ATGGCCTCCTTGTCCACCCAGATTACAACAAGGTCTCCCATGAACGCATGTCGTCGAATCTTGCC

[0092] GGTGTCGGCGAAGCTGTCTCTGCCTGAGTGCTTTACCATCTCCGCCTGGATGTCCAGTTCTAAAA

[0093] ATCTGTCACTCAACTACCAGGTTTCCGAGCGTAAGCTGTCCAAGGCCACCGGAATCCGAGTGGC

[0094] AACGGTGGACGCGCCTCAGGTCCACGACCAGGATGATTCGACAGTCCATCAGGGCCATGACGCT

[0095] GTGAATAACATTGAGGACCCCATTGAATACATTCGGACTCTACTAAGAACCACCGGCGACGGACG

[0096] CATTTCCGTTTCTCCTTACGACACTGCTTGGGTGGCTCTCATAAAGGATCTTGAAGGAAGAAATG

[0097] CCCCACAATTCCCTAGCTCTCTCGAATGGATTGTCCAGAACCAGCTGGAGGATGGCTCCTGGGGT

[0098] GATGAAAAGTTTTTCTGCATCTATGATCGACTGGTCAACACTATCGCCTGTGTCGTTGCGTTGAGA

[0099] TCGTGGTCTGTTCACGCCGAGAAGGTCATGAAGGGTGTGACTTACATCAAGGAAAACGTCGACA

[0100] AGCTGAAAGAGGGTAACGTAGAGCACATGACCTGTGGCTTTGAGGTGGTTTTTCCGGCTCTGCT

[0101] GCAACGAGCCAAGTCTCTGGGTATCGAGGAACTGCCCTACGATGCTGAGGTCATTCGTGAGATCT

[0102] ACCATACCCGAGAGCAGAAATTGAAGCGAATTCCGCTGGAAATCATGCACAAGGTGCCCACTAG

[0103] TCTGCTTTTTTCGCTTGAGGCTGGGCTGGAGAACCTGGAGTGGGACAAGTTACTCAAACTGCAG

[0104] AGCGCAGACGGGAGCTTTCTGACGAGTCCATCGTCCACTGCGTTTGCTTTCATGCAGACCAAAG

[0105] ACGAGAAGTGTTATCAATTCATCAAGAATACCGTGGACACCTTCAACGGCGGCGCTCCCCACACC

[0106] TACCCCGTGGACGTGTTTGGACGACTGTGGGCCATTGACCGACTTCAGAGACTGGGTATTTCTAG

[0107] ATTCTTCGAGAGTGAAATTGCTGATTGCATAGGCCACATTCACAAATTCTGGACCGACAAGGGCG

[0108] TCTTCTCCGGCCGGGAGTCTGATTTCTGTGACATCGACGACACTAGCATGGGTGTTCGCCTCATGC

[0109] GGATGCATGGATACAACGTTGATCCCAACGTCTTGCGAAACTTCCAACAAAAGGACGGCAAGTT

[0110] CTCGTGCTACGGAGGACAGATGATTGAGTCGGCTTCCCCCATTTATAATTTGTATCGGGCCTCGCA

[0111] GCTCCGTTTTCCTGGCGAGGAAATTCTCGAAGAGGCCAACAAGTTTGCATTCGAGTTTCTACAAG

[0112] AGAAACTCGCAAACAACCAGATTCTGGACAAGTGGGTTATCTCAAAGCATTTGCCAAACGAGAT

[0113] CAAGCTCGGACTCGAAATGCCTTGGTATGCTACCCTCCCTCGAGTTGAGGCGAAGCACTACCTCC

[0114] AGTACTACGCCGGCAGTGGTGATGTCTGGATTGGAAAGACGCTGTACCGAATGCCCGAAATCTCA

[0115] AACGACACCTACCACGAGCTGGCCAAGGACGGCTTTAAGCGATGCCAGTCTCAGCACCAGTTCG

[0116] AGTGGATTTACATGCAAGAGTGGTACGAGAGTTGCAACGTGGAGGAGTTCGGGATCTCCCGGAA

[0117] GGATCTCCTCCTGGCTTACTTTCTTGCCACTTCTTCCATTTTCGAGGTTGAGAGGACAAATGAACG

[0118] AATCGCATGGGCTAAGAGCCAGATCATCAGCAAGATGATCACGTCGTTCTTCAAGAAGGAGACG

[0119] ACATCGTCTGACGCCAAGTGCTCACTCCTCAACGCTCTTAAGAACATTAATGGGCTTAATTCGACT

[0120] TCCTCTTCTAAACGAGAAGACGGTGCCGCTTCTATGGTGGTAGAGACTCTGACCCAGTTTTTGGA

[0121] AGGTTTCGATCGATACACTCGACATCAACTGAAAAACGCCTGGTCGCAATGGCTCACCAAGCTTC

[0122] AGCAGGGAGAGGGCGATGGAGGAGCAGACGCCGAGCTTCTCACTAACACCTTGAACATCTGTGC

[0123] CGGTCATATCGCCTTTCGCGAGGAGATTCTCAGTCACAACGAGTACAAGACCCTGTCCAACCTAA

[0124] CGTCTAAGATTTGTCAGCATCTCTCCTTCATCCAGAACGAAAAGGAGATGGAGGTCGAGGCCCCC

[0125] AAGTACTCCATCAAGGACAAAGAATTGGAACAGGACATGCAGGCCCTGGCGAAGCTGGTGCTTG

[0126] AAACATCAGTCGGCATCAACAGAAACATCAAGAAGTCTTTCCTGGCTGTTATGAAAACATACTAT

[0127] TACAGAGCTTACTATGGAAGCGAAACCATTGATTCGCACATGTTCAAGGTGCTATTTGAGCCCGT

[0128] GCCTTAG

[0129] SEQ ID NO:2

[0130] ATGTCGTTGACTTTCAATGCCGGTGTTGTCAGATTCTCTTCCCACAGAGTTCGATCTACCAAGGAC

[0131] TGTTTTACAGTTTATGGCTTCCCCATGATTGCCAACAAGGCCGCGTTTGCCGTGAAGTGCAGCTTA

[0132] ACTCCCACGGATCTCATGGGAAGGGTAGAGGAAAAGTTCAAGGGCAAAAACGGCAATTCTCTGG

[0133] CAGCGTCCACAACGGTCGAGAGCGCAGATATTCCATCAAACCTGTGTATCATTGACACGCTTCAG

[0134] CGTTTGGGTGTGGACCGATACTTCCAGACTGAGATCAACGCCATACTCGAGGACACATACCGCCT

[0135] CTGGGAGCGAAAGGACAAGGATATCTATAGTGATGCCACCACCCACGCCATGGCGTTTCGACTTC

[0136] TTCGAGTGAAGGGATACGAGGTGTCCTCGGAGGAACTGGCTCCCTACGCGGACCAGGAGTGTGT

[0137] CAATGTGCAAACGGCCGACGTGGCTACGGTGATTGAGCTGTACCGGGCTGCCCAGGTGCGCATC

[0138] TCCGAAGAGGAGTCGTCACTTAAAAAGCTGCATGCTTGGACTACAACCTTCCTGAAATACCAGCT

[0139] GCAATCCAACTCCATTCCCGAGAAGAAGCTACACAAGCTTGTGGAATACTACCTCAAAAACTACC

[0140] ACGGGATTCTGGACCGGATGGGTGTTCGAATGGACCTCGATCTGTTTGACATTTCTCATTACAGAA

[0141] CCCTGCAGGCTAGTGATCGGTTTTCTTCGCTCCGAAACGAAGACTTCCTGGAGTTCGCCCGGCAG

[0142] GACTTCAACATCTGCCAGGCCAAGCATCAGAAGGAACTGCAACAACTGCAGCGATGGTACGCAG

[0143] ACTGCCGTTTGGATACACTCAAGTTTGGGAGAGATGTTGTAAGAGTAGCGAACTTCCTCACCAGT

[0144] GCCATTTTTGGCGAGCCGGAGCTGTCTGACGCCCGACTGATTTTCGCCAAGCACATTGTTTTGGT

[0145] GACCTGCATCGATGAGTTCTTCGACCACGGTGGCTCCAAGGAAGAATCTTACAAAATTCTAGAAC

[0146] TCGTGGAGGAGTGGAAGGAGAAGCCTACCGGAGAGTACGGCTGTGAGGAGGTTGAGATCTTATT

[0147] TACCGCTGTCTACTCCACTGTCAACGAGCTCGCCGAGATGGCTCATGTGGAGCAGGGCCGATCTG

[0148] TCAAGGAGTTTTTAGTCAAACTGTGGGTCCAAATCTTGTCGATCTTCAAGATCGAACTTGACACC

[0149] TGGTCGGACGATACTGAACTCACCCTCGATAGTTATCTCAACAACTCGTGGGTGTCTATTGGATGT

[0150] CGAATCTGTATCCTGATGTCCATGCAGTTCGCAGGTGTCAAGCTGAGCGACGAAATGCTGCTGTC

[0151] AGAGGAGTGCGTCGATCTTTGCCGACACGTGTCAATGGTCGACCGTTTGCTTAACGACGTCCAGA

[0152] CCTTTGAAAAAGAGCGTAAAGAGAACACTGGTAACTCGGTTTCACTGTTGCAAGCAGCTGCTGA

[0153] ACGTGAGGGACGAGCAATTACAGAAGAGGAGGCCATCACCCAGATCAAGGAATTGGCTGAGTAT

[0154] CATCGAAGAAAGCTCATGCAGATTGTCTACAAGACTGACACCATCTTTCCTCGCAAATGCAAGGA

[0155] TATGTTTCTCAAGGTGTGTAGAATCGGATGCTACCTGTATGCTAGCGGAGACGAGTTCACCACCCC

[0156] TCAGCAGATGATGGAGGATATGAAGTCTCTTGTGTACCAGCCCCTTACTGTTGACGACATGTCTGC

[0157] TAAGGAGTTGACTAGCGTCCGAAATTAG

[0158] SEQ ID NO:3

[0159] ATGAAGACCGTGGTGATCATCGACGCCCTGCGAACCCCCATCGGCAAGTACAAGGGCTCTCTGTC

[0160] TCAAGTGTCTGCCGTGGACCTGGGCACCCACGTGACCACTCAGCTGCTGAAGAGACACTCTACC

[0161] ATCTCTGAGGAGATTGACCAAGTGATCTTCGGCAACGTGCTGCAAGCCGGCAACGGACAGAACC

[0162] CCGCCCGACAGATCGCCATCAACTCTGGCCTGTCTCACGAGATCCCCGCTATGACCGTGAACGAG

[0163] GTGTGTGGCTCTGGCATGAAGGCCGTGATCCTGGCCAAGCAGCTGATTCAGCTGGGCGAGGCCG

[0164] AGGTGCTGATCGCCGGCGGCATCGAGAACATGTCTCAAGCCCCCAAGCTGCAACGATTCAACTA

[0165] CGAGACCGAGTCTTACGACGCCCCCTTCTCTTCTATGATGTACGACGGCCTGACCGACGCCTTCT

[0166] CTGGCCAAGCTATGGGCCTGACCGCCGAGAACGTGGCCGAGAAGTACCACGTGACCCGAGAGG

[0167] AGCAAGATCAGTTCTCTGTGCATTCCCAACTGAAGGCCGCCCAAGCCCAAGCCGAGGGCATCTT

[0168] CGCCGACGAGATCGCCCCCCTGGAGGTGTCTGGCACCCTGGTGGAGAAGGACGAGGGCATCCG

[0169] ACCCAACTCTTCTGTGGAGAAGCTGGGCACCCTGAAGACCGTGTTCAAGGAGGACGGCACCGT

[0170] GACCGCCGGCAACGCCTCTACCATCAACGACGGCGCCTCTGCCCTGATCATCGCCTCTCAAGAGT

[0171] ACGCCGAGGCCCACGGCCTGCCCTACCTGGCCATCATCCGAGACTCTGTGGAGGTGGGCATCGA

[0172] CCCCGCCTACATGGGCATCTCTCCCATCAAGGCCATTCAGAAGCTGCTGGCCCGAAATCAGCTGA

[0173] CCACCGAGGAGATCGACCTCTATGAGATCAACGAGGCCTTCGCCGCCACCTCTATCGTGGTGCAG

[0174] CGAGAGCTGGCCCTGCCCGAGGAGAAGGTGAACATCTACGGCGGAGGCATCTCTCTGGGCCACG

[0175] CCATCGGCGCCACCGGCGCCCGACTGCTGACCTCTCTGTCTTATCAGCTGAATCAGAAGGAGAAG

[0176] AAGTACGGCGTGGCCTCTCTGTGTATCGGCGGAGGCCTCGGCCTGGCCATGCTGCTGGAGCGAC

[0177] CTCAGCAGAAGAAGAACTCTCGATTCTATCAGATGTCTCCCGAGGAGCGACTCGCCTCTCTGCTG

[0178] AACGAGGGACAGATCTCTGCCGACACCAAGAAGGAGTTCGAGAACACCGCCCTGTCTTCTCAGA

[0179] TCGCCAACCACATGATCGAGAATCAGATCTCTGAGACCGAGGTGCCTATGGGCGTGGGCCTGCAC

[0180] CTGACCGTGGACGAGACCGACTACCTGGTGCCTATGGCCACCGAGGAGCCCTCTGTGATTGCCG

[0181] CCCTGTCTAACGGCGCCAAGATCGCCCAAGGCTTCAAGACCGTGAATCAGCAGCGACTGATGCG

[0182] AGGACAGATCGTGTTCTACGACGTGGCCGACGCCGAGTCTCTGATCGACGAGCTGCAAGTGCGA

[0183] GAGACCGAGATCTTTCAGCAAGCCGAGCTGTCTTACCCCTCTATCGTGAAGCGAGGCGGAGGCC

[0184] TGCGAGATCTGCAATACCGAGCCTTCGACGAGTCTTTCGTGTCTGTGGACTTCCTGGTGGACGTG

[0185] AAGGACGCTATGGGCGCCAACATCGTGAACGCCATGCTGGAGGGCGTGGCCGAGCTGTTCCGAG

[0186] AGTGGTTCGCCGAGCAGAAGATCCTGTTCTCTATCCTGTCTAACTACGCCACCGAGTCTGTGGTG

[0187] ACCATGAAGACCGCCATCCCTGTGTCTCGACTGTCTAAGGGCTCTAACGGCCGAGAGATCGCCGA

[0188] GAAGATCGTGCTGGCCTCTCGATACGCCTCTCTGGACCCCTACCGAGCCGTGACCCACAACAAG

[0189] GGCATCATGAACGGCATCGAGGCCGTGGTGCTGGCCACCGGCAACGACACCCGAGCCGTGTCTG

[0190] CCTCTTGTCACGCCTTCGCCGTGAAGGAGGGCCGATACCAAGGCCTGACCTCTTGGACCCTGGA

[0191] CGGCGAGCAGCTGATTGGCGAGATCTCTGTGCCCCTGGCTCTGGCCACTGTGGGCGGAGCTACTA

[0192] AGGTGCTGCCCAAGTCTCAAGCCGCTGCCGACCTGCTGGCCGTGACCGACGCTAAGGAGCTGTC

[0193] TCGAGTGGTGGCCGCCGTGGGCCTGGCTCAGAACCTGGCCGCCCTGCGAGCCCTGGTGTCTGAG

[0194] GGCATTCAGAAGGGCCACATGGCCCTGCAAGCCCGATCTCTGGCCATGACTGTGGGCGCTACCG

[0195] GCAAAGAGGTGGAGGCCGTGGCTCAGCAGCTGAAGCGACAGAAGACCATGAACCAAGACCGA

[0196] GCCCTGGCCATCCTGAACGACCTGCGAAAGCAGTAA

[0197] SEQ ID NO:4

[0198] ATGTCGCAACCCCAGAACGTTGGAATCAAAGCCCTCGAGATCTACGTGCCTTCTCGAATTGTCAA

[0199] CCAGGCTGAGCTCGAGAAGCACGACGGTGTCGCTGCTGGCAAGTACACCATTGGTCTTGGTCAG

[0200] ACCAACATGGCCTTTGTCGACGACAGAGAGGACATCTATTCCTTTGCCCTGACCGCCGTCTCTCG

[0201] ACTGCTCAAGAACAACAACATCGACCCTGCATCTATTGGTCGAATCGAGGTTGGTACTGAAACCC

[0202] TTCTGGACAAGTCCAAGTCCGTCAAGTCTGTGCTCATGCAGCTCTTTGGCGAGAACAGCAACATT

[0203] GAGGGTGTGGACAACGTCAACGCCTGCTACGGAGGAACCAACGCCCTGTTCAACGCTATCAACT

[0204] GGGTTGAGGGTCGATCTTGGGACGGCCGAAACGCCATCGTCGTTGCCGGTGACATTGCCCTCTAC

[0205] GCAAAGGGCGCTGCCCGACCCACCGGAGGTGCCGGCTGTGTTGCCATGCTCATTGGCCCCGACG

[0206] CTCCCCTGGTTCTTGACAACGTCCACGGATCTTACTTCGAGCATGCCTACGATTTCTACAAGCCTG

[0207] ATCTGACCTCCGAGTACCCCTATGTTGATGGCCACTACTCCCTGACCTGTTACACAAAGGCCCTCG

[0208] ACAAGGCCTACGCTGCCTACAACGCCCGAGCCGAGAAGGTCGGTCTGTTCAAGGACTCCGACAA

[0209] GAAGGGTGCTGACCGATTTGACTACTCTGCCTTCCACGTGCCCACCTGCAAGCTTGTCACCAAGT

[0210] CTTACGCTCGACTTCTCTACAACGACTACCTCAACGACAAGAGCCTGTACGAGGGCCAGGTCCCC

[0211] GAGGAGGTTGCTGCCGTCTCCTACGATGCCTCTCTCACCGACAAGACCGTCGAGAAGACCTTCCT

[0212] TGGTATTGCCAAGGCTCAGTCCGCCGAGCGAATGGCTCCTTCTCTCCAGGGACCCACCAACACCG

[0213] GTAACATGTACACCGCCTCTGTGTACGCTTCTCTCATCTCTCTGCTGACTTTTGTCCCCGCTGAGC

[0214] AGCTGCAGGGCAAGCGAATCTCTCTCTTCTCTTACGGATCTGGTCTTGCTTCCACTCTTTTCTCTC

[0215] TGACCGTCAAGGGAGACATTTCTCCCATCGTCAAGGCCTGCGACTTCAAGGCTAAGCTCGATGAC

[0216] CGATCCACCGAGACTCCCGTCGACTACGAGGCTGCCACCGATCTCCGAGAGAAGGCCCACCTCA

[0217] AGAAGAACTTTGAGCCCCAGGGAGACATCAAGCACATCAAGTCTGGCGTCTACTACCTCACCAA

[0218] CATCGATGACATGTTCCGACGAAAGTACGAGATCAAGCAGTAG

[0219] SEQ ID NO:5

[0220] ATGACCCAGTCTGTGAAGGTGGTTGAGAAGCACGTTCCTATCGTCATTGAGAAGCCCAGCGAGA

[0221] AGGAGGAGGACACCTCTTCTGAAGACTCCATTGAGCTGACTGTCGGAAAGCAGCCCAAGCCCGT

[0222] GACCGAGACCCGTTCTCTGGACGACCTAGAGGCTATCATGAAGGCAGGTAAGACCAAGCTTCTG

[0223] GAGGACCACGAGGTTGTCAAGCTCTCTCTCGAGGGCAAGCTTCCTTTGTATGCTCTTGAGAAGCA

[0224] GCTTGGTGACAACACCCGAGCTGTTGGCATCCGACGATCTATCATCTCCCAGCAGTCTAATACCA

[0225] AGACTTTAGAGACCTCAAAGCTTCCTTACCTGCACTACGACTACGACCGTGTTTTTGGAGCCTGT

[0226] TGCGAGAACGTTATTGGTTACATGCCTCTCCCCGTTGGTGTTGCTGGCCCCATGAACATTGATGGC

[0227] AAGAACTACCACATTCCTATGGCCACCACTGAGGGTTGTCTTGTTGCCTCAACCATGCGAGGTTG

[0228] CAAGGCCATCAACGCCGGTGGCGGTGTTACCACTGTGCTTACTCAGGACGGTATGACACGAGGT

[0229] CCTTGTGTTTCCTTCCCCTCTCTCAAGCGGGCTGGAGCCGCTAAGATCTGGCTTGATTCCGAGGA

[0230] GGGTCTCAAGTCCATGCGAAAGGCCTTCAACTCCACCTCTCGATTTGCTCGTCTCCAGTCTCTTC

[0231] ACTCTACCCTTGCTGGTAACCTGCTGTTTATTCGATTCCGAACCACCACTGGTGATGCCATGGGCA

[0232] TGAACATGATCTCCAAGGGCGTCGAACACTCTCTGGCCGTCATGGTCAAGGAGTACGGCTTCCCT

[0233] GATATGGACATTGTGTCTGTCTCGGGTAACTACTGCACTGACAAGAAGCCCGCAGCGATCAACTG

[0234] GATCGAAGGCCGAGGCAAGAGTGTTGTTGCCGAAGCCACCATCCCTGCTCACATTGTCAAGTCT

[0235] GTTCTCAAAAGTGAGGTTGACGCTCTTGTTGAGCTCAACATCAGCAAGAATCTGATCGGTAGTGC

[0236] CATGGCTGGCTCTGTGGGAGGTTTCAATGCACACGCCGCAAACCTGGTGACCGCCATCTACCTTG

[0237] CCACTGGCCAGGATCCTGCTCAGAATGTCGAGTCTTCCAACTGCATCACGCTGATGAGCAACGTC

[0238] GACGGTAACCTGCTCATCTCCGTTTCCATGCCTTCTATCGAGGTCGGTACCATTGGTGGAGGTACT

[0239] ATTTTGGAGCCCCAGGGGGCTATGCTGGAGATGCTTGGCGTGCGAGGTCCTCACATCGAGACCCC

[0240] CGGTGCCAACGCCCAACAGCTTGCTCGCATCATTGCTTCTGGAGTTCTTGCAGCGGAGCTTTCGC

[0241] TGTGTTCTGCTCTTGCTGCCGGCCATCTTGTGCAAAGTCATATGACCCACAACCGGTCCCAGGCT

[0242] CCTACTCCGGCCAAGCAGTCTCAGGCCGATCTGCAGCGTCTACAAAACGGTTCGAATATTTGCAT

[0243] ACGGTCATAG

[0244] SEQ ID NO:6

[0245] GGAGGCGGCGGATCTGGCGGAGGAGGCTCTGGAGGCGGAGGATGCGGCCTGGAGCAGTACGCC

[0246] AATCAGCTGGCCGATCAGATCATCAAGGAGGCCACCGAGGGCTGT(RIAD)

[0247] CTGCGAGAGTGTGAGCTGTACGTGCAGAAGCACAACATTCAGGCTCTGCTGAAGGACTCTATTG

[0248] TGCAGCTGTGTACCGCTCGACCTGAGCGACCCATGGCTTTTCTGCGAGAGTACTTTGAGCGACTG

[0249] GAGAAGGAGGAGGCCAAG (RIDD)

[0250] SEQ ID NO: 7

[0251] ATGTCCAAGGCGAAATTCGAAAGCGTGTTCCCCCGAATCTCCGAGGAGCTGGTGCAGCTGCTGC

[0252] GAGACGAGGGTCTGCCCCAGGATGCCGTGCAGTGGTTTTCCGACTCACTTCAGTACAACTGTGT

[0253] GGGTGGAAAGCTCAACCGAGGCCTGTCTGTGGTCGACACCTACCAGCTACTGACCGGCAAGAAG

[0254] GAGCTCGATGACGAGGAGTACTACCGACTCGCGCTGCTCGGCTGGCTGATTGAGCTGCTGCAGG

[0255] CGTTTTTCCTCGTGTCGGACGACATTATGGATGAGTCCAAGACCCGACGAGGCCAGCCCTGCTGG

[0256] TACCTCAAGCCCAAGGTCGGCATGATTGCCATCAACGATGCTTTCATGCTAGAGAGTGGCATCTAC

[0257] ATTCTGCTTAAGAAGCATTTCCGACAGGAGAAGTACTACATTGACCTTGTCGAGCTGTTCCACGA

[0258] CATTTCGTTCAAGACCGAGCTGGGCCAGCTGGTGGATCTTCTGACTGCCCCCGAGGATGAGGTTG

[0259] ATCTCAACCGGTTCTCTCTGGACAAGCACTCCTTTATTGTGCGATACAAGACTGCTTACTACTCCT

[0260] TCTACCTGCCCGTTGTTCTAGCCATGTACGTGGCCGGCATTACCAACCCCAAGGACCTGCAGCAG

[0261] GCCATGGATGTGCTGATCCCTCTCGGAGAGTACTTCCAGGTCCAGGACGACTACCTTGACAACTT

[0262] TGGAGACCCCGAGTTCATTGGTAAGATCGGCACCGACATCCAGGACAACAAGTGCTCCTGGCTCGTTAACAAAGCCCTTCAGAAGGCCACCCCCGAGCAGCGACAGATCCTCGAGGACAACTACGGCGTCAAGGACAAGTCCAAGGAGCTCGTCATCAAGAAACTGTATGATGACATGAAGATTGAGCAGGACTACCTTGACTACGAGGAGGAGGTTGTTGGCGACATCAAGAAGAAGATCGAGCAGGTTGACGAGAGCCGAGGCTTCAAGAAGGAGGTGCTCAACGCTTTCCTCGCCAAGATTTACAAGCGACAGAAGTAG

[0263] SEQ ID NO: 8

[0264] ATGGTCGCTCAGACCTTCAACCTGGACACCTACCTGTCCCAGCGACAGCAGCAGGTTGAGGAGGCTCTGTCTGCTGCTCTGGTCCCTGCTTACCCTGAGCGAATCTACGAGGCCATGCGATACTCCCTCCTGGCTGGTGGTAAGCGACTGCGACCTATCCTCTGCCTCGCTGCTTGTGAGCTGGCTGGTGGCTCTGTTGAGCAGGCTATGCCTACCGCTTGCGCTCTGGAGATGATTCACACCATGTCTCTGATCCACGACGACCTCCCCGCTATGGACAACGACGACTTTCGACGAGGCAAGCCTACCAACCACAAGGTCTTCGGCGAGGACATCGCCATTCTGGCTGGTGACGCTCTGCTGGCTTACGCTTTCAGCACATCGCCTCCCAGACCCGAGGTGTTCCTCCTCAGCTGGTTCTCCAGGTCATCGCTCGAATCGGTCACGCTGTCGCTGCTACCGGTCTGGTTGGTGGTCAGGTTGTCGACCTGGAGTCCGAGGGTAAGGCTATCTCCCTGGAGACCCTCGAGTACATTCACTCCCACAAGACCGGCGCCCTGCTGGAGGCTTCTGTTGTTTCTGGCGGTATCCTGGCCGGCGCTGACGAGGAGCTGCTGGCTCGACTGTCTCACTACGCTCGAGACATTGGCCTGGCCTTTCAGATCGTCGACGACATTCTGGACGTGACCGCTACCTCTGAGCAGCTGGGTAAGACCGCTGGTAAGGACCAGGCTGCTGCTAAGGCTACCTACCCTTCCCTGCTGGGTCTGGAGGCTTCCCGACAGAAGGCTGAGGAGCTGATTCAGTCCGCCAAGGAGGCTCTGCGACCTTACGGTTCTCAGGCTGAGCCTCTCCTGGCTCTGGCTGACTTTATTACCCGACGACAGCACTAA

[0265] SEQ ID NO: 9

[0266]

[0267] SEQ ID NO: 10

[0268] ATATGCACTGGCAAATCTCGGATCGGATAATCGGAGTGTCTCCGTAATCTTTGTTGCGATACTTGTACGCGTCAGTAGTTAGCAAGCTCGATACAGTTCATTGTGGATTTACTAAGCGAGCATTTCCATCCCAAGTAACCTTGTGTTTGTTTTATTAGTAATATTCGTGTAAGCGTACATTCATTTCAAAGGTTATTCTTTCACAGCAAAGTTATAATTAAATGAATGTATATGCAGAAAAAAAAGTTTACATACGCAATAACATCAGAAGTCTACAACCATTGTTTCTACAAGTACAAGTGCAAGTACACCCGTAAGGGTTGGCGAGCTCATTCTTAAATCAAGACTACTTGTAGTTGTAGCTATCGTGTACCACGTGGTGTCTTCGTGCGATATCAAATTTCCAGCATAGCCGCGTATGACAAGCACCATCGTTTTTGCATGTTAGGCTAAATTGCAGGCTGTGCACAGGCAGTGATTACTGTACTGTCTTATTGTGGTACGATGAGTGGACATGATATAGTACCACTGCATCTCTCTTGGTACAGAAGCTAACTTTGGCCCCTTACACACATTCCATGACGTGATGATTTCTCCATTTGAAAGGTGGTGTCATACCTTGCATAATCATAGCAGGGGGATCCATGAAGGATACCCCGAGCCAGCCATCGGCCTATATAAAATAACAAAATCCACTGGATCAGGACTGCTGCATCCTCAGGATTCTCACAGTGGTCCAAGTACATATTTTCCGAGCCCGTTACTTTTATTTCTTATTACCCCTATCCCTGGCATTATCGTCCATGTAATTGGACCGTTACATTTAGACACGATACATACGACATTAGCGAGCAGGTTTGGAAGAAAATAAGGTTTCGTGATTGTGGACGAGGTTCCAGAAGAGTTTAATTTATCCTTCCATTTCTACTTCATCTTGTCTGGTGCTCTCAACATCTACAACTGGTCTCTCACTCAAGTCGACCACAAC

[0269] SEQ ID NO: 11

[0270] ATGTCTGAAGACCACCCAGCCATCCACCCACCCTCCGAGTTCAAGGACAACCACCCCCACTTCGGAGGCCCCCACCTCGACTGTCTGCAGGACTACCACCAGCTGCACAAGGAGTCCATTGAGGACCCCAAGGCCTTCTGGAAGAAGATGGCCAACGAGCTCATCTCCTGGTCAACCCCCTTTGAAACTGTGCGATCTGGCGGCTTCGAGCACGGCGACGTGGCCTGGTTCCCCGAGGGCCAGCTCAACGCCTCCT

[0271] ACAACTGTGTGGATCGACACGCCTTTGCCAACCCCGACAAGCCCGCCATCATTTTTGAGGCCGAT

[0272] GAGCCGGGCCAGGGCCGAATCGTCACCTACGGCGAACTGCTGCGACAGGTGTCTCAGGTCGCAG

[0273] CCACCCTGCGATCCTTCGGCGTCCAGAAGGGCGATACTGTGGCCGTCTACCTGCCCATGATCCCC

[0274] GAGGCCATTGTCACTCTGCTGGCCATCACCCGAATTGGCGCTGTCCACTCGGTCATCTTCGCCGG

[0275] CTTCTCCTCCGGTTCTCTGCGAGACCGAATCAACGACGCCAAGTCCAAGGTTGTCGTCACCACCG

[0276] ACGCCTCCATGCGAGGAGGCAAGACCATCGACACCAAGAAGATTGTCGATGAAGCCTTGCGAGA

[0277] CTGCCCCTCTGTTACCCACACCCTGGTCTTCCGACGAGCAGGTGTCGAGAACCTGGCCTGGACTG

[0278] AGGGCCGGGACTTCTGGTGGCACGAGGAGGTCGTCAAGCACCGACCCTACCTTGCCCCCGTCCC

[0279] CGTTGCCTCCGAGGACCCCATCTTCCTGCTTTACACCTCTGGATCCACCGGCACCCCCAAGGGTC

[0280] TGGCCCACGCTACCGGTGGCTACCTGCTTGGTGCTGCCCTGACCGCCAAGTACGTGTTTGACATC

[0281] CACGGAGACGACAAGCTGTTCACCGCTGGAGACGTTGGCTGGATCACCGGCCACACCTACGTGC

[0282] TCTACGGTCCTCTGATGCTCGGAGCCACCACTGTTGTGTTCGAGGGAACCCCTGCCTACCCCTCC

[0283] TTCTCGCGATACTGGGACATTGTCGACGACCACAAGATCACCCACTTCTACGTGGCTCCCACCGC

[0284] CCTGCGTCTCCTGAAGCGGGCCGGCACCCATCACATTAAGCACGACCTGTCGTCTCTGCGAACCC

[0285] TCGGCTCTGTGGGTGAGCCCATTGCCCCCGACGTGTGGCAGTGGTACAACGACAACATTGGCCG

[0286] AGGCAAGGCCCACATCTGTGACACCTACTGGCAGACCGAGACTGGCTCGCATATCATTGCCCCCA

[0287] TGGCCGGCGTGACCCCCACCAAGCCCGGTTCTGCTTCCCTGCCTGTCTTTGGAATTGATCCCGTTA

[0288] TCATTGATCCCGTGTCTGGCGAGGAGCTCAAGGGTAACAACGTTGAGGGTGTTCTTGCCCTGCGA

[0289] TCTCCCTGGCCCTCCATGGCCCGAACCGTGTGGAACACCCACGAGCGATACATGGAGACCTACCT

[0290] GCGGCCCTACCCCGGCTACTACTTCACCGGTGATGGTGCTGCCCGAGACAATGACGGCTTTTACT

[0291] GGATCCGAGGCCGAGTCGACGACGTTGTCAACGTTTCTGGCCACCGTCTTTCCACCGCCGAGATT

[0292] GAGGCTGCTCTCATTGAGCACGCTCAGGTGTCTGAGTCTGCCGTTGTTGGTGTCCATGACGATCT

[0293] GACTGGCCAGGCCGTCAACGCCTTTGTGGCTCTCAAGAACCCCGTCGAGGATGTGGACGCTCTG

[0294] CGAAAGGAGCTTGTTGTGCAGGTGCGAAAGACCATTGGACCCTTTGCTGCTCCCAAGAATGTCAT

[0295] CATCGTGGACGATCTGCCCAAGACTCGGTCTGGCAAGATCATGCGACGAATTCTGCGAAAGGTG

[0296] CTTGCTGGCGAGGAGGACCAGCTCGGAGACATTTCCACTCTTGCTAACCCCGACGTTGTCCAGA

[0297] CCATCATTGAGGTTGTTCACTCGTTGAAAAAGTAA

[0298] SEQ ID NO: 12

[0299] ATGACTGTCAGAGACGCCCTCAACACCGCACTGCGAGAGGAGATGGACCGAAACGATAATGTTT

[0300] TCATCATGGGTGAGGAGGTCGGCCAGTACAACGGTGCCTACAAGGTCACCAAGGGCCTTCTCGA

[0301] CAAGTTCGGCGAGAAGCGAGTGGTTGACACCCCTATCACCGAGATGGGTTTCGCCGGTGTTTGT

[0302] GTCGGTGCCGCCCTGGCCGGTCTCACCCCCGTCTGCGAGTTCATGACCTGGAACTTCGCCATGCA

[0303] GGCCATTGATCAGATCATCAATTCCGGTGCCAAGACCTACTACATGTCCGGAGGTACCCAGCAGT

[0304] GCAATGTCACCTTCCGAGGTCCTAACGGTGCCGCCGCTGGTGTTGCTGCCCAACACTCTCAGGAT

[0305] TTCACCGGGTGGTACGGCCAGATTCCCGGTCTCAAGGTCGTCTCTCCCTACAGCTCTGAGGATGC

[0306] CAAGGGTCTGCTCAAGGCCGCCATCCGAGACCCCAACGTGACTGTTTTCCTCGAGAACGAGATC

[0307] ATGTACGGAGAGTCTTTCCCCATGTCTGAGGAGGCCATGTCCCCCGACTTCGTTCTGCCCCTTGG

[0308] AAAGGCCAAGATTGAGCGAGAGGGTAAGGATATCACTCTTGTCGGTCACTCCCGAAACGTCGAG

[0309] ACCGCCCTCAAGGCCGCCGACCTCCTCAAGAAGCACCACAACGTCGATGCCGAGGTCATTAACC

[0310] TGCGAACTGTCAAGCCTCTCGACACTGAGACCATTTTCAACTCCATCAAGAAGACTAACCGACTT

[0311] GTCTCTGTCGAGGCTGGCTTCCCCGCCTTTGGCATGGGCTCCGAGCTCTGTGGTGTCGTCAACGA

[0312] CTCCTGGGCCTGGGATTACCTTGATGCCCCCATCCAGCGAGTTACCGGAGCTGAGGTTCCCACTC

[0313] CTTACGCCATTGAGCTTGAGAACTTCGCCTTCCCCACACCCGAGATTGTTGTCAAGGCTGCCAAG

[0314] GACGCCCTCTACATTGAGGAGTAG

[0315] SEQ ID NO:13

[0316] ATGATTTCTGCTATTCGTCCCGCCGTTCGATCTTCCGTTCGTGTTGCCCCTATGGCCAACACCGCCT

[0317] TCCGGGCCTACTCTACCCAGGATGGTCTTAAGGAGCGATTCGCCGAGCTCATCCCCGAGAACGTC

[0318] GAGAAGATCAAGAAGCTCCGAAAGGAGAAGGGTAACACCGTCATCGGCGAGGTCATCCTCGAC

[0319] CAGGCTTACGGTGGTATGCGAGGTATTAAGGGTCTCGTCTGGGAGGGATCCGTCCTCGACCCCGA

[0320] GGAGGGTATCCGATTCCGAGGTCTGACTATCCCCGACCTCCAGAAGCAGCTCCCCCACGCCCCTG

[0321] GCGGAAAGGAGCCTCTCCCCGAGGGTCTTTTCTGGCTCCTGCTCACCGGCGAGATCCCCACTGAT

[0322] GCTCAGGTCAAGGGTCTGTCCGCTGACTGGGCCTCTCGAGCCGAGATCCCCAAGCATGTTGAGG

[0323] AGCTCATCGACCGATGCCCCCCCACCCTCCACCCCATGGCTCAGCTCGGTATTGCCGTCAACGCT

[0324] CTGGAGTCCGAGTCTCAGTTCACCAAGGCTTACGAGAAGGGTGTTAACAAGAAGGAGTACTGGC

[0325] AGTACACCTACGAGGATTCCATGAACCTCATTGCCAAGCTCCCCGTCATTGCTTCTCGAATCTACC

[0326] GAAACCTTTTCAAGGACGGAAAGATTGTTGGCTCCATTGACAACTCTCTTGACTACTCTGCTAAC

[0327] TTCGCCTCTCTGCTCGGCTTTGGCGACAACAAGGAGTTCATTGAGCTTCTGCGACTCTACCTCAC

[0328] CATCCACGCTGACCACGAGGGAGGTAACGTCTCTGCCCACACCACCAAGCTTGTTGGTTCTGCTC

[0329] TCTCCTCTCCCTTCCTCTCTCTGTCCGCTGGTCTCAACGGTCTTGCCGGTCCTCTCCACGGCCGAG

[0330] CTAACCAGGAGGTCCTTGAGTGGATTCTCGAGATGAAGTCCAAGATTGGCTCTGATGTCACCAAG

[0331] GAGGACATTGAGAAGTACCTCTGGGATACCCTTAAGGCCGGTCGAGTCGTCCCCGGTTACGGAC

[0332] ACGCCGTTCTCCGAAAGACCGATCCTCGATACACCGCCCAGCGAGAGTTCGCCCTCGAGCACAT

[0333] GCCCGACTACGACCTCTTCCACCTCGTTTCCACCATCTACGAGGTTGCCCCCAAGGTTCTCACCG

[0334] AGCACGGCAAGACCAAGAACCCCTGGCCCAATGTGGACTCCCACTCCGGTGTCCTCCTCCAGTA

[0335] CTACGGTCTCACTGAGCAGTCTTACTACACTGTTCTCTTCGGTGTTTCCCGAGCTATCGGTGTCCT

[0336] GCCCCAGCTCATCATGGACCGAGCTTACGGTGCTCCCATCGAGCGACCCAAGTCCTTCTCTACCG

[0337] AGAAGTACGCTGAGCTCGTTGGCCTCAAGCTCTAA

[0338] SEQ ID NO: 14

[0339] ATGACTGGCACCTTACCCAAGTTCGGCGACGGAACCACCATTGTGGTTCTTGGAGCCTCCGGCGA

[0340] CCTCGCTAAGAAGAAGACCTTCCCCGCCCTCTTCGGCCTTTACCGAAACGGCCTGCTGCCCAAA

[0341] AATGTTGAAATCATCGGCTACGCACGGTCGAAAATGACTCAGGAGGAGTACCACGAGCGAATCA

[0342] GCCACTACTTCAAGACCCCCGACGACCAGTCCAAGGAGCAGGCCAAGAAGTTCCTTGAGAACA

[0343] CCTGCTACGTCCAGGGCCCTTACGACGGTGCCGAGGGCTACCAGCGACTGAATGAAAAGATTGA

[0344] GGAGTTTGAGAAGAAGAAGCCCGAGCCCCACTACCGTCTTTTCTACCTGGCTCTGCCCCCCAGC

[0345] GTCTTCCTTGAGGCTGCCAACGGTCTGAAGAAGTATGTCTACCCCGGCGAGGGCAAGGCCCGAA

[0346] TCATCATCGAGAAGCCCTTTGGCCACGACCTGGCCTCGTCACGAGAGCTCCAGGACGGCCTTGC

[0347] TCCTCTCTGGAAGGAGTCTGAGATCTTCCGAATCGACCACTACCTCGGAAAGGAGATGGTCAAG

[0348] AACCTCAACATTCTGCGATTTGGCAACCAGTTCCTGTCCGCCGTGTGGGACAAGAACACCATTTC

[0349] CAACGTCCAGATCTCCTTCAAGGAGCCCTTTGGCACTGAGGGCCGAGGTGGATACTTCAACGAC

[0350] ATTGGAATCATCCGAGACGTTATTCAGAACCATCTGTTGCAGGTTCTGTCCATTCTAGCCATGGAG

[0351] CGACCCGTCACTTTCGGCGCCGAGGACATTCGAGATGAGAAGGTCAAGGTGCTCCGATGTGTCG

[0352] ACATTCTCAACATTGACGACGTCATTCTCGGCCAGTACGGCCCCTCTGAAGACGGAAAGAAGCC

[0353] CGGATACACCGATGACGATGGCGTTCCCGATGACTCCCGAGCTGTGACCTTTGCTGCTCTCCATCT

[0354] CCAGATCCACAACGACAGATGGGAGGGTGTTCCTTTCATCCTCCGAGCCGGTAAGGCTCTGGAC

[0355] GAGGGCAAGGTCGAGATCCGAGTGCAGTTCCGAGACGTGACCAAGGGCGTTGTGGACCATCTGC

[0356] CTCGAAATGAGCTCGTCATCCGAATCCAGCCCTCCGAGTCCATCTACATGAAGATGAACTCCAAG

[0357] CTGCCTGGCCTTACTGCCAAGAACATTGTCACCGACCTGGATCTGACCTACAACCGACGATACTC

[0358] GGACGTGCGAATCCCTGAGGCTTACGAGTCTCTCATTCTGGACTGCCTCAAGGGTGACCACACC A

[0359] ACTTTGTGCGAAACGACGAGCTGGACATTTCCTGGAAGATTTTCACCGATCTGCTGCACAAGATT

[0360] GACGAGGACAAGAGCATTGTGCCCGAGAAGTACGCCTACGGCTCTCGTGGCCCCGAGCGACTCA

[0361] AGCAGTGGCTCCGAGACCGAGGCTACGTGCGAAACGGCACCGAGCTGTACCAATGGCCTGTCAC

[0362] CAAGGGCTCCTCGTGA

[0363] SEQ ID NO: 15

[0364] ATGACTGACACTTCAAACATCAAGCCTGTCGCTGACATTGCCCTCATCGGTCTCGCCGTCATGGG

[0365] CCAGAACCTGATCCTCAACATGGCCGACCACGGTAAGTATCAATTGACTCAAGACGCACCAGCA

[0366] AGATACAGAGCATACCCAGCAATCGCTCCTCTGATAATCGCCATTGTAACACTACGTTGGTTAGAT

[0367] TGATCTAAGGTCGTTGCTGGTTCCATGCACTTCCACTTGCTCATATGAAGGGAGTCAAACTCTATT

[0368] TTGATAGTGTCCTCTCCCATCCCCGAAATGTCGCATTGTTGCTAACAATAGGCTACGAGGTTGTTG

[0369] CCTACAACCGAACCACCTCCAAGGTCGACCACTTCCTCGAGAACGAGGCCAAGGGTGAGTATCC

[0370] GTCCAGCTATGCTGTTTACAGCCATTGACCCCACCTTCCCCCACAATTGCTACGTCACCATTAAAA

[0371] AACAAAATTACCGGTATCGGCAAGCTAGACTTTCATGCAACCTACGCAGGGTAACAAGTTGAGTT

[0372] TCAGCCGTGCACCTTACAGGAAAACCAGTCATACGCCGAGGCAGTGTGAAAGCGAAAGCACAC

[0373] AGCCTACGGTGATTGATTGCATTTTTTTGACATAGGAGGGAAACACGTGACATGGCAAGTGCCCA

[0374] ACACGAATACTAACAAACAGGAAAGTCCATTATTGGTGCTCACTCTATCAAGGAGCTGTGTGCTC

[0375] TGCTGAAGCGACCCCGACGAATCATTCTGCTCGTTAAGGCCGGTGCTGCTGTCGATTCTTTCATCG

[0376] AACAGCTCCTGCCCTATCTCGATAAGGGTGATATCATCATTGACGGTGGTAACTCCCACTTCCCCG

[0377] ACTCCAACCGACGATACGAGGAGCTTAACGAGAAGGGAATCCTCTTTGTTGGTTCCGGTGTTTCC

[0378] GGCGGTGAGGAGGGTGCCCGATACGGTCCCTCCATCATGCCCGGTGGAAACAAGGAGGCCTGGC

[0379] CCCACATTAAGAAGATTTTCCAGGACATCTCTGCTAAGGCTGATGGTGAGCCCTGCTGTGACTGG

[0380] GTCGGTGACGCTGGTGCCGGCCACTTTGTCAAGATGGTTCACAACGGTATTGAGTATGGTGACAT

[0381] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0382] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0383] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0384] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0385] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0386] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0387] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0388] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0389] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0390] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0391] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC

[0392] CGATCTGCCAAGCTCCCCGCTAACCTGCTCCAGGCCCAGCGAGACTACTTCGGCGCCCACACCTA

[0393] CCAGCTCCTCGATGGTGATGGAAAGTGGATCCACACCAACTGGACCGGCCGAGGTGGTGAGGTT

[0394] TCTTCTTCCACTTACGATGCTTAA

[0395] SEQ ID NO: 16

[0396] ATGTTACGACTACGAACCATGCGACCCACACAGACCAGCGTCAGGGCGGCGCTTGGGCCCACCG

[0397] CCGCGGCCCGAAACATGTCCTCCTCCAGCCCCTCCAGCTTCGAATACTCGTCCTACGTCAAGGGC

[0398] ACGCGGGAAATCGGCCACCGAAAGGCGCCCACAACCCGTCTGTCGGTTGAGGGCCCCATCTACG

[0399] TGGGCTTCGACGGCATTCGTCTTCTCAACCTGCCGCATCTCAACAAGGGCTCGGGATTCCCCCTC

[0400] AACGAGCGACGGGAATTCAGACTCAGTGGTCTTCTGCCCTCTGCCGAAGCCACCCTGGAGGAAC

[0401] AGGTCGACCGAGCATACCAACAATTCAAAAAGTGTGGCACTCCCTTAGCCAAAAACGGGTTCTG

[0402] CACCTCGCTCAAGTTCCAAAACGAGGTGCTCTACTACGCCCTGCTGCTCAAGCACGTTAAGGAG

[0403] GTCTTCCCCATCATCTATACACCGACTCAGGGAGAAGCCATTGAACAGTACTCGCGGCTGTTCCG

[0404] GCGGCCCGAAGGCTGCTTCCTCGACATCACCAGTCCCTACGACGTGGAGGAGCGTCTGGGAGCG

[0405] TTTGGAGACCATGACGACATTGACTACATTGTCGTGACTGACTCCGAGGGTATTCTCGGAATTGG

[0406] AGACCAAGGAGTGGGCGGTATTGGTATTTCCATCGCCAAGCTGGCTCTCATGACTCTATGTGCTG

[0407] GAGTCAACCCCTCACGAGTCATTCCTGTGGTTCTGGATACGGGAACCAACAACCAGGAGCTGCT

[0408] GCACGACCCCCTGTATCTCGGCCGACGAATGCCCCGAGTGCGAGGAAAGCAGTACGACGACTTC

[0409] ATCGACAACTTTGTGCAGTCTGCCCGAAGGCTGTATCCCAAGGCGGTGATCCATTTCGAGGACTT

[0410] TGGGCTCGCTAACGCACACAAGATCCTCGACAAGTATCGACCGGAGATCCCCTGCTTCAACGAC

[0411] GACATCCAGGGCACTGGAGCCGTCACTTTGGCCTCCATCACGGCCGCTCTCAAGGTGCTGGGCA

[0412] AAAATATCACAGATACTCGAATTCTCGTGTACGGAGCTGGTTCGGCCGGCATGGGTATTGCTGAA

[0413] CAGGTCTATGATAACCTGGTTGCCCAGGGTCTCGACGACAAGACTGCGCGACAAAACATCTTTCT

[0414] CATGGACCGACCGGGTCTACTGACCACCGCACTTACCGACGAGCAGATGAGCGACGTGCAGAAG

[0415] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0416] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0417] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0418] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0419] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0420] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0421] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0422] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0423] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0424] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0425] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0426] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC

[0427] ATGTCCACCACCGCTACTCGAGGCCTGTCCACCAAGATCAAGGTCAAGAACCCCATTGTCGAGCT

[0428] CGATGGTGATGAGATGACCCGAATCATCTGGAAGTCCATCAAGGACAAGCTCATTCTGCCCTATCT

[0429] CGACATTGATCTTAAGTACTACGATCTGGGCATCGAGTACCGAGACCAGACTAACGACCAGGTGA

[0430] CCATTGACGCCGCCGAGGCCATCAAGAAGTACCAGGTCGGTGTCAAGTGCGCCACCATCACCCC

[0431] CGACGAGGCCCGAGTCAAGGAGTTTGGCCTCAAGAAGATGTGGCTGTCGCCCAACGGTACCATC

[0432] CGAAACATTCTCGGCGGTACTGTTTTCCGAGAGCCCATTGTCATTCCCGCCGTCCCCCGGCTTGTG

[0433] CCCGGATGGAAGGAGCCTATCATCATTGGTCGACACGCCCACGGCGACCAGTACAAGGCCCAGG

[0434] ATGCCGTCATCCCCGGCGCCGGTGAGCTGACTCTTAACTTCAAGCCCGCTAACGGAGGCGACGA

[0435] GCAGGTCATCAAGGTGTACACCTACGACGCCCCTGGTGTCGCCATGGCCATGTACAACACTGACG

[0436] AGTCCATCACCGGCTTTGCCTACTCTTCATTCAACCTGGCTCTGCAGAAGAAGCTGCCCCTGTAC

[0437] ATGTCTACCAAGAACACCATCCTTAAGAAGTACGACGGCCGATTCAAGGACATTTTCCAGGAGAT

[0438] TTACGACAAG GAGTACAAGG ATAAGTTTGA TGCTGCCGGC ATTTGGTACG AGCACCGACT CATTG

[0439] ATGACATGGT CGCCCAGATG ATCAAGTCTA AGGGAGGCTT CATCATGGCC CTCAAGAACT ACGAC

[0440] GGAGACGTGC AGTCCGACAT TGTTGCCCAG GGCTTTGGCT CTCTCGGTCT CATGACCTCT GTTCT

[0441] CGTCACCCCC GACGGAAAGA CCTTTGAGTC CGAGGCCGCC CACGGCACCG TGACTCGACA CTAC

[0442] CGACAGCACC AGCAGGGCAA GGAGACCTCT ACCAACTCCA TTGCCTCCAT CTTCGCCTGG ACCC

[0443] GAGGCCTCAT CCAGCGAGGC ATTCTCGACG AGACCCCTGA GGTGACCAAG TTTGCCGAGG CTCT

[0444] CGAGAAGGCC ACCGTCGACA CTGTTGACAA GGACGGCATT ATGACCAAGG ATCTGGCTCT GGCC

[0445] GGTGGCAAGA CCGACCGATC CTCGTATGTG CTGACCGAGG AGTTTATCGA CGCTGTGGCC AACA

[0446] GACTGAAGAAGGACCTGGCTTAG

[0447] SEQ ID NO: 18

[0448] ATGCGACTAC TCATCCGCCG AACCGGTATA ACACGGCCCC ACAGCGTGCA AGCGCGCCGA TCAC

[0449] ATGGATTCGG CTTCTCTCGA CCGAGATATT GCATGCAGAA CTGCTTCCCG ACCGCCAGTC CGCCC

[0450] ACTACGTCCAGGAGTCGACCTCTCTGTCATCTCTGGTGTGGGACAAGCCTCTGGAAAACGTTCTG

[0451] ATCGTCAAAAAACCCTGGGACCACAATGTGCGCGAGTCGCTCATCCAGATGGCATCTCACATCCA

[0452] GCGCCGGTACCCCCGAGTCAACATTCTGGTGGAGGAACATGTGGCCGACGAGGTCCAGAAGCAG

[0453] ATTGGAGCCGCAGGCGTGACCGCCATCCACACGGGGCCAGGAGAGGTGCTGAGAAACAAGACG

[0454] GATCTGCTCGTGACTCTGGGAGGCGACGGAACTATTCTACATGCCACCTCCATGTTTGCTTCCGG

[0455] AGAAGTGCCGCCGGTGCTGTCCTTTTCGCTGGGGACTCTGGGTTTCCTGCTGCCGTTTGATTTCA

[0456] AGGACTTCAAAACTGCATTCGACATGGTGTACTCGTCGCAGGCCTCGGTGGTCAACCGCGCCCG

[0457] CCTAGCATGTCAGAAAATGTCCATTCGCAAGGAAATCACCCACTTGCCCTCCCAATCGCACATTG

[0458] AACACAACTCAACCCATGTCTACGGCAATCCCGACGACTACAATCTTAGCCCACTAACCTACGCC

[0459] ATGAACGACATCAACATCCACCGTGGAGCTGAGCCGCATCTCACCAAGCTCGACATCCACGTTGA

[0460] CGGCGAGTTCATCACCCGAGCCATTGCTGACGGTGTCACCATCGCCACACCCACGGGCTCCACG

[0461] GAGTGGTTTACCGCGAGAGCGAGGAGGAGAATTCGCCGGCGGCGGCGGCGGCGGCGGCGGCGGC

[0462] GAGTGGTTTACCGCGAGAGCGAGGAGGAGAATTCGCCGGCGGCGGCGGCGGCGGCGGCGGCGGC

[0463] GAGTGGTTTACCGCGAGAGCGAGGAGGAGAATTCGCCGGCGGCGGCGGCGGCGGCGGCGGCGGC

[0464] GAGTGGTTTACCGCGAGAGCGAGGAGGAGAATTCGCCGGCGGCGGCGGCGGCGGCGGCGGCGGC

[0465] GAGTGGTTTACCGCGAGAGCGAGGAGGAGAATTCGCCGGCGGCGGCGGCGGCGGCGGCGGCGGC

[0466] GAGTGGTTTACCGCGAGAGCGAGGAGGAGAATTCGCCGGCGGCGGCGGCGGCGGCGGCGGCGGC

Claims

1. A method for constructing a recombinant strain with high sclareol production, characterized by, The construction method comprises the following steps: (1) introducing a sclareol synthase pathway into a host strain to obtain a recombinant strain A, wherein the host strain is Yarrowia lipolytica; Or, the construction method further comprises the following step: (2) using peroxisomal acetyl-CoA for the mevalonate pathway in the recombinant strain A to obtain a recombinant strain B; Or, the construction method further comprises the following step: (3) strengthening the mevalonate pathway and weakening at least one of the competing pathways to squalene and lipid synthesis in the recombinant strain B to obtain a recombinant strain C; Or, the construction method further comprises the following step: (4) overexpressing acetyl-CoA and the cofactor NADPH in the recombinant strain C to obtain a recombinant strain D with high sclareol production.

2. The construction method of claim 1, wherein, In step (1), the introduction of the sclareol synthase pathway is overexpression of the pyrophosphate lysadalenediol ester synthase gene tPvLPPS and the sclareol synthase gene PvSCS at the E1 and B1 sites of the genome of the host strain; the nucleotide sequence of the pyrophosphate lysadalenediol ester synthase gene tPvLPPS is shown in SEQ ID NO: 1, and the nucleotide sequence of the sclareol synthase gene PvSCS is shown in SEQ ID NO:

2.

3. The construction method of claim 2, wherein, The C-terminus of the tPvLPPS and PvTPS genes is connected to a pair of polypeptide interaction tags RIAD and RIDD, and the nucleotide sequence of the RIAD and RIDD is shown in SEQ ID NO:

6.

4. The construction method of claim 1, wherein, In step (2), the way of using peroxisomal acetyl-CoA for the mevalonate pathway is to sequentially integrate the mvaE, ERG13 and tHMGR genes with a C-terminal targeting tag ePTS1 into different sites of the host strain genome, wherein the different sites are: 1) DNA fragment P TEFin -mvaE-ePTS1-T XPR2 integrated into the A1 site, the nucleotide sequence of the mvaE gene being as set forth in SEQ ID NO: 3; 2) DNA fragment P TEFin - ERG13-ePTS1-T XPR2 integrated into the A3 site, the nucleotide sequence of the ERG13 gene being as shown in SEQ ID NO: 4; 3) DNA fragment P TEFin -tHMGR-ePTS1-T XPR2 integrated into the A08 site, the nucleotide sequence of which is shown as SEQ ID NO:

5.

5. The construction method of claim 1, wherein, In step (3), the way of strengthening the mevalonate pathway is at least one selected from 1) to 8): 1) DNA fragment P TEFin -ERG20-tHMGR-T XPR2 integrated into host D17 site, nucleotide sequence of ERG20 is SEQ ID NO: 7; 2) DNA fragment P TEFin -SsGGPPS-T XPR2 integrated into the host MFE1 site, the nucleotide sequence of SsGGPPS is shown as SEQ ID NO: 8; 3) DNA fragment P TEFin -tPvLPPS-RIDD-T XPR2 integrated into the host XPR2 locus, the nucleotide sequence of tPvLPPS is shown in SEQ ID NO: 1; 4) DNA fragment P TEFin - PvSCS-RIAD-T XPR2 integrated into the host A2 site, the nucleotide sequence of PvSCS is shown in SEQ ID NO: 2; 5) The DNA fragment P TEFin - tHMGR-T XPR2 integrated into the host F17 site; 6) DNA fragment P TEFin -tHMGR-T XPR2 -P EXP -PaGGPPS-T LIP integrated into the host F17 site, the nucleotide sequence of PaGGPPS is shown as SEQ ID NO: 9; 7) replacing the DNA segment P ERG11 in the host with the DNA segment P ERG9 , the nucleotide sequence of which is shown in SEQ ID NO: 10; ERG11 in SEQ ID NO: 10; 8) the DNA fragment P TEFin -mvaE-T LIP integrated into the host C4 site, the nucleotide sequence of mvaE is shown as SEQ ID NO: 3; and / or, in step (3) the way to weaken the competing pathway to squalene synthesis is to replace the promoter P ERG9 to the promoter P ERG11 .

6. The construction method of claim 1, wherein, In step (4), the overexpression of acetyl-CoA is overexpression of at least one of acetyl-CoA synthase ACS, pyruvate dehydrogenase PDH and knock-out of citrate synthase CIT1; The nucleotide sequence of the ACS is shown in SEQ ID NO: 11; The nucleotide sequence of the PDH is shown in SEQ ID NO: 12; The nucleotide sequence of the CIT1 is shown in SEQ ID NO:

13.

7. The construction method of claim 1, wherein, In step (4), the overexpression of the cofactor NADPH is overexpression of at least one of zwf, gnd, malate enzyme mae1, isocitrate dehydrogenase IDH and NAD kinase DADK in the pentose phosphate pathway; The nucleotide sequence of the zwf is shown in SEQ ID NO: 14; The nucleotide sequence of the gnd is shown in SEQ ID NO: 15; The nucleotide sequence of the mae1 is shown in SEQ ID NO: 16; The nucleotide sequence of the IDH is shown in SEQ ID NO: 17; The nucleotide sequence of the DADK is shown in SEQ ID NO:

18.

8. A recombinant Yarrowia lipolytica strain obtained by the construction method according to any one of claims 1-7.

9. Use of a recombinant strain of Yarrowia lipolytica according to claim 8 for the production of sclareol.

10. Use according to claim 9, characterized in that, The substrate for the production of sclareol is waste oil, preferably waste edible oil.

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