A recombinant strain with high sclareol production and a construction method and use thereof
By introducing the perillaldehyde biosynthetic pathway and optimizing the mevalonic acid metabolic pathway into the Yersinia lipolytica strain, the problem of low yield in existing strains was solved, and a high-yielding recombinant strain SC37 was achieved. This strain is suitable for efficient production using waste oil as a substrate and has both environmental and economic benefits.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI RECOM BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-07-22
- Publication Date
- 2026-06-12
Smart Images
Figure CN121065228B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial metabolic engineering technology, specifically relating to a high-yield recombinant strain of perilla frutescens, its construction method, and its uses. Background Technology
[0002] Waste cooking oil (WCO) is a byproduct of frying oil production, containing triglycerides, polar compounds, and non-volatile compounds. Most WCO is dumped into sewage pipes, causing water pollution. Therefore, recycling and reusing waste oil as a raw material for producing value-added chemicals is of great significance for preventing environmental pollution and can also generate additional economic value from WCO.
[0003] As an oil-producing yeast, *Yarrowia lipolytica* is characterized by its salt tolerance, low-temperature tolerance, and ability to grow in low-pH environments. Metabolically, it has the advantage of utilizing various substrates such as glucose, waste cooking oil, ethanol, and glycerol, making it an excellent chassis cell for synthesizing natural products. Secondly, *Yarrowia lipolytica* possesses a high-throughput tricarboxylic acid (TCA) cycle, providing abundant acetyl-CoA precursors for terpene synthesis; its abundant lipid droplets and subcellular structures in the cytoplasm also provide storage sites for hydrophobic products. Furthermore, *Yarrowia lipolytica* exhibits a high pentose phosphate pathway (PPP) metabolic flux, providing necessary cofactors for product synthesis. These advantages make *Yarrowia lipolytica* an excellent chassis strain for metabolic engineering.
[0004] Perillyl alcohol (also known as hard-tailed butterfly) is mainly derived from the plant perillyl alcohol. It is a secondary metabolite of the plant, commonly found as a white crystalline powder. It is readily soluble in organic solvents such as ethanol and acetone, but sparingly soluble in water. Its chemical molecule contains C 20 H 36 O2, with a molecular weight of 308.5, has the chemical structure shown below. Perillyl alcohol is mainly used in the synthesis of natural ambergris such as perillyl lactone and ambroxan, and can also be used in the formulation of fragrances. It has important applications in the pharmaceutical and cosmetic industries.
[0005]
[0006] Chinese patent (CN 117264792A) discloses a recombinant *Yersinia lipolytica* strain with high perillaldehyde production. This recombinant *Yersinia lipolytica* strain was constructed by overexpressing the mutant gene *CaLPPSR227Q* of lysine pyrophosphate diol ester synthase, the perillaldehyde synthase gene *SsTPS*, and the geranyl-geranyl pyrophosphate synthase gene *SaGGPP* in *Yersinia lipolytica po1f*, and expressing 3-hydroxy-3-methylglutaryl-CoA reductase *tHMGR* and the isopentenyl pyrophosphate isomerase gene *idi*. In a 5L fermenter, this strain achieved a maximum perillaldehyde yield of only 8510 mg / L, indicating that the yield still needs improvement. Therefore, the development of a high-yield recombinant perillaldehyde strain is urgently needed. Summary of the Invention
[0007] In order to solve the above-mentioned problems in the existing technology, the purpose of this invention is to provide a high-yielding recombinant strain of perillaldehyde, its construction method and uses.
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] This invention provides a method for constructing a recombinant strain that produces high levels of perillaldehyde, the method comprising the following steps:
[0010] (1) The perillol biosynthesis pathway was introduced into the host strain to obtain recombinant strain A, wherein the host strain was Yersinia lipolytica;
[0011] Alternatively, the construction method may further include the following steps: (2) using acetyl-CoA of the peroxisome in the recombinant strain A via the mevalonate pathway to obtain recombinant strain B;
[0012] Alternatively, the construction method further includes the following steps: (3) strengthening the mevalonate pathway in recombinant strain B and weakening at least one of the competing pathways to squalene and lipid synthesis to obtain recombinant strain C;
[0013] Alternatively, the construction method may further include the following steps: (4) overexpressing acetyl-CoA and cofactor NADPH in recombinant strain C to obtain recombinant strain D with high production of perillyl alcohol.
[0014] Further, in step (1), the biosynthetic pathway of perillyl pyrophosphate is introduced by overexpressing the lysine pyrophosphate diol ester synthase gene tPvLPPS and the perillyl pyrophosphate synthase gene PvSCS at the E1 and B1 sites of the host strain genome; the nucleotide sequence of the lysine pyrophosphate diol ester synthase gene tPvLPPS is shown in SEQ ID NO: 1, and the nucleotide sequence of the perillyl pyrophosphate synthase gene PvSCS is shown in SEQ ID NO: 2.
[0015] Furthermore, the C-terminus of the tPvLPPS and PvTPS genes is linked to a pair of polypeptide interaction tags RIAD and RIDD, the nucleotide sequences of which are shown in SEQ ID NO: 6.
[0016] Further, the method of using acetyl-CoA from the peroxisome for the mevalonate pathway in step (2) is as follows: the mvaE, ERG13, and tHMGR genes with the C-terminal targeting tag ePTS1 are sequentially integrated into different sites in the host strain genome, wherein the different sites are:
[0017] 1) DNA fragment P TEFin -mvaE-ePTS1-T XPR2 The mvaE gene is integrated into the A1 site, and its nucleotide sequence is shown in SEQ ID NO: 3;
[0018] 2) DNA fragment P TEFin -ERG13-ePTS1-T XPR2 The nucleotide sequence of the ERG13 gene, which is integrated into the A3 site, is shown in SEQ ID NO: 4;
[0019] 3) Transfer DNA fragment P TEFin -tHMGR-ePTS1-T XPR2 The nucleotide sequence of the tHMGR, integrated into the A08 site, is shown in SEQ ID NO: 5.
[0020] Furthermore, the method of strengthening the mevalonate pathway described in step (3) is selected from at least one of (1) to (8):
[0021] 1) DNA fragment P TEFin -ERG20-tHMGR-T XPR2 The nucleotide sequence of ERG20, which integrates into the host D17 site, is as shown in SEQ ID NO: 7.
[0022] 2) DNA fragment P TEFin -SsGGPPS-T XPR2 The nucleotide sequence of SsGGPPS, which integrates into the host MFE1 site, is shown in SEQ ID NO: 8.
[0023] 3) Transfer DNA fragment P TEFin -tPvLPPS-RIDD-T XPR2 The nucleotide sequence of tPvLPPS, which integrates into the host XPR2 site, is shown in SEQ ID NO: 1;
[0024] 4) Transfer DNA fragment P TEFin-PvSCS-RIAD-T XPR2 The nucleotide sequence of PvSCS, which integrates into the host A2 site, is shown in SEQ ID NO: 2;
[0025] 5) Transfer DNA fragment P TEFin -tHMGR-T XPR2 It integrates into the host F17 site;
[0026] 6) Transfer DNA fragment P TEFin -tHMGR-T XPR2 -P EXP -PaGGPPS-T LIP The nucleotide sequence of PaGGPPS, which integrates into the host F17 site, is shown in SEQ ID NO: 9;
[0027] 7) Transfer DNA fragment P ERG11 Replace P in the host ERG9 P ERG11 The nucleotide sequence is shown in SEQ ID NO: 10;
[0028] 8) Transfer DNA fragment P TEFin -mvaE-T LIP Upon integration into the host C4 site, the nucleotide sequence of mvaE is shown in SEQ ID NO: 3;
[0029] And / or, in step (3), the competitive pathway to squalene synthesis is weakened by reducing the promoter P of squalene synthase. ERG9 Replace with promoter P ERG11 .
[0030] Further, the overexpression of acetyl-CoA in step (4) is at least one of overexpression of acetyl-CoA synthase ACS, pyruvate dehydrogenase PDH, and citrate synthase CIT1 knockout;
[0031] The nucleotide sequence of the ACS is shown in SEQ ID NO: 11;
[0032] The nucleotide sequence of the PDH is shown in SEQ ID NO: 12;
[0033] The CIT1 nucleotide sequence is shown in SEQ ID NO: 13.
[0034] Further, the overexpression of cofactor NADPH in step (4) is at least one of zwf, gnd, malic acid oxidase mae1, isocitrate dehydrogenase IDH and NAD kinase DADK in the overexpression pentose phosphate pathway;
[0035] The nucleotide sequence of the zwf is shown in SEQ ID NO: 14;
[0036] The nucleotide sequence of the described gnd is shown in SEQ ID NO: 15;
[0037] The nucleotide sequence of the mae1 is shown in SEQ ID NO: 16;
[0038] The nucleotide sequence of the IDH is shown in SEQ ID NO: 17;
[0039] The nucleotide sequence of the DADK is shown in SEQ ID NO: 18.
[0040] The present invention also provides a recombinant strain of Yersinia lipophila obtained according to the above-described construction method.
[0041] The present invention also provides the use of the above-mentioned recombinant strain of Yersinia lipophila in the production of perillaldehyde.
[0042] Furthermore, the substrate for producing perillaldehyde is waste oil.
[0043] Furthermore, the waste oil is waste cooking oil.
[0044] The present invention has achieved the following beneficial effects:
[0045] This invention provides a recombinant *Yarrowia lipolytica* strain SC37 that produces high levels of perillaldehyde. First, a perillaldehyde biosynthetic pathway was constructed. Then, acetyl-CoA from peroxisomes was directed to the mevalonate pathway. Next, the intracellular mevalonate metabolic pathway was optimized, and precursor acetyl-CoA and cofactor NADPH were introduced to obtain the recombinant strain SC37. This invention provides a recombinant strain SC37 that produces high levels of perillaldehyde using waste oil as a substrate. By introducing and optimizing the expression of the perillaldehyde biosynthetic pathway, perillaldehyde yields reached 2.5 g / L in shake-flask batch fermentation and 20.18 g / L in bioreactor fed-batch fermentation. The recombinant strain SC37 of this invention has significant value in reducing environmental pollution and increasing the economic benefits of waste oil, and possesses promising application prospects.
[0046] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.
[0047] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description
[0048] Figure 1 The yield of perillaldehyde in recombinant strain SC01 was measured when waste cooking oil and glucose were used as carbon sources.
[0049] Figure 2 The yield of perillaldehyde from recombinant strains SC05, SC15, and SC16 was determined.
[0050] Figure 3 The yield of perillol from recombinant strains SC20, SC25, and SC37 was determined.
[0051] Figure 4 To determine the yield of perillaldehyde in a batch-fed bioreactor using recombinant strain SC38. Detailed Implementation
[0052] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.
[0053] The waste oil used in the specific embodiments of this invention is all waste edible oil.
[0054] The following experiments, where no temperature is specified, are reactions conducted under normal temperature conditions, which is room temperature, or 25±5℃.
[0055] The initial strains sampled in the following examples were based on Yersinia lipolytica po1f (genotype ATCC MYA-2613,ura3-302,leu2-270,xpr2-322,axp-2,leu2-270) that the company previously purchased from the strain preservation bank.
[0056] Example 1: Construction of a high-yield recombinant strain of perillaldehyde
[0057] 1) Overexpression of the lysine pyrophosphate synthase gene (tPvLPPS) and the perillyl alcohol synthase gene (PvSCS) derived from Prunella vulgaris.
[0058] First, the donor DNA expression cassettes tPvLPPS and PvSCS were overexpressed on the Yersinia lipolyticis genome. TEFin -tPvLPPS-T XPR2 P TEFin -PvSCS-T XPR2 The donor DNA fragment includes 1000 bp of homologous arms upstream and downstream of the E1 and B1 sites amplified using Yersinia lipolyticis po1f as a template (gene integration site reference Wheeldon et al., ACSSynth. Biol. 2017, 6, 402-409).
[0059] Using CRISPR-Cas9 technology (transformation process referenced Wheeldon et al., ACS Synth. Biol. 2017, 6, 402-409), donor DNA and gRNA were chemically transformed into the starting strain po1f, plated on YNB plates, incubated statically, screened, and verified by colony PCR. The recombinant strain SC01 was obtained by subculturing in YPD+5-FOA solid medium to induce plasmid loss.
[0060] Specifically, using *Yersinia lipolyticis* po1f as a template, 1000bp homologous arm fragments (E1-LH, E1-RH, B1-LH, B1-RH) upstream and downstream of the neutral sites E1 and B1 on the chromosome, as well as the promoter P, were amplified. TEFin Termination of sub-T XPR2 The genes tPvLPPS (SEQ ID NO: 1) and PvSCS (SEQ ID NO: 2) were obtained after codon optimization, and synthases were constructed to express donor DNA separately: E1-LH and P... TEFin -tPvLPPS-T XPR2 E1-RH and B1-LH, P TEFin -PvSCS-T XPR2 After PCR fusion of B1-RH, the resulting plasmids pCRISPRyl-E1-tPvLPPS and pCRISPRyl-B1-PvSCS, containing donor DNA, were ligated into the pCRISPRyl plasmid. The C-terminus of the PvLPPS and PvTPS genes is linked to a simple polypeptide interaction tag pair, RIAD and RIDD, enabling efficient assembly of the two enzymes in vivo. The nucleotide sequences of RIAD and RIDD are shown in SEQ ID NO: 6. Using pCASyl as a template, PAM sequences containing E1 and B1 sites were amplified using primer pairs E01-sg-F / E01-sg-R and B01-sg-F / B01-sg-R, respectively. Ligation of these sequences yielded plasmids pCASyl-E1 and pCASyl-B1, containing integration site PAM sequences. Take 500 ng each of plasmids pCRISPRyl-E1-tPvLPPS and pCASyl-E1 (or pCRISPRyl-B1-PvSCS and pCASyl-B1) containing donor DNA, and transform them into Yersinia lipolytica po1f strain using chemical transformation. Spread the transformed samples on YNB selection plates and incubate them statically at 30°C for 3-4 days. The transformants are then cultured overnight in liquid YPD medium and verified by colony PCR. The verified transformants are inoculated into antibiotic-free YPD liquid and then streaked on YPD plates containing 5-FOA (5-fluoroorotic acid) for subculturing to achieve plasmid loss. The strain after plasmid loss is named recombinant strain SC01.
[0061] The recombinant strain SC01 was activated on YPD medium and cultured at 220 rpm for 16–20 h, then transferred to 25 mL YPD medium / 100 mL shake flasks. Glucose and waste oil were used as carbon sources, respectively. Initial OD... 600 Fermentation was carried out at 0.2 μg / mL, 30°C, and 220 rpm for 120 h, and the yield of perillyl alcohol was determined. The experimental results are as follows: Figure 1 As shown, the yield of perillaldehyde was significantly higher when glucose was used as the carbon source than when waste oil was used as the carbon source.
[0062] 2) Improve the utilization rate of waste oil and increase the production of perillaldehyde.
[0063] To further increase the utilization rate of waste oil, recombinant strain SC05 was obtained by overexpressing mvaE (SEQ ID NO:3), ERG13 (SEQ ID NO:4), and tHMGR (SEQ ID NO:5), which contain peroxisome-localizing peptides, based on recombinant strain A. These three genes can synthesize mevalonic acid from acetyl-CoA after β-oxidation of long-chain fatty acids in peroxisomes. Subsequently, the synthesized mevalonic acid crosses the peroxisome into the cytoplasm for the synthesis of perillyl alcohol. When waste oil is used as a carbon source, recombinant strain SC05 significantly increased the yield of perillyl alcohol, reaching 150 mg / L. Figure 2 ).
[0064] Specifically, the process was as follows: First, using the *Yarrowia lipolyticis* po1f genome as a template, 1000 bp homologous arm sequences upstream and downstream of the integration site, the promoter, terminator, and structural genes were amplified and ligated into the pCRISPRyl plasmid to obtain a plasmid containing donor DNA. Subsequently, the pCASyl plasmid containing the PAM sequence of the integration site was amplified and ligated using appropriate primer pairs. Both the pCRISPRyl and pCASyl plasmids (500 ng each) were transformed into the recombinant strain SC01 via chemical transformation. The transformed strains were plated on YNB selection plates and incubated at 30°C for 3–4 days. Colony PCR confirmed correctness. The strains were then passaged on YPD+5-FOA plates to induce plasmid loss. The strains with plasmid loss were preserved and used for the next round of gene editing. Other genome editing procedures described below followed a similar workflow.
[0065] After fermentation and extraction analysis, the recombinant strain SC05 showed a significant increase in perillaldehyde production when using waste oil as a carbon source, reaching 150 mg / L. Figure 2 The study found that directing mitochondrial acetyl-CoA to mevalonic acid significantly increases the production of perillaldehyde.
[0066] 3) Strengthening the MVA pathway increases the yield of perillaldehyde.
[0067] Based on the recombinant strain SC05, the mevalonic acid (MVA) pathway was strengthened while the squalene synthesis pathway was weakened; using the CRISPR / Cas9 system, key genes in the MVA pathway were placed in P TEFin After the promoter was modified and integrated into different sites in the genome, recombinant strain SC15 was obtained. Subsequently, using SC15 as the starting strain, the promoter P of squalene synthase was modified by weakening the bypass metabolic pathway. ERG9 Replace with a weaker promoter P ERG11 The recombinant strain SC16 was obtained.
[0068] The enhanced mevalonic acid pathway includes one or more of 1) to 8), and this embodiment uses the following seven: 1) to 4), 6) to 8).
[0069] 1) DNA fragment P TEFin -ERG20-tHMGR-T XPR2 The nucleotide sequence of ERG20, which integrates into the host D17 site, is as shown in SEQ ID NO: 7.
[0070] 2) DNA fragment P TEFin -SsGGPPS-T XPR2 The nucleotide sequence of SsGGPPS, which integrates into the host MFE1 site, is shown in SEQ ID NO: 8.
[0071] 3) Transfer DNA fragment P TEFin -tPvLPPS-RIDD-T XPR2 The nucleotide sequence of tPvLPPS, which integrates into the host XPR2 site, is shown in SEQ ID NO: 1;
[0072] 4) Transfer DNA fragment P TEFin -PvSCS-RIAD-T XPR2 The nucleotide sequence of PvSCS, which integrates into the host A2 site, is shown in SEQ ID NO: 2;
[0073] 5) Transfer DNA fragment P TEFin -tHMGR-T XPR2 It integrates into the host F17 site;
[0074] 6) Transfer DNA fragment P TEFin -tHMGR-T XPR2 -P EXP -PaGGPPS-T LIP The nucleotide sequence of PaGGPPS, which integrates into the host F17 site, is shown in SEQ ID NO: 9;
[0075] 7) Transfer DNA fragment P ERG11 Replace P in the hostERG9 P ERG11 The nucleotide sequence is shown in SEQ ID NO: 10;
[0076] 8) Transfer DNA fragment P TEFin -mvaE-T LIP When integrated into the host C4 site, the nucleotide sequence of mvaE is shown in SEQ ID NO: 3.
[0077] After fermentation, extraction, and testing, the results are as follows: Figure 2 As shown, the recombinant strain SC15, which was enhanced with MVA, produced 800 mg / L of perillyl alcohol. The recombinant strain SC16 further increased the perillyl alcohol production to 1.2 g / L.
[0078] Based on recombinant strain SC16, the copy number of PvLPPS and PvTPS genes was further increased, i.e., one PvLPPS gene and one PvTPS gene were overexpressed in the genome to obtain recombinant strain SC20. After fermentation extraction and detection, the results are as follows... Figure 3 As shown, the yield of perillyl alcohol increased to 2.1 g / L after increasing the copy number of the PvLPPS and PvTPS genes.
[0079] 4) Enhance the supply of precursor acetyl-CoA and reducing agent NADPH to improve the ability of chassis cells to synthesize products.
[0080] Based on recombinant strain SC20, the supply of the precursor acetyl-CoA was enhanced. Using the CRISPR / Cas9 system, donor DNA expression cassettes for acetyl-CoA synthase ACS (SEQ ID NO: 11), pyruvate dehydrogenase PDH (SEQ ID NO: 12), and citrate synthase CIT1 knockout (SEQ ID NO: 13) were constructed and integrated into different sites of the genome via chemical transformation. After fermentation extraction and detection, the results are as follows: Figure 3 As shown, the integration expression of ACS and the knockout of CIT1 were most conducive to the accumulation of styraxol. This recombinant strain was named SC25, and the yield reached 2.3 g / L.
[0081] Based on recombinant strain SC25, the supply of reducing NADPH was enhanced. Using the CRI SPR / Cas9 system, donor DNA expression cassettes containing the 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 were constructed and integrated into different sites on the genome. After fermentation and extraction, the results are as follows: Figure 3As shown, after integrating and expressing the zwf, gnd, and IDH genes, the yield of perillaldehyde reached 2.5 g / L. This recombinant strain was named SC37 (i.e., recombinant strain D).
[0082] Example 2: Feed-in-batch fermentation of recombinant strains
[0083] To test the high-density fermentation capacity of the recombinant strain, a shake-flask fed-batch experiment was conducted. The ura and leu genes (used only for labeling) were added back to SC37 for screening, and the strain was named recombinant strain SC38.
[0084] (1) Feed-in-batch fermentation of recombinant strains in shake flasks
[0085] Recombinant strain SC38 was subjected to fed-batch fermentation in shake flasks using YPD medium, 50 mL / 250 mL shake flasks, inoculated with initial OD. 600 =0.2. After fermentation at 30℃ and 220rpm for 5 days, the yield of perillaldehyde reached 2.5g / L.
[0086] (2) Feed-in-process batch fermentation of recombinant strains in parallel bioreactor
[0087] The fed-batch fermentation system used a 5L bioreactor system with a total fermentation volume of 2.5L. YPD medium was used in the fermentation, with a volume of 2.5L, and OD was inoculated. 600 =0.4, pH 5.5. The fed-batch culture medium used was 10×YPD medium with a glucose feed concentration of 800 g / L. In the fed-batch fermentation, exponential feeding was started when the glucose was depleted, and enzyme samples were taken at 4-hour intervals to detect OD. 600 The residual sugar in the culture medium was measured, and the yield of perillol was measured every 12 hours. The highest yield of recombinant strain SC38 was 20.18 g / L. Figure 4 ).
[0088] In summary, this invention provides a recombinant *Yarrowia lipolytica* strain SC37 that produces high levels of perillaldehyde. First, the perillaldehyde biosynthetic pathway was constructed. Then, acetyl-CoA from the peroxisome was directed to the mevalonate pathway. Next, the intracellular mevalonate metabolic pathway was optimized, and the precursor acetyl-CoA and cofactor NADPH were introduced to obtain the recombinant strain SC37. This invention provides a recombinant strain SC37 that produces high levels of perillaldehyde using waste oil as a substrate. By introducing and optimizing the expression of the perillaldehyde biosynthetic pathway, perillaldehyde yields reached 2.5 g / L in shake-flask batch fermentation and 20.18 g / L in bioreactor fed-batch fermentation. The recombinant strain SC37 of this invention has significant value in reducing environmental pollution and increasing the economic benefits of waste oil, and possesses promising application prospects.
[0089] The nucleotide sequence involved in this invention 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] ACCATACCCGAGAGCAGAAATTGAAGCGAATTCCGCTCGGAATCATGCACAAGGTCACAGGTGCCCACTAG
[0103] TCTGCTTTTTTCGCTTGAGGCTGGGCTGGAGAACCTGGAGTGGGACAAGTTACTCAAACTGCAG
[0104] AGCGCAGACGGGAGCTTTCTGACGAGTCCATCGTCCACTGCGTTTTGCTTTCATGCAGACCAAAG
[0105] ACGAGAAGTGTTATCAATTCATCAAGAATACCGTGGACACCTTCAACGGCGGCGCTCCCCACACC
[0106] TACCCCGTGGACGTTGTTTGGACGACTGTGGGCCATTGACCGACTTCAGAGACTGGGTATTTCTAG
[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] AGCTGCAGGGCAAGCGAATCTCTCTCTCTCTTACGGATCTGGTCTTGCTTCCACTCTTTTCTCTC
[0215] TGACCGTCAAGGGAGACATTTCTCCCATCGTCAAGGCCTGCGACTTCAAGGCTAAGCTCGATGAC
[0216] CGATCCACCGAGACTCCCGTCGACTACGAGGCTGCCACCGATCTCCGAGAGAAGGCCCACCTCA
[0217] AGAAGAACTTTGAGCCCCAGGGAGACATCAAGCACATCAAGTCTGGCGTCTACTACCTCACACAA
[0218] CATCGATGACATGTTCCGACGAAAGTACGAGATCAAGCAGTAG
[0219] SEQ ID NO:5
[0220] ATGACCCAGTCTGTGAAGGTGGTTGAGAAGCACGTTCCTATCGTCATTGAGAAGCCCAGCGAGA
[0221] AGGAGGAGGACACCTCTTCTGAAGACTCCATTGAGCTGACTGTCGGAAAGCAGCCCAAGCCCCGT
[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] ATGGTCGCTCAGACCTTCAACCTGGACACCTACCTGTCCCAGCGACAGCAGCAGGTTGAGGAGGCTCTGTCTGCTGCTCTGGTCCCTGCTTACCCTGAGCGAATCTACGAGGCCATGCGATACTCCCTCCTGGCTGGTGGTAAGCGACTGCGACCTATCCTCTGCCTCGCTGCTTGTGAGCTGGCTGGTGGCTCTGTTGAGCAGGCTATGCCTACCGCTTGCGCTCTGGAGATGATTCACACCATGTCTCTGATCCACGACGACCTCCCCGCTATGGACAACGACGACTTTCGACGAGGCAAGCCTACCAACCACAAGGTCTTCGGCGAGGACATCGCCATTCTGGCTGGTGACGCTCTGCTGGCTTACGCTTTCGAGCACATCGCCTCCCAGACCCGAGGTGTTCCTCCTCAGCTGGTTCTCCAGGTCATCGCTCGAATCGGTCACGCTGTCGCTGCTACCGGTCTGGTTGGTGGTCAGGTTGTCGACCTGGAGTCCGAGGGTAAGGCTATCTCCCTGGAGACCCTCGAGTACATTCACTCCCACAAGACCGGCGCCCTGCTGGAGGCTTCTGTTGTTTCTGGCGGTATCCTGGCCGGCGCTGACGAGGAGCTGCTGGCTCGACTGTCTCACTACGCTCGAGACATTGGCCTGGCCTTTCAGATCGTCGACGACATTCTGGACGTGACCGCTACCTCTGAGCAGCTGGGTAAGACCGCTGGTAAGGACCAGGCTGCTGCTAAGGCTACCTACCCTTCCCTGCTGGGTCTGGAGGCTTCCCGACAGAAGGCTGAGGAGCTGATTCAGTCCGCCAAGGAGGCTCTGCGACCTTACGGTTCTCAGGCTGAGCCTCTCCTGGCTCTGGCTGACTTTATTACCCGACGACAGCACTAA
[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] GGACGTGCGAATCCCTGAGGCTTACGAGTCTCTCATTCTGGACTGCCTCAAGGGTGACCACACCA
[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] GCAGCTTATCTGCGAGGCTTACGACCTCATGAAGCGAGGTGCTGGTTTCACCAATGAGGAGATTG
[0382] GAGACGTTTTCGCCAAGTGGAACAACGGTATCCTCGACTCCTTCCTCATTGAGATCACCCGAGAC
[0383] ATCTTCAAGTACGACGACGGCTCTGGAACTCCTCTCGTTGAGAAGATCTCCGACACTGCTGGCCA
[0384] GAAGGGTACTGGAAAGTGGACCGCTATCAACGCTCTTGACCTTGGTATGCCCGTCACCCTGATCG
[0385] GTGAGGCCGTCTTCGCTCGATGCCTTTCTGCCCTCAAGCAGGAGCGTGTCCGAGCTTCCAAGGTT
[0386] CTTGATGGCCCCGAGCCCGTCAAGTTCACTGGTGACAAGAAGGAGTTTGTCGACCAGCTCGAGC
[0387] AGGCCCTTTACGCCTCCAAGATCATCTCTTACGCCCAGGGTTTCATGCTTATCCGAGAGGCCGCCA
[0388] AGACCTACGGCTGGGAGCTCAACAACGCCGGTATTGCCCTCATGTGGCGAGGTGGTTGCATCATC
[0389] CGATCCGTCTTCCTTGCTGACATCACCAAGGCTTACCGACAGGACCCCAACCTCGAGAACCTGCT
[0390] GTTCAACGACTTCTTCAAGAACGCCATCTCCAAGGCCAACCCCTCTTGGCGAGCTACCGTGGCCA
[0391] AGGCTGTCACCTGGGGTGTTCCCACTCCCGCCTTTGCCTCGGCTCTGGCTTTCTACGACGGTTAC
[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] CCGTTTGCCAAGGACAAGGCCAATTACGAGGGAGTGGACACCAAGACTCTGGAGCACGTGGTTG
[0416] CTGCCGTCAAGCCCCATATTCTCATTGGATGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTC
[0417] GTCAAGGAGATGCTCAAACACACCCCTCGACCCATCATTCTCCCTCTTTCCAACCCCACACGTCT
[0418] TCATGAGGCTGTCCCTGCAGATCTGTACAAGTGGACCGACGGCAAGGCTCTGGTTGCCACCGGC
[0419] TCGCCCTTTGACCCAGTCAACGGCAAGGAGACGTCTGAGAACAATAACTGCTTTGTTTTCCCCGG
[0420] AATCGGGCTGGGAGCCATTCTGTCTCGATCAAAGCTCATCACCAACACCATGATTGCTGCTGCCA
[0421] TCGAGTGCCTCGCCGAACAGGCCCCCATTCTCAAGAACCACGACGAGGGAGTACTTCCCGACGT
[0422] AGCTCTCATCCAGATCATTTCGGCCCGGGTGGCCACTGCCGTGGTTCTTCAGGCCAAGGCTGAGG
[0423] GCCTAGCCACTGTCGAGGAAGAGCTCAAGCCCGGCACCAAGGAACATGTGCAGATTCCCGACAA
[0424] CTTTGACGAGTGTCTCGCCTGGGTCGAGACTCAGATGTGGCGGCCCGTCTACCGGCCTCTCATCC
[0425] ATGTGCGGGATTACGACTAG
[0426] SEQ ID NO:17
[0427] ATGTCCACCACCGCTACTCGAGGCCTGTCCACCAAGATCAAGGTCAAGAACCCCATTGTCGAGCT
[0428] CGATGGTGATGAGATGACCCGAATCATCTGGAAGTCCATCAAGGACAAGCTCATTCTGCCCTATCT
[0429] CGACATTGATCTTAAGTACTACGATCTGGGCATCGAGTACCGAGACCAGACTAACGACCAGGTGA
[0430] CCATTGACGCCGCCGAGGCCATCAAGAAGTACCAGGTCGGTGTCAAGTGCGCCACCATCACCCC
[0431] CGACGAGGCCCGAGTCAAGGAGTTTGGCCTCAAGAAGATGTGGCTGTCGCCCAACGGTACCATC
[0432] CGAAACATTCTCGGCGGTACTGTTTTCCGAGAGCCCATTGTCATTCCCGCCGTCCCCCGGCTTGTG
[0433] CCCGGATGGAAGGAGCCTATCATCATTGGTCGACACGCCCACGGCGACCAGTACAAGGCCCAGG
[0434] ATGCCGTCATCCCCGGCGCCGGTGAGCTGACTCTTAACTTCAAGCCCGCTAACGGAGGCGACGA
[0435] GCAGGTCATCAAGGTGTACACCTACGACGCCCCTGGTGTCGCCATGGCCATGTACAACACTGACG
[0436] AGTCCATCACCGGCTTTGCCTACTCTTCATTCAACCTGGCTCTGCAGAAGAAGCTGCCCCTGTAC
[0437] ATGTCTACCAAGAACACCATCCTTAAGAAGTACGACGGCCGATTCAAGGACATTTTCCAGGAGAT
[0438] TTACGACAAGGAGTACAAGGATAAGTTTGATGCTGCCGGCATTTGGTACGAGCACCGACTCATTG
[0439] ATGACATGGTCGCCCAGATGATCAAGTCTAAGGGAGGCTTCATCATGGCCCTCAAGAACTACGAC
[0440] GGAGACGTGCAGTCCGACATTGTTGCCCAGGGCTTTGGCTCTCTCGGTCTCATGACCTCTGTTCT
[0441] CGTCACCCCCGACGGAAAGACCTTTGAGTCCGAGGCCGCCCACGGCACCGTGACTCGACACTAC
[0442] CGACAGCACCAGCAGGGCAAGGAGACCTCTACCAACTCCATTGCCTCCATCTTCGCCTGGACCC
[0443] GAGGCCTCATCCAGCGAGGCATTCTCGACGAGACCCCTGAGGTGACCAAGTTTGCCGAGGCTCT
[0444] CGAGAAGGCCACCGTCGACACTGTTGACAAGGACGGCATTATGACCAAGGATCTGGCTCTGGCC
[0445] GGTGGCAAGACCGACCGATCCTCGTATGTGCTGACCGAGGAGTTTATCGACGCTGTGGCCAACA
[0446] GACTGAAGAAGGACCTGGCTTAG
[0447] SEQ ID NO:18
[0448] ATGCGACTACTCATCCGCCGAACCGGTATAACACGGCCCCACAGCGTGCAAGCGCGCCGATCCAC
[0449] ATGGATTCGGCTTCTCTCGACCGAGATATTGCATGCAGAACTGCTTCCCGACCGCCAGTCGCCCC
[0450] ACTACGTCCAGGAGTCGACCTCTCTGTCATCTCTGGTGTGGGACAAGCCTCTGGAAAACGTTCTG
[0451] ATCGTCAAAAAACCCTGGGACCACAATGTGCGCGAGTCGCTCATCCAGATGGCATCTCACATCCA
[0452] GCGCCGGTACCCCCGAGTCAACATTCTGGTGGAGGAACATGTGGCCGACGAGGTCCAGAAGCAG
[0453] ATTGGAGCCGCAGGCGTGACCGCCATCCACACGGGGCCAGGAGAGGTGCTGAGAAACAAGACG
[0454] GATCTGCTCGTGACTCTGGGAGGCGACGGAACTATTCTACATGCCACCTCCATGTTTGCTTCCGG
[0455] AGAAGTGCCGCCGGTGCTGTCCTTTTCGCTGGGGACTCTGGGTTTCCTGCTGCCGTTTGATTTCA
[0456] AGGACTTCAAAACTGCATTCGACATGGTGTACTCGTCGCAGGCCTCGGTGGTCAACCGCGCCCG
[0457] CCTAGCATGTCAGAAAATGTCCATTCGCAAGGAAATCACCCACTTGCCCTCCCAATCGCACATTG
[0458] AACACAACTCAACCCATGTCTACGGCAATCCCGACGACTACAATCTTAGCCCACTAACCTACGCC
[0459] ATGAACGACATCAACATCCACCGTGGAGCTGAGCCGCATCTCACCAAGCTCGACATCCACGTTGA
[0460] CGGCGAGTTCATCACCCGAGCCATTGCTGACGGTGTCACCATCGCCACACCCACGGGCTCCACG
[0461] GCCTACTCGCTGTCGTCTGGCGGCTCCATTGTGCATCCCCGAGTCGCCTGCATTCTGCTGACCCCC
[0462] ATCTGTCCGCGATCGCTGTCATTCCGGCCTCTCATTTTCCCAGCCACCTCCAAAATATGCATCACC
[0463] GCCTCGTCCGAATCTCGAGGTAGAGGCGCCGAGCTGTCTGTCGACGGAATCGCCAAGGGTCTGG
[0464] TTCGACCCAGCGACAAGATTCTGGTCGAAAGCGAAACCGGCCACAACTCGGGCATCTGGTGCGT
[0465] GGCCAAGACAGACAGAGACTGGGTCAGTGGCCTCAACGGGTTACTGGGCTTCAATAGCAGTTTT
[0466] GGCAAGGGCGGGGAGGCGTCAGGCGATGTTGCTTAG
Claims
1. A method for constructing a recombinant strain that produces high levels of perillaldehyde, characterized in that, The construction method includes the following steps: (1) The perillol biosynthesis pathway was introduced into the host strain to obtain recombinant strain A, wherein the host strain was Yersinia lipolytica; (2) In recombinant strain A, acetyl-CoA of the peroxisome was used for the mevalonate pathway to obtain recombinant strain B; (3) In recombinant strain B, the mevalonic acid pathway was strengthened and the competitive pathway to squalene was weakened to obtain recombinant strain C; (4) Overexpressing acetyl-CoA and cofactor NADPH in recombinant strain C yields recombinant strain D with high production of perillyl alcohol; In step (1), the biosynthetic pathway of perillyl pyrophosphate is introduced by overexpressing the lysine pyrophosphate diol ester synthase gene tPvLPPS and the perillyl pyrophosphate synthase gene PvSCS at the E1 and B1 sites of the host strain genome; the nucleotide sequence of the lysine pyrophosphate diol ester synthase gene tPvLPPS is shown in SEQ ID NO: 1, and the nucleotide sequence of the perillyl pyrophosphate synthase gene PvSCS is shown in SEQ ID NO:
2. The C-terminus of the tPvLPPS and PvTPS genes is linked to a pair of polypeptide interaction tags RIAD and RIDD, the nucleotide sequences of which are shown in SEQ ID NO:
6. Step (2) describes the method of using acetyl-CoA from the peroxisome for the mevalonate pathway as follows: the mvaE, ERG13, and tHMGR genes with the C-terminal targeting tag ePTS1 are sequentially integrated into different sites in the host strain genome. These 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 The nucleotide sequence of the ERG13 gene, which is integrated into the A3 site, is shown in SEQ ID NO: 4; 3) Transfer DNA fragment P TEFin -tHMGR-ePTS1-T XPR2 The nucleotide sequence of the tHMGR, integrated into the A08 site, is shown in SEQ ID NO: 5; The method for enhancing the mevalonic acid pathway described in step (3) is as follows: 1) DNA fragment P TEFin -ERG20-tHMGR-T XPR2 The nucleotide sequence of ERG20, which integrates into the host D17 site, is as shown in SEQ ID NO:
7. 2) DNA fragment P TEFin -SsGGPPS-T XPR2 The nucleotide sequence of SsGGPPS, which integrates into the host MFE1 site, is shown in SEQ ID NO:
8. 3) Transfer DNA fragment P TEFin -tPvLPPS-RIDD-T XPR2 The nucleotide sequence of tPvLPPS, which integrates into the host XPR2 site, is shown in SEQ ID NO: 1; 4) Transfer DNA fragment P TEFin -PvSCS-RIAD-T XPR2 The nucleotide sequence of PvSCS, which integrates into the host A2 site, is shown in SEQ ID NO: 2; 5) Transfer DNA fragment P TEFin -tHMGR-T XPR2 -P EXP -PaGGPPS-T LIP The nucleotide sequence of PaGGPPS, which integrates into the host F17 site, is shown in SEQ ID NO: 9; 6) Transfer DNA fragment P ERG11 Replace P in the host ERG9 P ERG11 The nucleotide sequence is shown in SEQ ID NO: 10; 7) Transfer DNA fragment P TEFin -mvaE-T LIP Upon integration into the host C4 site, the nucleotide sequence of mvaE is shown in SEQ ID NO: 3; In step (3), the way to weaken the competitive pathway to squalene synthesis is to reduce the promoter P of squalene synthase. ERG9 Replace with promoter P ERG11 ; The overexpression of acetyl-CoA mentioned in step (4) refers to the overexpression of acetyl-CoA synthase ACS, pyruvate dehydrogenase PDH, and the knockout 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 CIT1 nucleotide sequence is shown in SEQ ID NO: 13; The overexpression of cofactor NADPH in step (4) refers to the overexpression of zwf, gnd, malic acid oxidase 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 described 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.
2. A recombinant strain of Yersinia lipophila obtained by the construction method according to claim 1.
3. The use of the recombinant strain of Yersinia lipolytica according to claim 2 in the production of perillaldehyde.
4. The use according to claim 3, characterized in that, The substrate for producing perillaldehyde is waste oil.
5. The use according to claim 3, characterized in that, The substrate for producing perillaldehyde is waste cooking oil.