A method for improving the production of malonic acid by Saccharomyces cerevisiae

Abstract

The present invention discloses a method for improving the production of malonic acid by Saccharomyces cerevisiae, belonging to the technical field of bioengineering. The present invention uses Saccharomyces cerevisiae BY4741 as the starting strain, and integrates the acetyl-CoA decarboxylase mutant gene and 3-hydroxyisobutyryl-CoA hydrolase mutant gene derived from Saccharomyces cerevisiae into the Delta site of the Saccharomyces cerevisiae genome. The Phe at the 121st position and Glu at the 124th position of the 3-hydroxyisobutyryl-CoA hydrolase of Saccharomyces cerevisiae are mutated, and then the Ser at the 659th and 1157th positions of the acetyl-CoA decarboxylase of Saccharomyces cerevisiae are mutated to Ala, and finally a Saccharomyces cerevisiae engineering strain with a relatively high malonic acid yield is obtained. After 7 days of shake-flask fermentation culture, the malonic acid yield can reach 80 mg / L.

Description

Technical Field

[0001] The present invention relates to a method for improving the production of malonic acid by Saccharomyces cerevisiae, belonging to the field of bioengineering. Background Art

[0002] Malonic acid, also known as carrot acid, malic acid or beet acid, has two functional groups, active methylene and carboxyl, in its molecular structure, so it can participate in various chemical reactions and is a very important organic synthesis intermediate. Malonic acid is also one of the top 30 chemicals that can be produced from biomass announced by the US Department of Energy.

[0003] With the rapid development of the domestic and international chemical industries at present, coupled with the extensive development of the uses of malonic acid and its downstream products, the output and quality of malonic acid have been increasing day by day. In recent years, with the increasingly mature market, consumers' requirements for product quality have become higher and higher. Therefore, the quality indicators of malonic acid also need to be continuously improved. This requires existing manufacturers to not only continuously improve their production capacity, but also accelerate the technological progress and innovation of the production process of malonic acid, as well as the research and development of downstream products of malonic acid, so as to promote the rapid development of the domestic and international malonic acid industries.

[0004] Currently, the industrial method for preparing malonic acid is usually to hydrolyze cyanoacetic acid or malonic acid ester. These methods involve cyanide ions in the preparation process. Cyanide ions are highly toxic and cause great harm to the environment. The reaction process is complex and requires complex and cumbersome purification procedures, resulting in low product yield, difficult control of raw material purity, and difficult treatment of three wastes. These problems greatly limit the production capacity of industrial production.

[0005] Based on the above problems, more and more researchers have chosen to biosynthesize malonic acid through cell factories. In previous studies, Sang et al. successfully constructed a malonic acid production pathway using alanine semialdehyde as a precursor in Escherichia coli by heterologously expressing β-alanine pyruvate transaminase, with a yield of 3.6 g / L. However, using Escherichia coli as a host still has problems such as poor cell tolerance and Escherichia coli toxicity. Based on the above problems, more and more researchers have chosen Saccharomyces cerevisiae as a biological cell factory. Saccharomyces cerevisiae is one of the simplest eukaryotes, with a clear genetic background, easy gene manipulation, strong genetic stability, strong vitality, strong acid tolerance and stress resistance. Moreover, Saccharomyces cerevisiae can produce various types of organic acids and there are many endogenous metabolic pathways that support the synthesis of organic acids. Additionally, Saccharomyces cerevisiae is one of the most commonly used strains for large-scale industrial fermentation production. This has made Saccharomyces cerevisiae attract great interest from researchers in malonic acid biosynthesis. Among them, Dietrich et al. found in Saccharomyces cerevisiae that by site-directed mutagenesis of the EHD3 gene, it can convert malonyl-CoA as a substrate into malonic acid, which makes it possible to accumulate malonic acid in Saccharomyces cerevisiae, but the yield is low. Summary of the Invention

[0006] Based on the above problems, the present invention constructs a malonic acid production pathway in Saccharomyces cerevisiae using a mutant EHD3 gene and a mutant acetyl-CoA decarboxylase gene (ACC1), and integrates it into the Saccharomyces cerevisiae delta locus to increase the malonic acid yield by increasing its copy number.

[0007] The present invention first provides a recombinant Saccharomyces cerevisiae capable of producing malonic acid. By constructing an overexpression integration cassette, a mutant of acetyl-CoA decarboxylase from Saccharomyces cerevisiae, a mutant of 3-hydroxyisobutyryl-CoA hydrolase, and a green fluorescent protein gene for screening the copy number of the delta locus are overexpressed and integrated into the genome of Saccharomyces cerevisiae BY4741. The fluorescence intensity of the green fluorescent protein expression is used to screen strains with relatively high copy numbers, thereby screening out recombinant Saccharomyces cerevisiae with high malonic acid production.

[0008] The first object of the present invention is to provide a recombinant Saccharomyces cerevisiae capable of producing malonic acid, wherein the recombinant Saccharomyces cerevisiae overexpresses a mutant of acetyl-CoA decarboxylase with the amino acid sequence shown in SEQ ID NO.3 and a mutant of 3-hydroxyisobutyryl-CoA hydrolase with the amino acid sequence shown in SEQ ID NO.6.

[0009] In one embodiment, the overexpression is to integrate the mutant of acetyl-CoA decarboxylase and the mutant of 3-hydroxyisobutyryl-CoA hydrolase into the Saccharomyces cerevisiae delta locus.

[0010] In one embodiment, the gene encoding the acetyl-CoA decarboxylase mutant is expressed under the initiation of the promoter UAS1, and the gene encoding the 3-hydroxyisobutyryl-CoA hydrolase mutant is expressed under the initiation of the promoter UAS3.

[0011] In one embodiment, the nucleotide sequence of the promoter UAS1 is as shown in SEQ ID NO.2, and the nucleotide sequence of the promoter UAS3 is as shown in SEQ ID NO.5.

[0012] In one embodiment, the recombinant Saccharomyces cerevisiae uses Saccharomyces cerevisiae BY4741 as the starting strain.

[0013] The second object of the present invention is to provide a method for constructing the recombinant Saccharomyces cerevisiae, which is to integrate the expression cassette IN-1 containing the gene encoding the acetyl-CoA decarboxylase mutant and the expression cassette IN-2 containing the gene encoding the 3-hydroxyisobutyryl-CoA hydrolase mutant into the Saccharomyces cerevisiae genome to construct the recombinant Saccharomyces cerevisiae MA-112.

[0014] In one embodiment, the expression cassette IN-1 consists of Delta1, gene GFP, promoter UAS1, gene ACC1**, terminator T ADH1 and the Ura1 segment, and the expression cassette IN-2 consists of Ura2, promoter UAS3, gene EHD3***, terminator T CYC1 and the three fragments of Delta2.

[0015] In one embodiment, the Saccharomyces cerevisiae includes Saccharomyces cerevisiae BY4741.

[0016] The third object of the present invention is to provide a method for producing malonic acid, which is to use the recombinant Saccharomyces cerevisiae for fermentation with glucose as the carbon source.

[0017] In one embodiment, the seed solution of the recombinant Saccharomyces cerevisiae is inoculated into the medium at an inoculation amount of 1-3% by volume and cultured at 28-30°C for 72-168 h.

[0018] In one embodiment, the medium includes 15-25 g / L of glucose, 5-15 g / L of yeast extract, and 15-25 g / L of peptone.

[0019] The fourth object of the present invention is to provide a method for improving the extracellular secretion of malonic acid by Saccharomyces cerevisiae, which is to overexpress the acetyl-CoA decarboxylase mutant with the amino acid sequence as shown in SEQ ID NO.3 and the 3-hydroxyisobutyryl-CoA hydrolase mutant with the amino acid sequence as shown in SEQ ID NO.6.

[0020] In one embodiment, the overexpression is achieved by integrating an acetyl-CoA decarboxylase mutant and a 3-hydroxyisobutyryl-CoA hydrolase mutant into the delta locus of Saccharomyces cerevisiae.

[0021] In one embodiment, the gene encoding the acetyl-CoA decarboxylase mutant is expressed under the initiation of the promoter UAS1, and the gene of the 3-hydroxyisobutyryl-CoA hydrolase mutant is expressed under the initiation of the promoter UAS3.

[0022] In one embodiment, the nucleotide sequence of the promoter UAS1 is as shown in SEQ ID NO.2, and the nucleotide sequence of the promoter UAS3 is as shown in SEQ ID NO.5.

[0023] In one embodiment, the recombinant Saccharomyces cerevisiae uses Saccharomyces cerevisiae BY4741 as the starting strain.

[0024] The present invention also provides the application of the recombinant Saccharomyces cerevisiae or the method for producing malonic acid in the preparation of malonic acid or its derivative products.

[0025] Beneficial effects:

[0026] In the present invention, an acetyl-CoA decarboxylase mutant with the amino acid sequence as shown in SEQ ID NO.3 and a 3-hydroxyisobutyryl-CoA hydrolase mutant with the amino acid sequence as shown in SEQ ID NO.6 are integrated into the genome of Saccharomyces cerevisiae, and the copy number of genes related to the malonic acid metabolic pathway is screened by combining GFP fluorescence signals. Finally, a recombinant Saccharomyces cerevisiae with high malonic acid production is obtained, and the malonic acid production of this recombinant Saccharomyces cerevisiae reaches 80 mg / L.

[0027] Recombinant Saccharomyces cerevisiae overexpressing unmutated acetyl-CoA decarboxylase and 3-hydroxyisobutyryl-CoA hydrolase cannot produce malonic acid, and the recombinant Saccharomyces cerevisiae constructed in the present invention achieves a malonic acid production of 80 mg / L. Description of the drawings

[0028] Figure 1 Malonic acid synthesis pathway and Delta locus integration method;

[0029] Figure 2 Plasmid map of pY26-UAS1-GPD-ACC1**;

[0030] Figure 3 Plasmid map of pY26-TEF1-UAS2-EHD3***;

[0031] Figure 4 Malonic acid production of different mutants of 3-hydroxyisobutyryl-CoA hydrolase Ehd3. Detailed implementation manners

[0032] The culture media involved in the following examples:

[0033] (1) SD-Ura defective medium: 20 g / L glucose, 1.7 g / L amino acid-free yeast nitrogen source, 5.0 g / L ammonium sulfate, 10 mL of 10× essential amino acid mixture, 10 mL of 10× HIS mixture, 10 mL of 10× LEU mixture. Add 2.0% agar powder to prepare solid medium, sterilize at 115 °C for 30 min. Do not add agar powder to the SD-Ura liquid medium.

[0034] (2) YPD liquid medium: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone. Add 2.0% agar powder to prepare solid medium, sterilize at 115 °C for 30 min.

[0035] The experimental methods involved in the following examples:

[0036] (1) Touchdown PCR:

[0037] Reaction system: The 50 μL PCR reaction system includes the following components: 200 ng template, 1.5 μL of each of the upstream and downstream primers at 10 μM, 25 μL of 2× PrimeSTAR MAX DNA polymerase, and make up to 50 μL with sterile water.

[0038] Reaction procedure: Pre-denaturation at 98 °C for 10 min; denaturation at 98 °C for 30 s, annealing at 60 °C for 15 s, extension at 72 °C for 30 s, 15 cycles; denaturation at 98 °C for 30 s, annealing at 55 °C for 15 s, extension at 72 °C for 30 s, 35 cycles; final extension at 72 °C for 10 min; keep at 4 °C until taken out.

[0039] (2) Malonic acid liquid chromatography-mass spectrometry detection:

[0040] Pretreatment: Centrifuge the fermentation sample at 12,000 rpm for 2 min to separate the fermentation broth from the cells, and treat the fermentation broth with a 0.22 μm filter membrane for liquid chromatography-mass spectrometry detection.

[0041] Liquid chromatography-mass spectrometry conditions: Detection wavelength: 200 - 400 nm, analytical column: BEH C18 (2.1x150 mm 1.7 μm), column temperature 45 °C, flow rate: 0.3 ml / min, injection volume: 5 μL, detector: Waters Acquity PDA (200 - 400 nm); mobile phase A is 0.1% (v / v) formic acid, and mobile phase B is acetonitrile.

[0042] Table 1 Primer sequence list involved in the following examples

[0043]

[0044] Plasmids involved in the following examples:

[0045] pY26-TEF-GPD: It has been published in the article, and the plasmid in this article is named pY26-GPD-TEF; see the article: Na Chen, Jingya Wang, Yunying Zhao* and Yu Deng*. Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer. Microbial Cell Factories, 2018, 17:67.

[0046] pGFP33: see the article: Zhao Gang, Cao Chunlei, Zhao Yunying, etc. Screening of membrane proteins and liposomal proteins related to ScRch1 localization in Saccharomyces cerevisiae [J]. Chinese Journal of Cell Biology, 2013(11): 75-83.

[0047] pGFP33-UAS1: The UAS1 sequence shown in nucleotide sequence SEQ ID NO.10 was cloned into the BamH I and Sph I sites of the pGFP33 plasmid.

[0048] pGFP33-UAS2: The UAS2 sequence shown in nucleotide sequence SEQ ID NO.11 was cloned into the BamH I and Sph I sites of the pGFP33 plasmid.

[0049] pGFP33-TEF1: The TEF1 sequence shown in nucleotide sequence SEQ ID NO.12 was cloned into the BamH I and Sph I sites of the pGFP33 plasmid.

[0050] Example 1 Construction of expression vectors for acetyl-CoA decarboxylase mutants and 3-hydroxyisobutyryl-CoA hydrolase mutants

[0051] (1) Construction of the mutant expression vector for acetyl-CoA decarboxylase

[0052] Using the genome of Saccharomyces cerevisiae BY4741 as a template and F1 / R1 as primers, the ACC1 gene fragment with the amino acid sequence shown in SEQ ID NO.1 was amplified by PCR. The ACC1 gene fragment was recovered by gel electrophoresis, and the ACC1 gene fragment and the plasmid pY26-TEF-GPD were double-digested with BglII / NotI respectively. After the digested products were purified and recovered, the plasmid pY26-TEF-GPD-ACC1 was constructed by overnight ligation with T4 ligase.

[0053] Using pGFP33-UAS1 as a template and F2 / R2 as primers, the promoter UAS1 with the nucleotide sequence shown in SEQ ID NO.2 was obtained by PCR amplification. The promoter UAS1 fragment was recovered by gel electrophoresis, and the promoter UAS1 fragment and the plasmid pY26-TEF-GPD-ACC1 were digested with SacI / NotI double enzymes respectively. After purifying and recovering the digestion products, plasmid pY26-UAS1-GPD-ACC1 was constructed by overnight ligation with T4 ligase.

[0054] According to the ACC1 gene sequence, primers F3 / R3 and F4 / R4 introducing S659A and S1157A mutations were designed and synthesized. Site-directed mutagenesis was performed on pY26-UAS1-GPD-ACC1 as a template by whole plasmid PCR. After DNA sequencing, the expression vector pY26-UAS1-GPD-ACC1 of the ACC1 mutant gene with the Ser codons at positions 659 and 1157 changed to Ala codons was obtained (the plasmid map is as Figure 2 shown), and the amino acid sequence of the acetyl-CoA decarboxylase mutant ACC1** is shown in SEQ ID NO.3.

[0055] (2) Construction of the mutant expression vector of 3-hydroxyisobutyryl-CoA hydrolase

[0056] Using the Saccharomyces cerevisiae BY4741 genome as a template and F5 / R5 as primers, the EHD3 gene fragment with the amino acid sequence shown in SEQ ID NO.4 was obtained by PCR amplification. The EHD3 gene fragment was recovered by gel electrophoresis, and the EHD3 gene fragment and the plasmid pY26-TEF-GPD were digested with SalI / HindⅢ double enzymes respectively. After purifying and recovering the digestion products, plasmid pY26-TEF-GPD-EHD3 was constructed by overnight ligation with T4 ligase.

[0057] Using pGFP33-UAS2 as a template and F6 / R6 as primers, the promoter UAS2 with the nucleotide sequence shown in SEQ ID NO.5 was amplified. The promoter UAS2 fragment was recovered by gel electrophoresis, and the promoter UAS2 fragment and the plasmid pY26-TEF-GPD-EHD3 were digested with SacI / BamHI double enzymes respectively. After purification and recovery, plasmid pY26-TEF-UAS2-EHD3 was constructed by overnight ligation with T4 ligase.

[0058] According to the gene sequence of EHD3, primers F7 / R7, F8 / R8, and F9 / R9 introducing F121I, E124S, and R3 / K7 / K14 / K18 / R22A mutations were designed and synthesized. Using pY26-TEF-UAS2-EHD3 as a template, site-directed mutagenesis of EHD3 was performed by whole plasmid PCR. After DNA sequencing, an expression vector pY26-TEF-UAS2-EHD3*** of the EHD3 mutant gene was obtained, in which the Phe codon at position 121 was changed to the Ile codon, the Glu codon at position 124 was changed to the Ser codon, and the Arg or Lys codons at positions 3, 7, 14, 18, and 22 were changed to Ala codons (the plasmid map is as shown in Figure 3 ), and the amino acid sequence of the 3-hydroxyisobutyryl-CoA hydrolase mutant EHD3*** is as shown in SEQ ID NO.6.

[0059] Example 2 Construction of integrated fragments

[0060] Using the Saccharomyces cerevisiae BY4741 genome as a template, and F10 / R10 and F11 / R11 as primers respectively, Delta1 with the nucleotide sequence shown in SEQ ID NO.7 and Delta2 with the nucleotide sequence shown in SEQ ID NO.8 were amplified by PCR;

[0061] Using the plasmid pGFP33-TEF1 as a template and F12 / R12 as primers, a GFP fragment with the nucleotide sequence shown in SEQ ID NO.9 was amplified by PCR;

[0062] Using the plasmid pY26-TEF-GPD as a template and F13 / R13 and F14 / R14 as primers respectively, Ura1 and Ura2 fragments were amplified by PCR;

[0063] Using the pY26-UAS1-GPD-ACC1** obtained in Example 1 as a template and F15 / R15 as primers, an amplified fragment I-ACC1** (including the promoter UAS1, the gene ACC1**, and the terminator ADH1) was obtained by PCR;

[0064] Using the pY26-TEF-UAS2-EHD3*** obtained in Example 1 as a template and F16 / R16 as primers, an amplified fragment I-EHD3*** (including the promoter UAS3, the gene EHD3***, and the terminator CYC1) was obtained by PCR.

[0065] Performing touchdown PCR on the four fragments of Delta1, GFP, I-EHD3***, and Ura1, and using the PCR product as a template and F10 / R16 as primers, IN-1 was amplified.

[0066] Perform touchdown PCR on the three fragments of Ura2, I-ACC1** and Delta2. Using the PCR product as a template and F14 / R11 as primers, IN-2 is amplified.

[0067] Example 3 Fluorescent Primary Screening of High-Copy Integrated Strains

[0068] Simultaneously transform the integration fragments IN-1 and IN-2 obtained in Example 2 into Saccharomyces cerevisiae BY4741 by the lithium acetate transformation method, and spread them on SD-Ura defective medium. Incubate at 30 °C for 2 - 3 days. The transformants picked are the successfully integrated recombinant bacteria (for the method of integration at the Delta site, see Figure 1 ).

[0069] Transfer the positive transformants into 2 mL of SD-Ura liquid medium as seeds. Incubate at 30 °C and 220 rpm overnight to obtain a seed solution. Inoculate the seed solution into 2 mL of SD-Ura liquid medium at an inoculation volume ratio of 10%. Incubate at 30 °C and 220 rpm until the OD600 reaches 0.7 - 0.9. Take 0.2 mL of the bacterial solution and place it in a 96-well plate, and measure the fluorescence intensity with a microplate reader. The fluorescence detection wavelength is 488 nm, and the excitation wavelength is 530 nm. After detection, 174 recombinant bacteria with higher fluorescence intensity (FLU / OD > 5000) are screened out.

[0070] Example 4 Tube Fermentation Re-Screening of High-Copy Integrated Strains

[0071] Inoculate the 174 integrated strains screened in Example 3 into 2 mL of YPD medium respectively. Incubate at 30 °C and 220 rpm overnight to obtain a seed solution. Transfer the seed solution into a test tube containing 9 mL of YPD medium at an inoculation volume ratio of 10%. Incubate on a shaker at 30 °C and 250 rpm for 7 d. Collect the fermentation broth at 168 h of fermentation, centrifuge at 13000 rpm for 5 min, take the supernatant, and filter it with a 0.22 μm filter membrane. The filtered supernatant is used for HPLC detection.

[0072] The malonic acid yield is detected by an Agilent high-performance liquid chromatograph, with a differential refractive index detector, a column temperature of 50 °C, and an injection volume of 20 μL. According to the results of the liquid phase, 3 integrated bacteria with the highest yield are screened out and then subjected to shake flask fermentation.

[0073] Example 5 Shake Flask Fermentation to Obtain High-Copy Integrated Strains with High Malonic Acid Yield

[0074] Inoculate the 3 integrated bacteria re-screened in Example 4 into 10 mL of YPD medium as seeds. Incubate at 30 °C and 220 rpm overnight to obtain a seed solution. Transfer the seed solution into a 250 mL shake flask containing 50 mL of YPD medium at an inoculation volume ratio of 2%. Incubate on a shaker at 30 °C and 220 rpm for 7 d to prepare the fermentation broth.

[0075] During the fermentation process, the fermentation broth was sampled every 12 h, centrifuged at 13,000 rpm for 5 min to obtain the supernatant, and the supernatant was filtered through a 0.22-μm filter membrane. The filtered supernatant was used for HPLC detection.

[0076] The malonic acid production was detected by an Agilent high-performance liquid chromatograph, with a differential refractive index detector, a column temperature of 50 °C, and an injection volume of 20 μL. A strain with high malonic acid production was screened out, and its production in the 48-well microculture medium fermentation was 13.5 mg / L (as Figure 4 shown). After 7 days of shake-flask fermentation culture, the malonic acid production was 80 mg / L, and it was named MA-112.

[0077] Comparative Example 1:

[0078] After site-directed mutagenesis of the E124 site of the Ehd3 protein in Saccharomyces cerevisiae cells, it can use malonyl-CoA as a substrate and be converted into malonic acid. This pathway makes it possible to accumulate malonic acid in Saccharomyces cerevisiae, but the production is low, less than 14 mg / L after 7 days of shake-flask fermentation culture.

[0079] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims. SEQUENCE LISTING <110> Jiangnan University <120> Method for improving the production of malonic acid by Saccharomyces cerevisiae <130> BAA211100A <160> 9 <170> PatentIn version 3.3 <210> 1 <211> 2233 <212> PRT <213> Artificial sequence <400> 1 Met Ser Glu Glu Ser Leu Phe Glu Ser Ser Pro Gln Lys Met Glu Tyr 1 5 10 15 Glu Ile Thr Asn Tyr Ser Glu Arg His Thr Glu Leu Pro Gly His Phe 20 25 30 Ile Gly Leu Asn Thr Val Asp Lys Leu Glu Glu Ser Pro Leu Arg Asp 35 40 45 Phe Val Lys Ser His Gly Gly His Thr Val Ile Ser Lys Ile Leu Ile 50 55 60 Ala Asn Asn Gly Ile Ala Ala Val Lys Glu Ile Arg Ser Val Arg Lys 65 70 75 80 Trp Ala Tyr Glu Thr Phe Gly Asp Asp Arg Thr Val Gln Phe Val Ala 85 90 95 Met Ala Thr Pro Glu Asp Leu Glu Ala Asn Ala Glu Tyr Ile Arg Met 100 105 110 Ala Asp Gln Tyr Ile Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr 115 120 125 Ala Asn Val Asp Leu Ile Val Asp Ile Ala Glu Arg Ala Asp Val Asp 130 135 140 Ala Val Trp Ala Gly Trp Gly His Ala Ser Glu Asn Pro Leu Leu Pro 145 150 155 160 Glu Lys Leu Ser Gln Ser Lys Arg Lys Val Ile Phe Ile Gly Pro Pro 165 170 175 Gly Asn Ala Met Arg Ser Leu Gly Asp Lys Ile Ser Ser Thr Ile Val 180 185 190 Ala Gln Ser Ala Lys Val Pro Cys Ile Pro Trp Ser Gly Thr Gly Val 195 200 205 Asp Thr Val His Val Asp Glu Lys Thr Gly Leu Val Ser Val Asp Asp 210 215 220 Asp Ile Tyr Gln Lys Gly Cys Cys Thr Ser Pro Glu Asp Gly Leu Gln 225 230 235 240 Lys Ala Lys Arg Ile Gly Phe Pro Val Met Ile Lys Ala Ser Glu Gly 245 250 255 Gly Gly Gly Lys Gly Ile Arg Gln Val Glu Arg Glu Glu Asp Phe Ile 260 265 270 Ala Leu Tyr His Gln Ala Ala Asn Glu Ile Pro Gly Ser Pro Ile Phe 275 280 285 Ile Met Lys Leu Ala Gly Arg Ala Arg His Leu Glu Val Gln Leu Leu 290 295 300 Ala Asp Gln Tyr Gly Thr Asn Ile Ser Leu Phe Gly Arg Asp Cys Ser 305 310 315 320 Val Gln Arg Arg His Gln Lys Ile Ile Glu Glu Ala Pro Val Thr Ile 325 330 335 Ala Lys Ala Glu Thr Phe His Glu Met Glu Lys Ala Ala Val Arg Leu 340 345 350 Gly Lys Leu Val Gly Tyr Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr 355 360 365 Ser His Asp Asp Gly Lys Phe Tyr Phe Leu Glu Leu Asn Pro Arg Leu 370 375 380 Gln Val Glu His Pro Thr Thr Glu Met Val Ser Gly Val Asn Leu Pro 385 390 395 400 Ala Ala Gln Leu Gln Ile Ala Met Gly Ile Pro Met His Arg Ile Ser 405 410 415 Asp Ile Arg Thr Leu Tyr Gly Met Asn Pro His Ser Ala Ser Glu Ile 420 425 430 Asp Phe Glu Phe Lys Thr Gln Asp Ala Thr Lys Lys Gln Arg Arg Pro 435 440 445 Ile Pro Lys Gly His Cys Thr Ala Cys Arg Ile Thr Ser Glu Asp Pro 450 455 460 Asn Asp Gly Phe Lys Pro Ser Gly Gly Thr Leu His Glu Leu Asn Phe 465 470 475 480 Arg Ser Ser Ser Asn Val Trp Gly Tyr Phe Ser Val Gly Asn Asn Gly 485 490 495 Asn Ile His Ser Phe Ser Asp Ser Gln Phe Gly His Ile Phe Ala Phe 500 505 510 Gly Glu Asn Arg Gln Ala Ser Arg Lys His Met Val Val Ala Leu Lys 515 520 525 Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr Thr Val Glu Tyr Leu Ile 530 535 540 Lys Leu Leu Glu Thr Glu Asp Phe Glu Asp Asn Thr Ile Thr Thr Gly 545 550 555 560 Trp Leu Asp Asp Leu Ile Thr His Lys Met Thr Ala Glu Lys Pro Asp 565 570 575 Pro Thr Leu Ala Val Ile Cys Gly Ala Ala Thr Lys Ala Phe Leu Ala 580 585 590 Ser Glu Glu Ala Arg His Lys Tyr Ile Glu Ser Leu Gln Lys Gly Gln 595 600 605 Val Leu Ser Lys Asp Leu Leu Gln Thr Met Phe Pro Val Asp Phe Ile 610 615 620 His Glu Gly Lys Arg Tyr Lys Phe Thr Val Ala Lys Ser Gly Asn Asp 625 630 635 640 Arg Tyr Thr Leu Phe Ile Asn Gly Ser Lys Cys Asp Ile Ile Leu Arg 645 650 655 Gln Leu Ser Asp Gly Gly Leu Leu Ile Ala Ile Gly Gly Lys Ser His 660 665 670 Thr Ile Tyr Trp Lys Glu Glu Val Ala Ala Thr Arg Leu Ser Val Asp 675 680 685 Ser Met Thr Thr Leu Leu Glu Val Glu Asn Asp Pro Thr Gln Leu Arg 690 695 700 Thr Pro Ser Pro Gly Lys Leu Val Lys Phe Leu Val Glu Asn Gly Glu 705 710 715 720 His Ile Ile Lys Gly Gln Pro Tyr Ala Glu Ile Glu Val Met Lys Met 725 730 735 Gln Met Pro Leu Val Ser Gln Glu Asn Gly Ile Val Gln Leu Leu Lys 740 745 750 Gln Pro Gly Ser Thr Ile Val Ala Gly Asp Ile Met Ala Ile Met Thr 755 760 765 Leu Asp Asp Pro Ser Lys Val Lys His Ala Leu Pro Phe Glu Gly Met 770 775 780 Leu Pro Asp Phe Gly Ser Pro Val Ile Glu Gly Thr Lys Pro Ala Tyr 785 790 795 800 Lys Phe Lys Ser Leu Val Ser Thr Leu Glu Asn Ile Leu Lys Gly Tyr 805 810 815 Asp Asn Gln Val Ile Met Asn Ala Ser Leu Gln Gln Leu Ile Glu Val 820 825 830 Leu Arg Asn Pro Lys Leu Pro Tyr Ser Glu Trp Lys Leu His Ile Ser 835 840 845 Ala Leu His Ser Arg Leu Pro Ala Lys Leu Asp Glu Gln Met Glu Glu 850 855 860 Leu Val Ala Arg Ser Leu Arg Arg Gly Ala Val Phe Pro Ala Arg Gln 865 870 875 880 Leu Ser Lys Leu Ile Asp Met Ala Val Lys Asn Pro Glu Tyr Asn Pro 885 890 895 Asp Lys Leu Leu Gly Ala Val Val Glu Pro Leu Ala Asp Ile Ala His 900 905 910 Lys Tyr Ser Asn Gly Leu Glu Ala His Glu His Ser Ile Phe Val His 915 920 925 Phe Leu Glu Glu Tyr Tyr Glu Val Glu Lys Leu Phe Asn Gly Pro Asn 930 935 940 Val Arg Glu Glu Asn Ile Ile Leu Lys Leu Arg Asp Glu Asn Pro Lys 945 950 955 960 Asp Leu Asp Lys Val Ala Leu Thr Val Leu Ser His Ser Lys Val Ser 965 970 975 Ala Lys Asn Asn Leu Ile Leu Ala Ile Leu Lys His Tyr Gln Pro Leu 980 985 990 Cys Lys Leu Ser Ser Lys Val Ser Ala Ile Phe Ser Thr Pro Leu Gln 995 1000 1005 His Ile Val Glu Leu Glu Ser Lys Ala Thr Ala Lys Val Ala Leu 1010 1015 1020 Gln Ala Arg Glu Ile Leu Ile Gln Gly Ala Leu Pro Ser Val Lys 1025 1030 1035 Glu Arg Thr Glu Gln Ile Glu His Ile Leu Lys Ser Ser Val Val 1040 1045 1050 Lys Val Ala Tyr Gly Ser Ser Asn Pro Lys Arg Ser Glu Pro Asp 1055 1060 1065 Leu Asn Ile Leu Lys Asp Leu Ile Asp Ser Asn Tyr Val Val Phe 1070 1075 1080 Asp Val Leu Leu Gln Phe Leu Thr His Gln Asp Pro Val Val Thr 1085 1090 1095 Ala Ala Ala Ala Gln Val Tyr Ile Arg Arg Ala Tyr Arg Ala Tyr 1100 1105 1110 Thr Ile Gly Asp Ile Arg Val His Glu Gly Val Thr Val Pro Ile 1115 1120 1125 Val Glu Trp Lys Phe Gln Leu Pro Ser Ala Ala Phe Ser Thr Phe 1130 1135 1140 Pro Thr Val Lys Ser Lys Met Gly Met Asn Arg Ala Val Ser Val 1145 1150 1155 Ser Asp Leu Ser Tyr Val Ala Asn Ser Gln Ser Ser Pro Leu Arg 1160 1165 1170 Glu Gly Ile Leu Met Ala Val Asp His Leu Asp Asp Val Asp Glu 1175 1180 1185 Ile Leu Ser Gln Ser Leu Glu Val Ile Pro Arg His Gln Ser Ser 1190 1195 1200 Ser Asn Gly Pro Ala Pro Asp Arg Ser Gly Ser Ser Ala Ser Leu 1205 1210 1215 Ser Asn Val Ala Asn Val Cys Val Ala Ser Thr Glu Gly Phe Glu 1220 1225 1230 Ser Glu Glu Glu Ile Leu Val Arg Leu Arg Glu Ile Leu Asp Leu 1235 1240 1245 Asn Lys Gln Glu Leu Ile Asn Ala Ser Ile Arg Arg Ile Thr Phe 1250 1255 1260 Met Phe Gly Phe Lys Asp Gly Ser Tyr Pro Lys Tyr Tyr Thr Phe 1265 1270 1275 Asn Gly Pro Asn Tyr Asn Glu Asn Glu Thr Ile Arg His Ile Glu 1280 1285 1290 Pro Ala Leu Ala Phe Gln Leu Glu Leu Gly Arg Leu Ser Asn Phe 1295 1300 1305 Asn Ile Lys Pro Ile Phe Thr Asp Asn Arg Asn Ile His Val Tyr 1310 1315 1320 Glu Ala Val Ser Lys Thr Ser Pro Leu Asp Lys Arg Phe Phe Thr 1325 1330 1335 Arg Gly Ile Ile Arg Thr Gly His Ile Arg Asp Asp Ile Ser Ile 1340 1345 1350 Gln Glu Tyr Leu Thr Ser Glu Ala Asn Arg Leu Met Ser Asp Ile 1355 1360 1365 Leu Asp Asn Leu Glu Val Thr Asp Thr Ser Asn Ser Asp Leu Asn 1370 1375 1380 His Ile Phe Ile Asn Phe Ile Ala Val Phe Asp Ile Ser Pro Glu 1385 1390 1395 Asp Val Glu Ala Ala Phe Gly Gly Phe Leu Glu Arg Phe Gly Lys 1400 1405 1410 Arg Leu Leu Arg Leu Arg Val Ser Ser Ala Glu Ile Arg Ile Ile 1415 1420 1425 Ile Lys Asp Pro Gln Thr Gly Ala Pro Val Pro Leu Arg Ala Leu 1430 1435 1440 Ile Asn Asn Val Ser Gly Tyr Val Ile Lys Thr Glu Met Tyr Thr 1445 1450 1455 Glu Val Lys Asn Ala Lys Gly Glu Trp Val Phe Lys Ser Leu Gly 1460 1465 1470 Lys Pro Gly Ser Met His Leu Arg Pro Ile Ala Thr Pro Tyr Pro 1475 1480 1485 Val Lys Glu Trp Leu Gln Pro Lys Arg Tyr Lys Ala His Leu Met 1490 1495 1500 Gly Thr Thr Tyr Val Tyr Asp Phe Pro Glu Leu Phe Arg Gln Ala 1505 1510 1515 Ser Ser Ser Gln Trp Lys Asn Phe Ser Ala Asp Val Lys Leu Thr 1520 1525 1530 Asp Asp Phe Phe Ile Ser Asn Glu Leu Ile Glu Asp Glu Asn Gly 1535 1540 1545 Glu Leu Thr Glu Val Glu Arg Glu Pro Gly Ala Asn Ala Ile Gly 1550 1555 1560 Met Val Ala Phe Lys Ile Thr Val Lys Thr Pro Glu Tyr Pro Arg 1565 1570 1575 Gly Arg Gln Phe Val Val Val Ala Asn Asp Ile Thr Phe Lys Ile 1580 1585 1590 Gly Ser Phe Gly Pro Gln Glu Asp Glu Phe Phe Asn Lys Val Thr 1595 1600 1605 Glu Tyr Ala Arg Lys Arg Gly Ile Pro Arg Ile Tyr Leu Ala Ala 1610 1615 1620 Asn Ser Gly Ala Arg Ile Gly Met Ala Glu Glu Ile Val Pro Leu 1625 1630 1635 Phe Gln Val Ala Trp Asn Asp Ala Ala Asn Pro Asp Lys Gly Phe 1640 1645 1650 Gln Tyr Leu Tyr Leu Thr Ser Glu Gly Met Glu Thr Leu Lys Lys 1655 1660 1665 Phe Asp Lys Glu Asn Ser Val Leu Thr Glu Arg Thr Val Ile Asn 1670 1675 1680 Gly Glu Glu Arg Phe Val Ile Lys Thr Ile Ile Gly Ser Glu Asp 1685 1690 1695 Gly Leu Gly Val Glu Cys Leu Arg Gly Ser Gly Leu Ile Ala Gly 1700 1705 1710 Ala Thr Ser Arg Ala Tyr His Asp Ile Phe Thr Ile Thr Leu Val 1715 1720 1725 Thr Cys Arg Ser Val Gly Ile Gly Ala Tyr Leu Val Arg Leu Gly 1730 1735 1740 Gln Arg Ala Ile Gln Val Glu Gly Gln Pro Ile Ile Leu Thr Gly 1745 1750 1755 Ala Pro Ala Ile Asn Lys Met Leu Gly Arg Glu Val Tyr Thr Ser 1760 1765 1770 Asn Leu Gln Leu Gly Gly Thr Gln Ile Met Tyr Asn Asn Gly Val 1775 1780 1785 Ser His Leu Thr Ala Val Asp Asp Leu Ala Gly Val Glu Lys Ile 1790 1795 1800 Val Glu Trp Met Ser Tyr Val Pro Ala Lys Arg Asn Met Pro Val 1805 1810 1815 Pro Ile Leu Glu Thr Lys Asp Thr Trp Asp Arg Pro Val Asp Phe 1820 1825 1830 Thr Pro Thr Asn Asp Glu Thr Tyr Asp Val Arg Trp Met Ile Glu 1835 1840 1845 Gly Arg Glu Thr Glu Ser Gly Phe Glu Tyr Gly Leu Phe Asp Lys 1850 1855 1860 Gly Ser Phe Phe Glu Thr Leu Ser Gly Trp Ala Lys Gly Val Val 1865 1870 1875 Val Gly Arg Ala Arg Leu Gly Gly Ile Pro Leu Gly Val Ile Gly 1880 1885 1890 Val Glu Thr Arg Thr Val Glu Asn Leu Ile Pro Ala Asp Pro Ala 1895 1900 1905 Asn Pro Asn Ser Ala Glu Thr Leu Ile Gln Glu Pro Gly Gln Val 1910 1915 1920 Trp His Pro Asn Ser Ala Phe Lys Thr Ala Gln Ala Ile Asn Asp 1925 1930 1935 Phe Asn Asn Gly Glu Gln Leu Pro Met Met Ile Leu Ala Asn Trp 1940 1945 1950 Arg Gly Phe Ser Gly Gly Gln Arg Asp Met Phe Asn Glu Val Leu 1955 1960 1965 Lys Tyr Gly Ser Phe Ile Val Asp Ala Leu Val Asp Tyr Lys Gln 1970 1975 1980 Pro Ile Ile Ile Tyr Ile Pro Pro Thr Gly Glu Leu Arg Gly Gly 1985 1990 1995 Ser Trp Val Val Val Asp Pro Thr Ile Asn Ala Asp Gln Met Glu 2000 2005 2010 Met Tyr Ala Asp Val Asn Ala Arg Ala Gly Val Leu Glu Pro Gln 2015 2020 2025 Gly Met Val Gly Ile Lys Phe Arg Arg Glu Lys Leu Leu Asp Thr 2030 2035 2040 Met Asn Arg Leu Asp Asp Lys Tyr Arg Glu Leu Arg Ser Gln Leu 2045 2050 2055 Ser Asn Lys Ser Leu Ala Pro Glu Val His Gln Gln Ile Ser Lys 2060 2065 2070 Gln Leu Ala Asp Arg Glu Arg Glu Leu Leu Pro Ile Tyr Gly Gln 2075 2080 2085 Ile Ser Leu Gln Phe Ala Asp Leu His Asp Arg Ser Ser Arg Met 2090 2095 2100 Val Ala Lys Gly Val Ile Ser Lys Glu Leu Glu Trp Thr Glu Ala 2105 2110 2115 Arg Arg Phe Phe Phe Trp Arg Leu Arg Arg Arg Leu Asn Glu Glu 2120 2125 2130 Tyr Leu Ile Lys Arg Leu Ser His Gln Val Gly Glu Ala Ser Arg 2135 2140 2145 Leu Glu Lys Ile Ala Arg Ile Arg Ser Trp Tyr Pro Ala Ser Val 2150 2155 2160 Asp His Glu Asp Asp Arg Gln Val Ala Thr Trp Ile Glu Glu Asn 2165 2170 2175 Tyr Lys Thr Leu Asp Asp Lys Leu Lys Gly Leu Lys Leu Glu Ser 2180 2185 2190 Phe Ala Gln Asp Leu Ala Lys Lys Ile Arg Ser Asp His Asp Asn 2195 2200 2205 Ala Ile Asp Gly Leu Ser Glu Val Ile Lys Met Leu Ser Thr Asp 2210 2215 2220 Asp Lys Glu Lys Leu Leu Lys Thr Leu Lys 2225 2230 <210> 2 <211> 548 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (455)..(455) <223> n is a, c, g, or t <400> 2 ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa 60 aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt atataaagac 120 ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta 180 tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata aacacacata 240 aacaaacaaa atgtcgagtt tatcattatc aatactgcca tttcaaagaa tacgtaaata 300 attaatagta gtgattttcc taactttagt tagtcaaaaa attagccttt taattctgct 360 gtaacccgta catgcccaaa atagggggcg ggttactcag aatatataac atcgtaggtg 420 tctgggtgaa cagtttattc ctggcatcca ctaantataa tggagcccgc tttttaagct 480 ggcatccaga aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc aaccatcagt 540 tcataggt 548 <210> 3 <211> 2233 <212> PRT <213> Artificial Sequence <400> 3 Met Ser Glu Glu Ser Leu Phe Glu Ser Ser Pro Gln Lys Met Glu Tyr 1 5 10 15 Glu Ile Thr Asn Tyr Ser Glu Arg His Thr Glu Leu Pro Gly His Phe 20 25 30 Ile Gly Leu Asn Thr Val Asp Lys Leu Glu Glu Ser Pro Leu Arg Asp 35 40 45 Phe Val Lys Ser His Gly Gly His Thr Val Ile Ser Lys Ile Leu Ile 50 55 60 Ala Asn Asn Gly Ile Ala Ala Val Lys Glu Ile Arg Ser Val Arg Lys 65 70 75 80 Trp Ala Tyr Glu Thr Phe Gly Asp Asp Arg Thr Val Gln Phe Val Ala 85 90 95 Met Ala Thr Pro Glu Asp Leu Glu Ala Asn Ala Glu Tyr Ile Arg Met 100 105 110 Ala Asp Gln Tyr Ile Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr 115 120 125 Ala Asn Val Asp Leu Ile Val Asp Ile Ala Glu Arg Ala Asp Val Asp 130 135 140 Ala Val Trp Ala Gly Trp Gly His Ala Ser Glu Asn Pro Leu Leu Pro 145 150 155 160 Glu Lys Leu Ser Gln Ser Lys Arg Lys Val Ile Phe Ile Gly Pro Pro 165 170 175 Gly Asn Ala Met Arg Ser Leu Gly Asp Lys Ile Ser Ser Thr Ile Val 180 185 190 Ala Gln Ser Ala Lys Val Pro Cys Ile Pro Trp Ser Gly Thr Gly Val 195 200 205 Asp Thr Val His Val Asp Glu Lys Thr Gly Leu Val Ser Val Asp Asp 210 215 220 Asp Ile Tyr Gln Lys Gly Cys Cys Thr Ser Pro Glu Asp Gly Leu Gln 225 230 235 240 Lys Ala Lys Arg Ile Gly Phe Pro Val Met Ile Lys Ala Ser Glu Gly 245 250 255 Gly Gly Gly Lys Gly Ile Arg Gln Val Glu Arg Glu Glu Asp Phe Ile 260 265 270 Ala Leu Tyr His Gln Ala Ala Asn Glu Ile Pro Gly Ser Pro Ile Phe 275 280 285 Ile Met Lys Leu Ala Gly Arg Ala Arg His Leu Glu Val Gln Leu Leu 290 295 300 Ala Asp Gln Tyr Gly Thr Asn Ile Ser Leu Phe Gly Arg Asp Cys Ser 305 310 315 320 Val Gln Arg Arg His Gln Lys Ile Ile Glu Glu Ala Pro Val Thr Ile 325 330 335 Ala Lys Ala Glu Thr Phe His Glu Met Glu Lys Ala Ala Val Arg Leu 340 345 350 Gly Lys Leu Val Gly Tyr Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr 355 360 365 Ser His Asp Asp Gly Lys Phe Tyr Phe Leu Glu Leu Asn Pro Arg Leu 370 375 380 Gln Val Glu His Pro Thr Thr Glu Met Val Ser Gly Val Asn Leu Pro 385 390 395 400 Ala Ala Gln Leu Gln Ile Ala Met Gly Ile Pro Met His Arg Ile Ser 405 410 415 Asp Ile Arg Thr Leu Tyr Gly Met Asn Pro His Ser Ala Ser Glu Ile 420 425 430 Asp Phe Glu Phe Lys Thr Gln Asp Ala Thr Lys Lys Gln Arg Arg Pro 435 440 445 Ile Pro Lys Gly His Cys Thr Ala Cys Arg Ile Thr Ser Glu Asp Pro 450 455 460 Asn Asp Gly Phe Lys Pro Ser Gly Gly Thr Leu His Glu Leu Asn Phe 465 470 475 480 Arg Ser Ser Ser Asn Val Trp Gly Tyr Phe Ser Val Gly Asn Asn Gly 485 490 495 Asn Ile His Ser Phe Ser Asp Ser Gln Phe Gly His Ile Phe Ala Phe 500 505 510 Gly Glu Asn Arg Gln Ala Ser Arg Lys His Met Val Val Ala Leu Lys 515 520 525 Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr Thr Val Glu Tyr Leu Ile 530 535 540 Lys Leu Leu Glu Thr Glu Asp Phe Glu Asp Asn Thr Ile Thr Thr Gly 545 550 555 560 Trp Leu Asp Asp Leu Ile Thr His Lys Met Thr Ala Glu Lys Pro Asp 565 570 575 Pro Thr Leu Ala Val Ile Cys Gly Ala Ala Thr Lys Ala Phe Leu Ala 580 585 590 Ser Glu Glu Ala Arg His Lys Tyr Ile Glu Ser Leu Gln Lys Gly Gln 595 600 605 Val Leu Ser Lys Asp Leu Leu Gln Thr Met Phe Pro Val Asp Phe Ile 610 615 620 His Glu Gly Lys Arg Tyr Lys Phe Thr Val Ala Lys Ser Gly Asn Asp 625 630 635 640 Arg Tyr Thr Leu Phe Ile Asn Gly Ser Lys Cys Asp Ile Ile Leu Arg 645 650 655 Gln Leu Ala Asp Gly Gly Leu Leu Ile Ala Ile Gly Gly Lys Ser His 660 665 670 Thr Ile Tyr Trp Lys Glu Glu Val Ala Ala Thr Arg Leu Ser Val Asp 675 680 685 Ser Met Thr Thr Leu Leu Glu Val Glu Asn Asp Pro Thr Gln Leu Arg 690 695 700 Thr Pro Ser Pro Gly Lys Leu Val Lys Phe Leu Val Glu Asn Gly Glu 705 710 715 720 His Ile Ile Lys Gly Gln Pro Tyr Ala Glu Ile Glu Val Met Lys Met 725 730 735 Gln Met Pro Leu Val Ser Gln Glu Asn Gly Ile Val Gln Leu Leu Lys 740 745 750 Gln Pro Gly Ser Thr Ile Val Ala Gly Asp Ile Met Ala Ile Met Thr 755 760 765 Leu Asp Asp Pro Ser Lys Val Lys His Ala Leu Pro Phe Glu Gly Met 770 775 780 Leu Pro Asp Phe Gly Ser Pro Val Ile Glu Gly Thr Lys Pro Ala Tyr 785 790 795 800 Lys Phe Lys Ser Leu Val Ser Thr Leu Glu Asn Ile Leu Lys Gly Tyr 805 810 815 Asp Asn Gln Val Ile Met Asn Ala Ser Leu Gln Gln Leu Ile Glu Val 820 825 830 Leu Arg Asn Pro Lys Leu Pro Tyr Ser Glu Trp Lys Leu His Ile Ser 835 840 845 Ala Leu His Ser Arg Leu Pro Ala Lys Leu Asp Glu Gln Met Glu Glu 850 855 860 Leu Val Ala Arg Ser Leu Arg Arg Gly Ala Val Phe Pro Ala Arg Gln 865 870 875 880 Leu Ser Lys Leu Ile Asp Met Ala Val Lys Asn Pro Glu Tyr Asn Pro 885 890 895 Asp Lys Leu Leu Gly Ala Val Val Glu Pro Leu Ala Asp Ile Ala His 900 905 910 Lys Tyr Ser Asn Gly Leu Glu Ala His Glu His Ser Ile Phe Val His 915 920 925 Phe Leu Glu Glu Tyr Tyr Glu Val Glu Lys Leu Phe Asn Gly Pro Asn 930 935 940 Val Arg Glu Glu Asn Ile Ile Leu Lys Leu Arg Asp Glu Asn Pro Lys 945 950 955 960 Asp Leu Asp Lys Val Ala Leu Thr Val Leu Ser His Ser Lys Val Ser 965 970 975 Ala Lys Asn Asn Leu Ile Leu Ala Ile Leu Lys His Tyr Gln Pro Leu 980 985 990 Cys Lys Leu Ser Ser Lys Val Ser Ala Ile Phe Ser Thr Pro Leu Gln 995 1000 1005 His Ile Val Glu Leu Glu Ser Lys Ala Thr Ala Lys Val Ala Leu 1010 1015 1020 Gln Ala Arg Glu Ile Leu Ile Gln Gly Ala Leu Pro Ser Val Lys 1025 1030 1035 Glu Arg Thr Glu Gln Ile Glu His Ile Leu Lys Ser Ser Val Val 1040 1045 1050 Lys Val Ala Tyr Gly Ser Ser Asn Pro Lys Arg Ser Glu Pro Asp 1055 1060 1065 Leu Asn Ile Leu Lys Asp Leu Ile Asp Ser Asn Tyr Val Val Phe 1070 1075 1080 Asp Val Leu Leu Gln Phe Leu Thr His Gln Asp Pro Val Val Thr 1085 1090 1095 Ala Ala Ala Ala Gln Val Tyr Ile Arg Arg Ala Tyr Arg Ala Tyr 1100 1105 1110 Thr Ile Gly Asp Ile Arg Val His Glu Gly Val Thr Val Pro Ile 1115 1120 1125 Val Glu Trp Lys Phe Gln Leu Pro Ser Ala Ala Phe Ser Thr Phe 1130 1135 1140 Pro Thr Val Lys Ser Lys Met Gly Met Asn Arg Ala Val Ala Val 1145 1150 1155 Ser Asp Leu Ser Tyr Val Ala Asn Ser Gln Ser Ser Pro Leu Arg 1160 1165 1170 Glu Gly Ile Leu Met Ala Val Asp His Leu Asp Asp Val Asp Glu 1175 1180 1185 Ile Leu Ser Gln Ser Leu Glu Val Ile Pro Arg His Gln Ser Ser 1190 1195 1200 Ser Asn Gly Pro Ala Pro Asp Arg Ser Gly Ser Ser Ala Ser Leu 1205 1210 1215 Ser Asn Val Ala Asn Val Cys Val Ala Ser Thr Glu Gly Phe Glu 1220 1225 1230 Ser Glu Glu Glu Ile Leu Val Arg Leu Arg Glu Ile Leu Asp Leu 1235 1240 1245 Asn Lys Gln Glu Leu Ile Asn Ala Ser Ile Arg Arg Ile Thr Phe 1250 1255 1260 Met Phe Gly Phe Lys Asp Gly Ser Tyr Pro Lys Tyr Tyr Thr Phe 1265 1270 1275 Asn Gly Pro Asn Tyr Asn Glu Asn Glu Thr Ile Arg His Ile Glu 1280 1285 1290 Pro Ala Leu Ala Phe Gln Leu Glu Leu Gly Arg Leu Ser Asn Phe 1295 1300 1305 Asn Ile Lys Pro Ile Phe Thr Asp Asn Arg Asn Ile His Val Tyr 1310 1315 1320 Glu Ala Val Ser Lys Thr Ser Pro Leu Asp Lys Arg Phe Phe Thr 1325 1330 1335 Arg Gly Ile Ile Arg Thr Gly His Ile Arg Asp Asp Ile Ser Ile 1340 1345 1350 Gln Glu Tyr Leu Thr Ser Glu Ala Asn Arg Leu Met Ser Asp Ile 1355 1360 1365 Leu Asp Asn Leu Glu Val Thr Asp Thr Ser Asn Ser Asp Leu Asn 1370 1375 1380 His Ile Phe Ile Asn Phe Ile Ala Val Phe Asp Ile Ser Pro Glu 1385 1390 1395 Asp Val Glu Ala Ala Phe Gly Gly Phe Leu Glu Arg Phe Gly Lys 1400 1405 1410 Arg Leu Leu Arg Leu Arg Val Ser Ser Ala Glu Ile Arg Ile Ile 1415 1420 1425 Ile Lys Asp Pro Gln Thr Gly Ala Pro Val Pro Leu Arg Ala Leu 1430 1435 1440 Ile Asn Asn Val Ser Gly Tyr Val Ile Lys Thr Glu Met Tyr Thr 1445 1450 1455 Glu Val Lys Asn Ala Lys Gly Glu Trp Val Phe Lys Ser Leu Gly 1460 1465 1470 Lys Pro Gly Ser Met His Leu Arg Pro Ile Ala Thr Pro Tyr Pro 1475 1480 1485 Val Lys Glu Trp Leu Gln Pro Lys Arg Tyr Lys Ala His Leu Met 1490 1495 1500 Gly Thr Thr Tyr Val Tyr Asp Phe Pro Glu Leu Phe Arg Gln Ala 1505 1510 1515 Ser Ser Ser Gln Trp Lys Asn Phe Ser Ala Asp Val Lys Leu Thr 1520 1525 1530 Asp Asp Phe Phe Ile Ser Asn Glu Leu Ile Glu Asp Glu Asn Gly 1535 1540 1545 Glu Leu Thr Glu Val Glu Arg Glu Pro Gly Ala Asn Ala Ile Gly 1550 1555 1560 Met Val Ala Phe Lys Ile Thr Val Lys Thr Pro Glu Tyr Pro Arg 1565 1570 1575 Gly Arg Gln Phe Val Val Val Ala Asn Asp Ile Thr Phe Lys Ile 1580 1585 1590 Gly Ser Phe Gly Pro Gln Glu Asp Glu Phe Phe Asn Lys Val Thr 1595 1600 1605 Glu Tyr Ala Arg Lys Arg Gly Ile Pro Arg Ile Tyr Leu Ala Ala 1610 1615 1620 Asn Ser Gly Ala Arg Ile Gly Met Ala Glu Glu Ile Val Pro Leu 1625 1630 1635 Phe Gln Val Ala Trp Asn Asp Ala Ala Asn Pro Asp Lys Gly Phe 1640 1645 1650 Gln Tyr Leu Tyr Leu Thr Ser Glu Gly Met Glu Thr Leu Lys Lys 1655 1660 1665 Phe Asp Lys Glu Asn Ser Val Leu Thr Glu Arg Thr Val Ile Asn 1670 1675 1680 Gly Glu Glu Arg Phe Val Ile Lys Thr Ile Ile Gly Ser Glu Asp 1685 1690 1695 Gly Leu Gly Val Glu Cys Leu Arg Gly Ser Gly Leu Ile Ala Gly 1700 1705 1710 Ala Thr Ser Arg Ala Tyr His Asp Ile Phe Thr Ile Thr Leu Val 1715 1720 1725 Thr Cys Arg Ser Val Gly Ile Gly Ala Tyr Leu Val Arg Leu Gly 1730 1735 1740 Gln Arg Ala Ile Gln Val Glu Gly Gln Pro Ile Ile Leu Thr Gly 1745 1750 1755 Ala Pro Ala Ile Asn Lys Met Leu Gly Arg Glu Val Tyr Thr Ser 1760 1765 1770 Asn Leu Gln Leu Gly Gly Thr Gln Ile Met Tyr Asn Asn Gly Val 1775 1780 1785 Ser His Leu Thr Ala Val Asp Asp Leu Ala Gly Val Glu Lys Ile 1790 1795 1800 Val Glu Trp Met Ser Tyr Val Pro Ala Lys Arg Asn Met Pro Val 1805 1810 1815 Pro Ile Leu Glu Thr Lys Asp Thr Trp Asp Arg Pro Val Asp Phe 1820 1825 1830 Thr Pro Thr Asn Asp Glu Thr Tyr Asp Val Arg Trp Met Ile Glu 1835 1840 1845 Gly Arg Glu Thr Glu Ser Gly Phe Glu Tyr Gly Leu Phe Asp Lys 1850 1855 1860 Gly Ser Phe Phe Glu Thr Leu Ser Gly Trp Ala Lys Gly Val Val 1865 1870 1875 Val Gly Arg Ala Arg Leu Gly Gly Ile Pro Leu Gly Val Ile Gly 1880 1885 1890 Val Glu Thr Arg Thr Val Glu Asn Leu Ile Pro Ala Asp Pro Ala 1895 1900 1905 Asn Pro Asn Ser Ala Glu Thr Leu Ile Gln Glu Pro Gly Gln Val 1910 1915 1920 Trp His Pro Asn Ser Ala Phe Lys Thr Ala Gln Ala Ile Asn Asp 1925 1930 1935 Phe Asn Asn Gly Glu Gln Leu Pro Met Met Ile Leu Ala Asn Trp 1940 1945 1950 Arg Gly Phe Ser Gly Gly Gln Arg Asp Met Phe Asn Glu Val Leu 1955 1960 1965 Lys Tyr Gly Ser Phe Ile Val Asp Ala Leu Val Asp Tyr Lys Gln 1970 1975 1980 Pro Ile Ile Ile Tyr Ile Pro Pro Thr Gly Glu Leu Arg Gly Gly 1985 1990 1995 Ser Trp Val Val Val Asp Pro Thr Ile Asn Ala Asp Gln Met Glu 2000 2005 2010 Met Tyr Ala Asp Val Asn Ala Arg Ala Gly Val Leu Glu Pro Gln 2015 2020 2025 Gly Met Val Gly Ile Lys Phe Arg Arg Glu Lys Leu Leu Asp Thr 2030 2035 2040 Met Asn Arg Leu Asp Asp Lys Tyr Arg Glu Leu Arg Ser Gln Leu 2045 2050 2055 Ser Asn Lys Ser Leu Ala Pro Glu Val His Gln Gln Ile Ser Lys 2060 2065 2070 Gln Leu Ala Asp Arg Glu Arg Glu Leu Leu Pro Ile Tyr Gly Gln 2075 2080 2085 Ile Ser Leu Gln Phe Ala Asp Leu His Asp Arg Ser Ser Arg Met 2090 2095 2100 Val Ala Lys Gly Val Ile Ser Lys Glu Leu Glu Trp Thr Glu Ala 2105 2110 2115 Arg Arg Phe Phe Phe Trp Arg Leu Arg Arg Arg Leu Asn Glu Glu 2120 2125 2130 Tyr Leu Ile Lys Arg Leu Ser His Gln Val Gly Glu Ala Ser Arg 2135 2140 2145 Leu Glu Lys Ile Ala Arg Ile Arg Ser Trp Tyr Pro Ala Ser Val 2150 2155 2160 Asp His Glu Asp Asp Arg Gln Val Ala Thr Trp Ile Glu Glu Asn 2165 2170 2175 Tyr Lys Thr Leu Asp Asp Lys Leu Lys Gly Leu Lys Leu Glu Ser 2180 2185 2190 Phe Ala Gln Asp Leu Ala Lys Lys Ile Arg Ser Asp His Asp Asn 2195 2200 2205 Ala Ile Asp Gly Leu Ser Glu Val Ile Lys Met Leu Ser Thr Asp 2210 2215 2220 Asp Lys Glu Lys Leu Leu Lys Thr Leu Lys 2225 2230 <210> 4 <211> 500 <212> PRT <213> Artificial Sequence <400> 4 Met Leu Arg Asn Thr Leu Lys Cys Ala Gln Leu Ser Ser Lys Tyr Gly 1 5 10 15 Phe Lys Thr Thr Thr Arg Thr Phe Met Thr Thr Gln Pro Gln Leu Asn 20 25 30 Val Thr Asp Ala Pro Pro Val Leu Phe Thr Val Gln Asp Thr Ala Arg 35 40 45 Val Ile Thr Leu Asn Arg Pro Lys Lys Leu Asn Ala Leu Asn Ala Glu 50 55 60 Met Ser Glu Ser Met Phe Lys Thr Leu Asn Glu Tyr Ala Lys Ser Asp 65 70 75 80 Thr Thr Asn Leu Val Ile Leu Lys Ser Ser Asn Arg Pro Arg Ser Phe 85 90 95 Cys Ala Gly Gly Asp Val Ala Thr Val Ala Ile Phe Asn Phe Asn Lys 100 105 110 Glu Phe Ala Lys Ser Ile Lys Phe Phe Thr Asp Glu Tyr Ser Leu Asn 115 120 125 Phe Gln Ile Ala Thr Tyr Leu Lys Pro Ile Val Thr Phe Met Asp Gly 130 135 140 Ile Thr Met Gly Gly Gly Val Gly Leu Ser Ile His Thr Pro Phe Arg 145 150 155 160 Ile Ala Thr Glu Asn Thr Lys Trp Ala Met Pro Glu Met Asp Ile Gly 165 170 175 Phe Phe Pro Asp Val Gly Ser Thr Phe Ala Leu Pro Arg Ile Val Thr 180 185 190 Leu Ala Asn Ser Asn Ser Gln Met Ala Leu Tyr Leu Cys Leu Thr Gly 195 200 205 Glu Val Val Thr Gly Ala Asp Ala Tyr Met Leu Gly Leu Ala Ser His 210 215 220 Tyr Val Ser Ser Glu Asn Leu Asp Ala Leu Gln Lys Arg Leu Gly Glu 225 230 235 240 Ile Ser Pro Pro Phe Asn Asn Asp Pro Gln Ser Ala Tyr Phe Phe Gly 245 250 255 Met Val Asn Glu Ser Ile Asp Glu Phe Val Ser Pro Leu Pro Lys Asp 260 265 270 Tyr Val Phe Lys Tyr Ser Asn Glu Lys Leu Asn Val Ile Glu Ala Cys 275 280 285 Phe Asn Leu Ser Lys Asn Gly Thr Ile Glu Asp Ile Met Asn Asn Leu 290 295 300 Arg Gln Tyr Glu Gly Ser Ala Glu Gly Lys Ala Phe Ala Gln Glu Ile 305 310 315 320 Lys Thr Lys Leu Leu Thr Lys Ser Pro Ser Ser Leu Gln Ile Ala Leu 325 330 335 Arg Leu Val Gln Glu Asn Ser Arg Asp His Ile Glu Ser Ala Ile Lys 340 345 350 Arg Asp Leu Tyr Thr Ala Ala Asn Met Cys Met Asn Gln Asp Ser Leu 355 360 365 Val Glu Phe Ser Glu Ala Thr Lys His Lys Leu Ile Asp Lys Gln Arg 370 375 380 Val Pro Tyr Pro Trp Thr Lys Lys Glu Gln Leu Phe Val Ser Gln Leu 385 390 395 400 Thr Ser Ile Thr Ser Pro Lys Pro Ser Leu Pro Met Ser Leu Leu Arg 405 410 415 Asn Thr Ser Asn Val Thr Trp Thr Gln Tyr Pro Tyr His Ser Lys Tyr 420 425 430 Gln Leu Pro Thr Glu Gln Glu Ile Ala Ala Tyr Ile Glu Lys Arg Thr 435 440 445 Asn Asp Asp Thr Gly Ala Lys Val Thr Glu Arg Glu Val Leu Asn His 450 455 460 Phe Ala Asn Val Ile Pro Ser Arg Arg Gly Lys Leu Gly Ile Gln Ser 465 470 475 480 Leu Cys Lys Ile Val Cys Glu Arg Lys Cys Glu Glu Val Asn Asp Gly 485 490 495 Leu Arg Trp Lys 500 <210> 5 <211> 545 <212> DNA <213> Artificial Sequence <400> 5 tcgagtttat cattataata ctgccatttc aaagaatacg taaataatta atagtagtga 60 ttttcctaac tttatttagt caaaaaatta gccttttaat tctgctgtaa cccgtacatg 120 cccgaaatag ggtgcgggtt acacagaata tataacatcg taggtgtctg ggtgaacagt 180 ttattcctgg catccactaa atataatgga gcccgctctt taagctggca tccagaaaaa 240 aaaagaatcc cagcaccaaa atattgtttt cttcaccaac catcagttca taggtctcat 300 tttcttacac cttctattac cttctgctct ctctgatttg gaaaaagctg aaaaaaaagg 360 ttgaaaccag ttccctgaaa ttattcccct acttgactaa taagtatata aagacggtag 420 gtattgattg taattctgta aatctatttc ttaaacttct taaattctac ttttatagtt 480 agtctttttt ttagttttaa aacaccaaga acttagtttc gaataaacac acataaacaa 540 acaaa 545 <210> 6 <211> 500 <212> PRT <213> Artificial Sequence <400> 6 Met Leu Ala Asn Thr Leu Ala Cys Ala Gln Leu Ser Ser Ala Tyr Gly 1 5 10 15 Phe Ala Thr Thr Thr Ala Thr Phe Met Thr Thr Gln Pro Gln Leu Asn 20 25 30 Val Thr Asp Ala Pro Pro Val Leu Phe Thr Val Gln Asp Thr Ala Arg 35 40 45 Val Ile Thr Leu Asn Arg Pro Lys Lys Leu Asn Ala Leu Asn Ala Glu 50 55 60 Met Ser Glu Ser Met Phe Lys Thr Leu Asn Glu Tyr Ala Lys Ser Asp 65 70 75 80 Thr Thr Asn Leu Val Ile Leu Lys Ser Ser Asn Arg Pro Arg Ser Phe 85 90 95 Cys Ala Gly Gly Asp Val Ala Thr Val Ala Ile Phe Asn Phe Asn Lys 100 105 110 Glu Phe Ala Lys Ser Ile Lys Phe Ile Thr Asp Ser Tyr Ser Leu Asn 115 120 125 Phe Gln Ile Ala Thr Tyr Leu Lys Pro Ile Val Thr Phe Met Asp Gly 130 135 140 Ile Thr Met Gly Gly Gly Val Gly Leu Ser Ile His Thr Pro Phe Arg 145 150 155 160 Ile Ala Thr Glu Asn Thr Lys Trp Ala Met Pro Glu Met Asp Ile Gly 165 170 175 Phe Phe Pro Asp Val Gly Ser Thr Phe Ala Leu Pro Arg Ile Val Thr 180 185 190 Leu Ala Asn Ser Asn Ser Gln Met Ala Leu Tyr Leu Cys Leu Thr Gly 195 200 205 Glu Val Val Thr Gly Ala Asp Ala Tyr Met Leu Gly Leu Ala Ser His 210 215 220 Tyr Val Ser Ser Glu Asn Leu Asp Ala Leu Gln Lys Arg Leu Gly Glu 225 230 235 240 Ile Ser Pro Pro Phe Asn Asn Asp Pro Gln Ser Ala Tyr Phe Phe Gly 245 250 255 Met Val Asn Glu Ser Ile Asp Glu Phe Val Ser Pro Leu Pro Lys Asp 260 265 270 Tyr Val Phe Lys Tyr Ser Asn Glu Lys Leu Asn Val Ile Glu Ala Cys 275 280 285 Phe Asn Leu Ser Lys Asn Gly Thr Ile Glu Asp Ile Met Asn Asn Leu 290 295 300 Arg Gln Tyr Glu Gly Ser Ala Glu Gly Lys Ala Phe Ala Gln Glu Ile 305 310 315 320 Lys Thr Lys Leu Leu Thr Lys Ser Pro Ser Ser Leu Gln Ile Ala Leu 325 330 335 Arg Leu Val Gln Glu Asn Ser Arg Asp His Ile Glu Ser Ala Ile Lys 340 345 350 Arg Asp Leu Tyr Thr Ala Ala Asn Met Cys Met Asn Gln Asp Ser Leu 355 360 365 Val Glu Phe Ser Glu Ala Thr Lys His Lys Leu Ile Asp Lys Gln Arg 370 375 380 Val Pro Tyr Pro Trp Thr Lys Lys Glu Gln Leu Phe Val Ser Gln Leu 385 390 395 400 Thr Ser Ile Thr Ser Pro Lys Pro Ser Leu Pro Met Ser Leu Leu Arg 405 410 415 Asn Thr Ser Asn Val Thr Trp Thr Gln Tyr Pro Tyr His Ser Lys Tyr 420 425 430 Gln Leu Pro Thr Glu Gln Glu Ile Ala Ala Tyr Ile Glu Lys Arg Thr 435 440 445 Asn Asp Asp Thr Gly Ala Lys Val Thr Glu Arg Glu Val Leu Asn His 450 455 460 Phe Ala Asn Val Ile Pro Ser Arg Arg Gly Lys Leu Gly Ile Gln Ser 465 470 475 480 Leu Cys Lys Ile Val Cys Glu Arg Lys Cys Glu Glu Val Asn Asp Gly 485 490 495 Leu Arg Trp Lys 500 <210> 7 <211> 258 <212> DNA <213> Artificial Sequence <400> 7 ggctggcaac taatagggac actaccaata tattatcata tacggtgtta gacgatgaca 60 taagatacga ggaactgtca tcgaagttag aggaagctga aatgcaagga ttgataatgt 120 aataggataa tgaaacatat aaaacggaat gaggaataat cgtaatatta gtatatagag 180 ataaagattc cattttgagg attcctatat cctcgaggag aacttctagt atattctgta 240 tacctgatat tatagcct 258 <210> 8 <211> 222 <212> DNA <213> Artificial sequence <400> 8 tgttggaata aaaatcaact atcatctact aactagtatt tacgttacta gtatattatc 60 atatacggtg ttagaagatg acgcaaatga tgagaaatag tcatctaaat tagtggaagc 120 tgaaacgcaa ggattgataa tgtaatagga tcaatgaata ttaacatata aaatgatgat 180 aataatattt atagaattgt gtagaattgc agattccctt tt 222 <210> 9 <211> 1696 <212> DNA <213> Artificial sequence <400> 9 agtacctcct cgctcagcat ctgcttcttc ccaaagatga acgcggcgtt atgtcactaa 60 cgacgtgcac caacttgcgg aaagtggaat cccgttccaa aactggcatc cactaattga 120 tacatctaca caccgcacgc cttttttctg aagcccactt tcgtggactt tgccatatgc 180 aaaattcatg aagtgtgata ccaagtcagc atacacctca ctagggtagt ttctttggtt 240 gtattgatca tttggttcat cgtggttcat taattttttt tctccattgc tttctggctt 300 tgatcttact atcatttgga tggatccctc attttcttac accttctatt accttctgct 360 ctctctgatt tggaaaaagc tgaaaaaaaa ggttgaaacc agttccctga aattattccc 420 ctacttgact aataagtata taaagacggt aggtattgat tgtaattctg taaatctatt 480 tcttaaactt cttaaattct acttttatag ttagtctttt ttttagtttt aaaacaccaa 540 gaacttagtt tcgaataaac acacataaac aaacaaaatg gcatgcatga gtaaaggaga 600 agaacttttc actggagtgg tcccagttct tgttgaatta gatggcgatg ttaatgggca 660 aaaattctct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa 720 ttttatttgc actactggga agctacctgt tccatggcca acacttgtca ctactttctc 780 ttatggtgtt caatgcttct caagataccc agatcatatg aaacagcatg actttttcaa 840 gagtgccatg cccgaaggtt atgtacagga aagaactata ttttacaaag atgacgggaa 900 ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt 960 aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaaatgg aatacaacta 1020 taactcacat aatgtataca tcatgggaga caaaccaaag aatggcatca aagttaactt 1080 caaaattaga cacaacatta aagatggaag cgttcaatta gcagaccatt atcaacaaaa 1140 tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc 1200 tgccctttcc aaagatccca acgaaaagag agatcacatg atccttcttg agtttgtaac 1260 agctgctagg attacacatg gcatggatga actatacaaa aagcttggcg taatcatggt 1320 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 1380 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 1440 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 1500 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 1560 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 1620 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 1680 aaaaggccag gaaccg 1696

Claims

1. A recombinant Saccharomyces cerevisiae for producing malonic acid, characterized in that it overexpresses an acetyl-CoA decarboxylase mutant with the amino acid sequence shown in SEQ ID NO.3 and a 3-hydroxyisobutyryl-CoA hydrolase mutant with the amino acid sequence shown in SEQ ID NO.

6.

2. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that the gene encoding the acetyl-CoA decarboxylase mutant is expressed under the initiation of the promoter UAS1, and the gene of the 3-hydroxyisobutyryl-CoA hydrolase mutant is expressed under the initiation of the promoter UAS3.

3. The recombinant Saccharomyces cerevisiae according to claim 2, characterized in that the nucleotide sequence of the promoter UAS1 is as shown in SEQ ID NO.2, and the nucleotide sequence of the promoter UAS3 is as shown in SEQ ID NO.

5.

4. The recombinant Saccharomyces cerevisiae according to any one of claims 1 to 3, characterized in that the recombinant Saccharomyces cerevisiae uses Saccharomyces cerevisiae BY4741 as the starting strain.

5. A method for producing malonic acid, characterized in that the method uses glucose as a carbon source and ferments with the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 4.

6. The method according to claim 5, characterized in that the seed liquid of the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 4 is inoculated into the culture medium at an inoculation amount of 1 to 3% by volume and cultured at 28 to 30 °C for 72 to 168 h.

7. A method for improving the extracellular secretion of malonic acid by Saccharomyces cerevisiae, characterized in that the method is to overexpress an acetyl-CoA decarboxylase mutant with the amino acid sequence shown in SEQ ID NO.3 and a 3-hydroxyisobutyryl-CoA hydrolase mutant with the amino acid sequence shown in SEQ ID NO.

6.

8. The method according to claim 7, characterized in that the gene encoding the acetyl-CoA decarboxylase mutant is expressed under the initiation of the promoter UAS1, and the gene encoding the 3-hydroxyisobutyryl-CoA hydrolase mutant is expressed under the initiation of the promoter UAS3.

9. The method according to claim 8, characterized in that the nucleotide sequence of the promoter UAS1 is as shown in SEQ IDNO.2, and the nucleotide sequence of the promoter UAS3 is as shown in SEQ ID NO.

5.

10. Use of the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 4 or the method according to any one of claims 5 to 9 in the preparation of a product containing malonic acid.

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