CN113583993B - Polyketide synthase PreuA and application thereof in preparation of lichen ruber acid - Google Patents

Abstract

The invention relates to polyketide synthase and application thereof in preparation of bryozoan rubrum, belonging to the technical field of microbial chemistry. The invention clones polyketide synthase PreuA related to synthesis of red-pink lichen acid from photomelasma fungi for the first time, and constructs a yeast mutant strain capable of efficiently producing the red-pink lichen acid based on a saccharomyces cerevisiae heterologous expression technology; and the prepared pinus roxburghii ruff acid is taken as an object, the inhibitory activity of the compound on seven crop pathogenic fungi is researched. Researches show that the Bruguic acid has different degrees of inhibition effects on pathogenic fungi of four crops, namely ring rot of apple, corn microsporum, potato verticillium wilt and sclerotinia sclerotiorum, wherein the Bruguium has strong antagonistic effect (MIC, 25)μg/mL). The invention greatly enriches the production source of the red mead bryozoan and has important scientific value and application prospect for expanding the derivatization way and researching and developing novel biopesticides.

Description

Polyketide synthase PreuA and application thereof in preparation of erucic acid

Technical Field

The invention belongs to the technical field of microbial chemistry, and particularly relates to polyketide synthase and application thereof in preparation of erucic acid.

Background

Fungal polyketides (polyketidesPKs) is a broad classThe important natural product group has diversity of chemical structure and biological activity, and is a high-quality resource library (Keller, 2019) discovered by novel drug lead compounds. Representative fungal PKs are lovastatin (lovastatin) which has cholesterol lowering efficacy, the fungal inhibitor griseofulvin (griseofulvin), the bacterial inhibitor rifamycin (rifamycin), the immunosuppressant mycophenolic acid (mycophenolic acid), actin and the angiogenesis inhibitor cytochalasin E (cytochalasin E), and the like (Hussain et al, 2017). Polyketide synthase (a key enzyme in the biosynthesis of PKs)polyketide synthase, PKS) has complex structure and unique catalytic mechanism, and can provide key tools and chemical molecular probes (Van Lanen) for researching molecular mechanisms of enzyme catalysis, molecular recognition and protein interaction&Shen,2008)。

Bruguic acid (structure shown in figure 1) is a dimer of polyketide bryoid (orsellinic acid, structure shown in figure 1), and is mostly isolated from natural lichen (lichen), and is first isolated from Pyricularia sp in fungi (Umezawa et al, 1974). Research reports that bryozoac acid has biological activities of anti-bacterial, anti-tumor, anti-oxidative and anti-diabetic (Choudhary et al, 2011 lopes et al, 2008 luo et al, 2009;

et al, 2016; seo et al, 2009), it can be used as a potential biological resource for natural antibacterial agents, antioxidants, etc. in the food and pharmaceutical industries. However, commercially available erucic acid red (CAS: 480-56-8) is currently produced by chemical synthesis (Fischer E).&Fischer H.,1913；Koller&Pfeiffer, 1933), the selling price is about 2000 yuan/mg, the yield is low, and the selling price is high, so that the application of the compound fertilizer in the fields of food, medicine and the like is limited. Therefore, how to overcome the defects of the existing preparation technology of the red bryozoac acid is an urgent problem to be solved in the technical field of the existing microbial chemistry.

Disclosure of Invention

The invention aims to overcome the defects of the prior art for preparing the red mead bryotic acid and provides polyketide synthase and application thereof in preparing the red mead bryotic acid.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a polyketide synthase PreuA, the amino acid sequence of which is shown in SEQ ID NO. 1.

In a second aspect, the invention provides a gene encoding the polyketide synthase PreuA of claim 1.

Further, it is preferable that the coding sequence of the gene is a nucleotide sequence shown in SEQ ID NO. 2.

In a third aspect, the present invention provides a recombinant vector containing the above gene.

The fourth aspect of the invention provides a recombinant gene engineering bacterium obtained by transforming the recombinant vector.

The fifth aspect of the invention provides the application of the polyketide synthase PreuA in the preparation of red mealworm acid.

The sixth aspect of the invention provides a method for constructing a yeast mutant strain capable of efficiently producing pinoceric acid, comprising the following steps:

transforming saccharomyces cerevisiae by using the recombinant vector of claim 4 by using a LiAc/PEG4000 mediated transformation method, uniformly coating the transformation liquid on an SC-Ura solid plate, and culturing in an incubator at 30 ℃ to obtain the yeast mutant strain.

The seventh aspect of the invention provides a method for preparing erucic acid, the yeast mutant strain constructed by the construction method comprises the following steps:

inoculating a yeast mutant strain into an SC-Ura liquid culture medium, and culturing at 30 ℃ in a shaking table; adding 1% YPD medium into the cultured 2d, and continuously culturing for 3-4d to obtain fermentation broth;

wherein, 1% of the YPD medium and the SC-Ura liquid medium have the same volume;

step (2), extracting the fermentation liquor obtained by culturing in the step (1) by using ethyl acetate with the same volume, and concentrating the extract liquor to obtain a crude extract;

and (3) purifying the crude extract obtained in the step (2) to obtain the pinoceric acid.

Further, preferably, in the step (3), the crude extract obtained in the step (2) is purified, and the purification method comprises the following steps: sequentially washing the crude extract with n-hexane, chloroform, dichloromethane, ethyl acetate and methanol, recovering ethyl acetate and methanol washing solution, concentrating, and mixing; wherein the volume of n-hexane, chloroform, dichloromethane, ethyl acetate and methanol is one tenth of that of SC-Ura liquid culture medium.

The eighth aspect of the invention provides application of the pinic acid prepared by the preparation method of the pinic acid in preparation of a medicine for resisting pathogenic fungi of crops, which is characterized in that the pathogenic fungi of the crops are apple ring rot, cotton wilt, corn microsporum, potato verticillium wilt, sclerotinia sclerotiorum, rice sheath blight and strawberry black spot.

In the invention, we firstly clone polyketide synthase PreuA related to synthesis of red mealworm acid from photomelania fungi (Preussia isomera) and construct a yeast mutant strain (the yield of the red mealworm acid is about 0.5 g/L) capable of efficiently producing the red mealworm acid based on a saccharomyces cerevisiae heterologous expression technology; the prepared pinkish red moss acid is taken as an object, and the inhibitory activity of the pinkish red moss acid on seven crop pathogenic fungi (ring rot apple, cotton fusarium wilt, corn microsporum, potato verticillium wilt, sclerotinia sclerotiorum, rice sheath blight and strawberry black spot pathogen) is researched. Researches show that the erucic acid has different degrees of inhibition effects on four crop pathogenic fungi, namely ring spot pathogenic bacteria of apple, corn, verticillium wilt of potato and sclerotinia sclerotiorum, wherein the erucic acid has strong antagonistic effect (MIC, 25 mu g/mL) on the ring spot pathogenic bacteria of apple. The invention constructs a yeast mutant strain capable of efficiently producing polyketone molecule pinoresinoic acid for the first time, greatly enriches the production source of the yeast mutant strain, and has important scientific value and application prospect for expanding the derivatization way and researching and developing novel biopesticides.

Compared with the prior art, the invention has the beneficial effects that:

1. at present, commercially available pinoceric acid (CAS: 480-56-8) is produced by a chemical synthesis mode, the selling price is about 2000 yuan/mg, the yield is low, and the selling price is high. The invention constructs a yeast mutant strain (0.5 g/L) capable of efficiently producing the red-pink moss acid based on a heterologous expression technology, enriches the production source of the red-pink moss acid, can greatly improve the yield of the red-pink moss acid, and has considerable application prospect.

2. The invention also researches the inhibiting activity of the pinoceric acid on seven crop pathogenic fungi (ring rot of apple, cotton, corn, potato, rape, rice sheath blight and strawberry black spot). Researches find that the erucic acid has strong antagonistic action (MIC, 25 mu g/mL) on the ring rot of apple, and is expected to develop novel biological pesticides.

Drawings

FIG. 1 is a chemical structural formula of bryozoac acid (lecanoric acid) and bryoic acid (orsellinic acid); wherein, (a) is bryozoac acid and (b) is bryozoac acid;

FIG. 2 is a plasmid map of a polyketide synthase PreuA heterologous expression vector YEpADH2 p-URA-PreuA;

FIG. 3 is the restriction enzyme digestion verification diagram of the polyketide synthase PreuA heterologous expression vector YEpADH2 p-URA-PreuA; wherein, M, trans 2K marker; line 1&2, vector YEpADH2p-URA-PreuA

FIG. 4 is the metabolite analysis of the polyketide synthase PreuA Saccharomyces cerevisiae heterologous expression mutant strain; wherein, (a) is empty vector Saccharomyces cerevisiae, (b) polyketide synthase PreuA Saccharomyces cerevisiae mutant strain;

FIG. 5 is a diagram showing the ultraviolet absorption of a target metabolite of a polyketide synthase PreuA Saccharomyces cerevisiae mutant strain;

FIG. 6 is a high resolution mass spectrogram of a target metabolite of a polyketide synthase PreuA Saccharomyces cerevisiae mutant strain; wherein, (a) is in negative ion mode, (b) is in positive ion mode;

FIG. 7 shows a compound bryozoan ¹ H nuclear magnetic spectrogram (deuterated methanol, 500 MHz);

FIG. 8 shows the compound bryozoanic acid red powder ¹³ C nuclear magnetic spectrum (deuterated methanol, 125 MHz).

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, but are all conventional products available by purchase.

In the invention, we clone polyketide synthase PreuA related to synthesis of lichen ruber from photomelania isomera (MK 300824.1 in GenBank accession number, and the strain is currently stored in the subject group of professor of the young dragon of the institute of medicine of the university of the south and the middle provinces), and construct a yeast mutant strain capable of efficiently producing polyketide molecular lichen ruber acid (the molecular structure of the compound is shown in figure 1) based on a saccharomyces cerevisiae heterologous expression technology. The general procedure of this experiment was: firstly, constructing a heterologous expression vector YEpADH2p-URA-PreuA of polyketide synthase PreuA based on the principle of homologous recombination (a plasmid map is shown in figure 2); then, transforming the constructed heterologous expression vector into saccharomyces cerevisiae by adopting a PEG4000/LiAc transformation method to obtain a positive yeast transformation strain, analyzing the metabolite of the positive yeast transformation strain and carrying out structural identification to find that the constructed yeast mutant strain can produce the pinoceric acid (about 0.5 g/L) with high yield; finally, the prepared pinosolic acid is taken as an object, and the inhibitory activity of the pinosolic acid on seven crop pathogenic fungi (ring rot of apple, cotton fusarium wilt, corn microsporum, potato verticillium wilt, sclerotinia sclerotiorum, rice sheath blight and strawberry black spot) is researched (the experimental result is shown in table 3). The specific experimental procedure is as follows.

1. Construction of polyketide synthase PreuA heterologous expression vector YEpADH2p-URA-PreuA

1.1 cloning of the polyketide synthase Gene preuA: the full length of the mRNA sequence of polyketide synthase preuA is 6594-bp, as shown in SEQ ID NO.2, the mRNA sequence is divided into 3 segments for cloning, and cloning primers are shown in Table 1.

PCR reaction system configuration (100. Mu.L): ddH ₂ O55.5 mu L;5 × Phusion HF buffer 20 μ L; dNTPs (2.5 mmol/L) 8. Mu.L; forward primer (10 pmol/. Mu.L) 5. Mu.L; reverse primer (10 pmol/. Mu.L) 5. Mu.L; DMSO 3 mu L; phusion DNA Ploymerase (2U/. Mu.L) 1. Mu.L; cDNA 2.5. Mu.L. The PCR procedure was: 3min at 98 ℃; 34cycles in total, 30s at 98 ℃, 30s at 58 ℃ and 2min at 72 ℃; 10min at 72 ℃.

Note: primers PreuA-E-1F and PreuA-E-1R are used in the first section; the second primer is PreuA-E-2F or PreuA-E-2R; the third primer segment is PreuA-E-3F or PreuA-E-3R.

The DNA fragment was recovered by using Thermo Scientific GeneJET gel recovery kit (K0692), the detailed procedures of which are described in the specification.

1.2 preparation of heterologous expression vector fragments: fragment 4 (6.2-kb) was obtained by digesting the plasmid YEpADH2p-FLAG-URA with restriction enzymes Nde I/Pme I. The plasmid YEpADH2p-FLAG-URA was presented to professor Istvan Molnar, university of Arizona, academy of agriculture and Life sciences.

Enzyme digestion system configuration (180. Mu.L): ddH ₂ O132. Mu.L, 10 XFastdigest Green Buffer 18. Mu.L, plasmid YEpADH2p-FLAG-URA 18. Mu.L, restriction enzyme (1) (Nde I) 6. Mu.L, restriction enzyme (2) (Pme I) 6. Mu.L. After digestion at 37 ℃ for 2h, the Thermo Scientific GeneJET gel recovery kit (K0692) was used, for details see the description.

1.3 construction and transformation of heterologous expression vectors: by adopting SE seamless cloning and assembly kit (Chun union organism, ZC 231), the preuA gene fragment 1-3 prepared by 1.1 and the vector fragment 4 prepared by 1.2 are recombined to construct a heterologous expression vector YEpADH2p-URA-PreuA.

Recombinant reaction system configuration (10 μ L): 5 XSE Cloning Buffer 2. Mu.L; fragment 1. Mu.L; fragment 2. Mu.L; fragment 3. Mu.L; fragment 4. Mu.L; SE recombination 1. Mu.L. The reaction was carried out at 37 ℃ for 0.5h, on ice for 2min, and T1 PhageResistant competent cells (Token organism, ZC 102) were transformed for the specific steps described in the specification.

1.4 extraction and validation of heterologous expression plasmids: the extraction of the Escherichia coli plasmid adopts Axygen ^○R AxyPrep Plasmid Miniprep Kit (AP-MN-P-250), see the description thereof for specific steps. The constructed heterologous expression vector is subjected to Eco RI/Eco RV enzyme digestion verification (7877/2530/1382/1067-bp) and sequencing, and the correctness of the vector sequence is confirmed (see the enzyme digestion verification of the vector in figure 3).

Enzyme digestion verification system configuration (10 μ L): ddH ₂ O5. Mu.L, 10 XFastdigest Green Buffer 1. Mu.L, heterologous expression plasmid 3. Mu.L, restriction enzyme (1) (Eco RI) 0.5. Mu.L, restriction enzyme (2) (Eco RV) 0.5. Mu.L. Carrying out enzyme digestion reaction at 37 ℃ for 0.5h, and then carrying out gel running.

2. Construction of polyketide synthase PreuA saccharomyces cerevisiae heterologous expression mutant strain and analysis and identification of metabolite thereof

2.1 construction of heterologous expression mutant of polyketide synthase PreuA Saccharomyces cerevisiae: the vector YEpADH2p-URA-PreuA is transformed into saccharomyces cerevisiae by a LiAc/PEG4000 mediated transformation method.

3600r/min of the prepared 100 mu L of yeast competent cells are centrifuged for 5min, the supernatant is discarded, and then 78 mu L of ddH is added into a tube in sequence ₂ O, 36. Mu.L LiAc (1 mol/L), 240. Mu.L PEG4000 solution (50% (w/v)), 3. Mu.L heterologous expression vector (YEpADH 2 p-URA-PreuA), and mixing the mixture uniformly; standing at 30 deg.C for 30min, and transferring to 42 deg.C for 30min; 3600r/min of the transformation liquid is centrifuged for 5min, the supernatant is discarded, 200 mu L of sterile water is used for resuspending the thalli, then the thalli are evenly coated on an SC-Ura solid plate, and the thalli are cultured for 2-4d in an incubator at 30 ℃. After the yeast transformed strain grows out, the yeast transformed strain is fermented and analyzed for metabolites.

Configuration of SC-Ura solid plate: YNB 6.7g/L, glucose 20g/L,0.77g/L-ura Do Supplement (Clontech, USA), 15g agar, 121 ℃ sterilization for 15min.

2.2 minor fermentation of Yeast mutants: two clones were picked randomly on the transformed plates, streaked on SC-Ura solid plates, and the plates were incubated overnight in a 30 ℃ incubator. Inoculating proper amount of thallus into 25mL SC-Ura liquid culture medium (using 125mL triangular flask), culturing at 30 deg.C for 1d at 220 r/min; at 2d, 25mL of 1% YPD medium was added, and the culture was continued for 3d, followed by secondary metabolite analysis.

Preparation of SC-Ura liquid culture Medium: YNB 6.7g/L, glucose 20g/L,0.77g/L-ura Do Supplement (Clontech, USA), sterilized at 121 ℃ for 15min.

1% preparation of YPD Medium: 10g/L yeast extract, 20g/L peptone and 10g/L glucose, and sterilizing at 121 ℃ for 15min.

2.3 analysis of metabolites of yeast mutants (chromatogram shown in FIG. 4): and extracting the saccharomyces cerevisiae fermentation liquor by using ethyl acetate with the same volume, and putting the ethyl acetate phase into a round-bottom flask for rotary evaporation. After the solvent was suspended, 800. Mu.L of methanol was added to dissolve the metabolite for LC-MS analysis. The chromatographic conditions were: reverse-phase C ₁₈ column (Kromasil 100-5-C18, 4.6X 250mm,5 μm); the mobile phase was water (a)/methanol (B, with 0.1% glacial acetic acid); the flow rate is 0.8mL/min; the sample injection volume is 5 mu L; a DAD detector with a detection wavelength of 300nm; the column temperature is 25 ℃; with gradient elution, the procedure was to start mobile phase ratio 5% B holding 5min, linearly increasing B from 5% to 100% within 30min, holding 100% B10 min, then linearly decreasing B from 100% to 5% within 1min, and holding 5% B4 min. The mass spectrum conditions were: a Q active detector; an electrospray ion (ESI) source; switching and collecting positive ions and negative ions; a first mass spectrum full scan (150-1000 m/z); the spraying voltage is 3.8kv; the temperature of the ion transmission tube is 325 ℃; a sheath gas flow rate 40arb; auxiliary gas flow rate 20arb; the auxiliary gas is heated to 350 ℃.

2.4 separation and purification of yeast mutant strain target metabolite: fermenting 3L yeast mutant strain capable of producing target metabolite in large scale with the same fermentation and extraction methods as 2.2 and 2.3 respectively to obtain 2.1g crude extract. And (3) washing the crude extract by sequentially adopting 150mL of normal hexane, chloroform, dichloromethane, ethyl acetate and methanol, recovering ethyl acetate and methanol washing liquid, concentrating and combining to obtain about 1.5g of the target monomeric compound.

2.5 structural analysis of yeast mutant strain target metabolite: the target metabolite is white needle crystal, the maximum ultraviolet absorption wavelength is 320nm (figure 5), and the structure is processed by High Resolution Mass Spectrum (HRMS) and one-dimensional nuclear magnetic resonance spectrum ( ¹ H-NMR、 ¹³ C-NMR), the molecular ion peak m/z by HRMS is 317.06784[ m-H ]]-and 319.08127[ m ] +H]+ (FIG. 6) molecular formula is C ₁₆ H ₁₄ O ₇ The nuclear magnetic data required for the structural analysis are shown in fig. 7 to 8 and table 2, and the structure is finally analyzed to be pinoceric acid (the molecular structure is shown in fig. 1).

3. Activity test of red pink bryozoac acid against crop pathogenic fungi

3.1 Broth orchidic acid and ketoconazole (positive control) were prepared as 1mg/mL stock solutions.

3.2 activating pathogenic fungi (ring rot of apple, blight of cotton, corn microsporum, verticillium wilt of potato, sclerotinia sclerotiorum, rice sheath blight and strawberry black spot) of target crops in a PDB (potato dextrose broth) culture medium for 2-3 days, and adding 1mL of bacterial liquid into 100mL of the PDB culture medium after activation to obtain diluted bacterial liquid.

Preparing a PDB culture medium: 23g of PDB powder (Beijing Oobozoxin Biotechnology, inc.) was added with 1000mL of distilled water and autoclaved at 121 ℃ for 15min.

3.3 taking ketoconazole as a positive control, adding 2uL of a target compound into 198uL of target bacterial liquid, and testing the antagonistic activity of the pinoceric acid on the seven crop pathogenic fungi by adopting a two-fold dilution method. (the results of the activity are shown in Table 3)

4. Conclusion

Experimental results show that a yeast mutant strain (about 0.5 g/L) capable of efficiently producing the red-pink moss acid is constructed by cloning polyketide synthase PreuA related to the synthesis of the red-pink moss acid from photomelania isomera for the first time and based on a saccharomyces cerevisiae heterologous expression technology; the prepared pinoceric acid has different degrees of inhibition effects on pathogenic fungi of four crops, namely ring rot of apple, corn microsporum, potato verticillium wilt and sclerotinia sclerotiorum, wherein the pinoceric acid has strong antagonistic effect (MIC, 25 mu g/mL) on the ring rot of apple.

Table 1: cloning-related primer information of polyketide synthase gene preuA

Table 2: of the compound bryozoan ¹ H and ¹³ c Nuclear magnetic data (500 MHz, CD3OD, delta in ppm, J in Hz)

No.	δC	δH(m,JHH,area)
			1	170.87,C
2	105.43,C
			3	144.90,C
4	112.97,CH	6.27(d,J＝3.1,1H)
			5	166.73,C
6	102.02,CH	6.20(d,J＝2.4,1H)
			7	165.12,C
8	24.44,CH 3	2.54(s,3H)
			1’	174.50,C
2’	117.30,C
			3’	144.75,C
4’	113.07,CH	6.63(d,J＝2.0,1H)
			5’	155.21,C
6’	109.41,CH	6.60(d,J＝2.3,1H)
			7’	164.78,C
8’	23.85,CH 3	2.56(s,3H)

Table 3: inhibiting activity of bryozoac acid on seven crop pathogenic fungi (Ketoconazole is used as positive control)

The polyketone molecule pinoceric acid has biological activities of antibiosis, antitumor, antioxidation, diabetes resistance and the like, so the polyketone molecule pinoceric acid can be used as a potential biological resource of natural antibacterial agents, antioxidants and the like in food and medicine industries. However, at present, commercially available pinkish moss acid (CAS: 480-56-8) is produced by a chemical synthesis mode, the selling price is about 2000 yuan/mg, the yield is low, and the selling price is high, so that the application of the pinkish moss acid in the fields of food, medicine and the like is limited. In the invention, a yeast mutant strain (about 0.5 g/L) capable of efficiently producing the red mealworm acid is constructed by cloning from photomelania isomerous fungus (Preussia isomerora) to polyketide synthase PreuA related to the synthesis of the red mealworm acid for the first time and based on a saccharomyces cerevisiae heterologous expression technology; the prepared pinoceric acid is taken as an object, and the antagonistic action of the pinoceric acid on a plurality of crop pathogenic fungi is researched. Researches show that the erucic acid has different degrees of inhibition effects on pathogenic fungi of four crops, namely ring rot of apple, corn microsporum, potato verticillium wilt and sclerotinia sclerotiorum, wherein the erucic acid has strong antagonistic effect (MIC of 25 mu g/mL) on the ring rot of apple. The invention greatly enriches the production source of the bryozoan, and has important scientific value and application prospect for expanding the derivatization way and researching and developing novel biopesticides.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> Zhongnan national university

<120> polyketide synthase PreuA and application thereof in preparation of lichic acid

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Met Ser Asn Ser Thr Arg Asp Tyr Pro Ile Ser Ala Ala Phe Phe Cys

1 5 10 15

Pro Gln Ser Arg Ala Pro Pro Ala Glu Tyr Leu His Ala Leu Tyr Ser

20 25 30

Phe Leu Ser Gln Asn Thr Leu Gly Lys Ala Phe Leu Arg His Ile Ala

35 40 45

Ser Leu Asp Glu Val Trp Pro Ile Phe Ser Glu Ala Arg Asp Asp Ile

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Leu Arg Leu Pro Asp Ala Arg Gln Asn Ile Asn Val Leu Val Asp Trp

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Ala Lys Gly Gly Ser Ser Thr Pro Ile Ala Glu Ala Arg Ser Gly Val

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Ile Ala Leu Pro Ser Val Phe Ile Val Gln Leu Gly Gln Tyr Phe Arg

100 105 110

Tyr Leu Glu Ala Asn Arg Leu Ser His Gly Asp Phe Ile Gly Gln Leu

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Lys Asp Ile Gly Gly Val His Gly Tyr Cys Gly Gly Ala Ala Ala Ala

130 135 140

Leu Ser Val Ala Cys Ala Ala Asp Glu Thr Gln Leu Ile Asp His Ala

145 150 155 160

Ala Val Leu Leu Arg Leu Phe Val Gly Ile Gly Cys Cys Ile Glu Ala

165 170 175

Val Asp Asp Trp Thr Thr Thr Glu Ser Thr Val Ile Ala Cys Arg Leu

180 185 190

Lys Tyr Glu Gly Gln Gly Asp Glu Leu Cys Ser Arg Phe Pro Gly Thr

195 200 205

Tyr Val Ser Ala Ile Thr Glu Pro Lys Ser Ile Ser Ile Thr Gly Asn

210 215 220

Ala Arg Thr Leu Ser Glu Leu Phe Asp Tyr Ala Val Gly Leu Gly Leu

225 230 235 240

Pro Thr His Lys Met Glu Ile Thr Gly Lys Ala His Asn Pro Glu Asn

245 250 255

Ala Glu Leu Ala Lys Asp Phe Ile Asn Leu Tyr Arg Arg Thr Pro Ala

260 265 270

Leu Gln Leu Pro Pro Thr Phe Lys Leu Gln Ala Thr Val Arg Ser Asn

275 280 285

Arg Thr Ala Glu Lys Leu Thr Asn Glu Gly Ile Ile Glu Asp Met Ile

290 295 300

Thr Met Ile Ile Ala Ser Gln Cys Asp Trp Asn Thr Leu Leu Thr Arg

305 310 315 320

Val Ala Glu Asp Met Lys Val Ser Gly Arg Pro Phe His Lys Met Val

325 330 335

Ser Phe Gly Met Asn Asp Cys Val Pro Val Thr Pro Phe Asn Arg Gln

340 345 350

Arg Leu Lys Thr Thr Lys Phe Glu Ala His Val Leu Ile Glu Pro Leu

355 360 365

Lys Pro Ser Arg Ile Ser Ala Ala Gln Tyr Pro Thr Phe Ser Asp Asp

370 375 380

Ala Ile Ala Ile Thr Gly Ala Ser Leu Arg Leu Pro Gly Ala Asn Asn

385 390 395 400

Leu Asp Glu Leu Trp Asp Leu Ile Ser Lys Gly Thr Asp Cys His Arg

405 410 415

Glu Ile Pro Lys Asp Arg Phe Asp Pro His Asn Ile Tyr Arg Thr Ser

420 425 430

Gln Ser Gly Phe Ser Lys Ala Gln Lys Tyr Phe Gly Asn Phe Leu Glu

435 440 445

Asp Ile Lys Gly Phe Asp Arg Ala Tyr Phe Ser Met Gly Val Arg Glu

450 455 460

Ala Ala Asn Ile Asp Pro Gln Gln Arg Leu Leu Leu Glu Leu Ala Val

465 470 475 480

Glu Ala Leu Glu Ala Ser Gly Tyr Leu Ala Asn His Val Arg Glu Ala

485 490 495

Gly Asp Pro Val Gly Cys Phe Val Gly Ala Ser Phe Ile Glu Tyr Leu

500 505 510

Glu Asn Thr Gly Ala His Pro Pro Thr Ala Tyr Thr Ala Pro Gly Thr

515 520 525

Ile Arg Ala Phe Leu Cys Gly Arg Leu Ser Tyr Tyr Phe Gly Trp Thr

530 535 540

Ala Pro Ala Glu Val Ile Asp Thr Ala Cys Ser Ala Ser Met Val Ala

545 550 555 560

Ile Asn Arg Ala Val Lys Ser Ile Gln Ala Gly Glu Cys Glu Met Ala

565 570 575

Leu Ala Gly Gly Val Asn Leu Ile Thr Gly Met Asn Asn Tyr Leu Asp

580 585 590

Leu Ala Lys Ala Gly Phe Leu Ser Pro Thr Gly Gln Cys Lys Pro Phe

595 600 605

Asp Gln Ser Gly Asp Gly Tyr Cys Arg Ser Asp Gly Ala Gly Phe Val

610 615 620

Val Leu Lys Lys Leu Ser Gln Ala Leu Val Asn Gly Asp Pro Ile Met

625 630 635 640

Gly Val Ile Pro Ser Ile Glu Thr Asn Gln Gly Gly Leu Ser Gly Ser

645 650 655

Leu Thr Val Pro Ser Ser Thr Ala Leu Gln Ala Leu Tyr Lys Arg Val

660 665 670

Leu Ser Lys Ser Gly Leu Glu Pro Ala Gln Ile Thr Tyr Val Glu Ala

675 680 685

His Gly Thr Gly Thr Gln Ala Gly Asp Pro Ile Glu Val Glu Ser Val

690 695 700

Arg Ala Val Leu Gly Asp Pro Thr Arg Ala His Ser Leu Ser Leu Gly

705 710 715 720

Ser Val Lys Gly Asn Ile Gly His Cys Glu Thr Gly Ala Gly Val Ala

725 730 735

Gly Leu Leu Lys Val Leu Ala Met Ile Lys His Gly Gly Ile Pro Pro

740 745 750

Leu Ala Ser His Lys Ala Leu Asn Pro Lys Ile Pro Ala Leu Glu Thr

755 760 765

His His Met Glu Ile Ala Lys Gln Leu Lys Pro Trp Asp Val Pro Leu

770 775 780

Arg Ala Ala Phe Val Asn Ser Tyr Gly Ala Ala Gly Ser Asn Ala Ala

785 790 795 800

Val Ile Cys Val Glu Pro Pro Pro Val Val Thr Asp Gly Ser Ser Leu

805 810 815

Ile Gly Thr Glu Pro Gln Lys Val Thr Leu Pro Val Ile Val Ser Gly

820 825 830

Ala Thr Arg Lys Ser Leu Val Leu Asn Ala Arg Ala Leu Ala Ser Tyr

835 840 845

Leu Ser Gln Asp Gly Ser His Leu Ser Ile His Asp Val Ala Phe Thr

850 855 860

Val Asn Gln Arg Arg Lys Arg Asn Arg Phe Cys Ala Glu Val Ser Gly

865 870 875 880

Thr Asp Leu Pro Ser Leu Val Gln Ser Leu Arg Ala Val Asp Ser Pro

885 890 895

Ser Phe Glu Ser Pro Gly Lys Ser Lys Pro Val Val Leu Val Phe Ser

900 905 910

Gly Gln Asn Thr Asn Ala Val Ala Leu Asp Arg Thr Ile Tyr Asp Thr

915 920 925

Tyr Pro Val Phe Lys Ala Tyr Ile Asp Ala Cys Asp Ser Glu Ile Val

930 935 940

Lys Leu Gly Phe Pro Ser Ile Met Glu Ala Ile Phe Gln Lys Glu Pro

945 950 955 960

Ile Ser Thr Ala Val Ala Leu Gln Gly Ser Ile Phe Ala Met Gln Tyr

965 970 975

Ala Cys Ala Arg Ser Trp Ile Asp Ala Gly Leu Lys Pro Arg Ala Ile

980 985 990

Ile Gly His Ser Phe Gly Glu Leu Thr Ala Leu Ala Val Ser Gly Ala

995 1000 1005

Leu Ser Leu Ala Asp Ser Leu Lys Leu Val Thr Cys Arg Gly His Leu

1010 1015 1020

Ile Asp Thr Lys Trp Gly Glu Glu Arg Gly Gly Met Leu Val Ile His

1025 1030 1035 1040

Ala Asp Val Ala Thr Val Glu Arg Phe Gln Ser Arg Phe Lys Ala Gln

1045 1050 1055

His Asp Gly Ala Glu Leu Glu Ile Ala Cys Tyr Asn Ser Pro Thr Thr

1060 1065 1070

Thr Val Val Ala Gly Pro Val Ala Tyr Met Asp Ala Ala Glu Gln Met

1075 1080 1085

Leu Ala Thr Asp Pro Asp Phe Gln Gly Leu Arg Lys Leu Arg Ile Val

1090 1095 1100

Thr Ser Asn Ala Phe His Ser Ser Leu Ser Asp Pro Ile Leu Ala Asp

1105 1110 1115 1120

Leu Asp Ser Met Ala Asp Thr Leu Thr Trp Asn Glu Pro Ser Ile Pro

1125 1130 1135

Leu Glu Ala Cys Thr Ser Glu Gly Leu Ala Ser Ile Lys Glu Trp Ser

1140 1145 1150

Ala Ser Arg His Thr Arg Gly Ser Val Tyr Phe Thr Lys Ala Val Glu

1155 1160 1165

Arg Ile Glu Gly Arg Leu Gly Ala Cys Ile Trp Val Glu Ala Gly Leu

1170 1175 1180

Asp Ser Ala Ile Ile Ala Met Ala Arg Lys Ala Ser Ser Lys Pro Asp

1185 1190 1195 1200

Ser Gln Val Phe Gln Ser Val Ser Thr Lys Ala Gly Ala Thr Ser Phe

1205 1210 1215

Ile Asp Gly Ile Val Asn Asn Leu Trp Arg Gln Gly Val Pro Leu Ser

1220 1225 1230

His Leu Asn Ala Leu Ser Ala Thr Val Lys Pro Asn Pro Val Trp Leu

1235 1240 1245

Pro Pro Tyr Gln Phe Glu Arg Glu Gln His Trp Thr Glu His Ile Asp

1250 1255 1260

Arg Ala Thr Glu Ala Ser Gln Ala Ser Thr Thr Ser Asp Thr Ile Gln

1265 1270 1275 1280

Ser Thr Pro Thr Gln Thr Val Gln Ser Pro Pro Lys Leu Ile Ser Arg

1285 1290 1295

Leu Ala Ser Leu Gln Tyr Gln Ile Asn Thr Gln Cys Glu Arg Phe Gln

1300 1305 1310

Lys Ile Thr Glu Gly His Ala Val Leu Tyr Glu Pro Leu Cys Pro Ala

1315 1320 1325

Ser Leu Tyr Met Glu Cys Val Val Met Ala Leu Gln Glu Leu Ala Gly

1330 1335 1340

Asp Leu Gly Ser Arg Thr Leu Asp Phe Glu Asn Leu Asp Phe His Ala

1345 1350 1355 1360

Gly Leu Gly Leu Gln Thr Asp Arg Arg Val Leu Leu Asp Leu Glu Glu

1365 1370 1375

Ala Arg Pro His Ser Trp Thr Phe Lys Val Gln Ser Thr Lys Ala Gly

1380 1385 1390

Ser Ser Arg Ser Leu Leu His Cys Ser Gly Arg Val Ile Leu Thr Glu

1395 1400 1405

Ser Ser Val Pro Thr Thr Phe Gln Arg Leu Val Asp Gly Pro Arg Ser

1410 1415 1420

Arg Leu Asp Gln Asp Lys Asp Ala Glu Lys Leu Met Ser Ser Arg Ala

1425 1430 1435 1440

Tyr Gly Leu Phe Ser Asn Ile Met Thr Tyr Ser Glu Phe Leu Lys Pro

1445 1450 1455

Ile Ser Ser Ile Ile Leu Arg Glu Asn Glu Ser Leu Ala Thr Ile Lys

1460 1465 1470

Leu Pro Pro Asn Gln Pro Gly Leu His Glu Ser Thr Ala Trp Lys Arg

1475 1480 1485

Cys Asp Ala Val Phe Leu Asp Gly Phe Ile Ser Ser Ser Gly Leu Leu

1490 1495 1500

Leu Asn Ser Ser Ser Val Val Gln Ser Gly His Val Leu Ile Ala Val

1505 1510 1515 1520

Gly Val Glu Arg Ala Ile Leu Thr Ala Ala Phe Gln Ala Ser Leu Ala

1525 1530 1535

Ser Ser Trp Gln Ala Tyr Ala Thr Phe Thr Met Val Gly Glu Thr His

1540 1545 1550

Ala Leu Cys Asp Val Phe Ala Cys Thr Pro Asp Gly Glu Val Val Ala

1555 1560 1565

Met Met Thr Gly Val Arg Phe Asn Lys Met Glu Ile Ser Lys Leu Ala

1570 1575 1580

Lys Ser Leu Ser Ser Val Asn Ala Ser Ser Pro Thr Gly Gly Arg Thr

1585 1590 1595 1600

Gln Pro Pro Ala Ala Pro Lys Thr Gln Ala Gln Pro Met Ala Ser Arg

1605 1610 1615

Pro Ser Pro Thr Pro Leu Gln Val Ser Phe Ala Thr Ala Glu Pro Ala

1620 1625 1630

Ala Pro Glu Pro Val Gln Gln Ser Thr Ala Ala Leu Ala Arg Asn Asp

1635 1640 1645

Ile Gly Pro Val Leu Lys Ser Leu Ile Ser Asn Tyr Thr Gly Leu Ile

1650 1655 1660

Glu Glu Asp Val Ser Glu Asp Ser Pro Leu Val Asp Leu Gly Leu Asp

1665 1670 1675 1680

Ser Leu Ser Ser Val Glu Phe Ala Ser Glu Ile Gly Thr Lys Phe Gly

1685 1690 1695

Val Thr Leu Asp Ala Asp Thr Val Gly Asp Leu Thr Leu His Ser Leu

1700 1705 1710

Cys Gln Arg Leu Ser Gly Thr Ser Asn Val Val Ser Gln Lys Met Ser

1715 1720 1725

Glu Thr Pro Ala Ala Ala Pro Val Lys Glu Leu Ile Glu Thr Val Pro

1730 1735 1740

Ser Pro Ile Val Thr Phe Ser Ser Pro Val Ser Asn Ser Ile Thr Ser

1745 1750 1755 1760

Val Leu Lys Ser Leu Leu Gly Ser Tyr Thr Gly Leu Gln Glu Glu Asp

1765 1770 1775

Met Pro Asp Asp Val Pro Leu Ile Asp Leu Gly Leu Asp Ser Leu Ser

1780 1785 1790

Ser Val Glu Phe Ala Ser Glu Leu Asn Asp Lys Met Gly Ala Asp Ile

1795 1800 1805

Asp Ser Ala Val Val Ala Asp Met Thr Leu Ser Ala Leu Glu Gln Gln

1810 1815 1820

Leu Gly Ala Ser Ala Thr Pro Pro Ser Thr Thr Gly Ser Ser Thr Pro

1825 1830 1835 1840

Gly Asp Ile Ser Thr Ala Ala Thr Thr Pro Tyr Ala Thr Gly Ala Ser

1845 1850 1855

Thr Pro Asp Tyr Leu Val His Gly Asn Lys Pro Ser Ile Ser Asn Gly

1860 1865 1870

Val Val Ala Ala Lys Asp Ser Tyr Gln Val Lys Thr Val Glu Tyr Lys

1875 1880 1885

Arg Val Ser Gly Val Pro Ile His Ala Asp Ile Tyr Val Pro Leu Val

1890 1895 1900

Gln Arg Val Ser Pro Met Pro Leu Ala Leu Met Ile His Gly Gly Gly

1905 1910 1915 1920

His Met Thr Leu Ser Arg Lys Ala Val Arg Pro Thr Gln Ala Lys Tyr

1925 1930 1935

Leu Leu Ser His Gly Phe Leu Pro Ile Ser Ile Asp Tyr Arg Leu Cys

1940 1945 1950

Pro Glu Val Asn Leu Ile Asp Gly Pro Ile Ala Asp Val Arg Asp Ala

1955 1960 1965

Tyr Val Trp Ala Cys Gln Asn Leu Gly Thr His Leu Ala Glu His Ser

1970 1975 1980

Ile Ser Val Asp Gly Gly Arg Val Val Val Val Gly Trp Ser Thr Gly

1985 1990 1995 2000

Gly His Leu Ala Met Ser Leu Gly Trp Ser Leu Glu Glu Ala Gly Val

2005 2010 2015

Pro Pro Pro Lys Ala Val Leu Ser Phe Tyr Ala Pro Val Asp Phe Glu

2020 2025 2030

Ser Gly Glu Leu Asp Asn Gln Lys Asn Pro Ala Leu Pro Lys Pro Arg

2035 2040 2045

Met Thr Leu Asp Gln Ile Thr Lys Ala Leu Pro Arg Thr Pro Val Thr

2050 2055 2060

Gln Tyr Gly Ala Ser Ser Thr Asp Glu Thr Asn Leu Gly Trp Leu His

2065 2070 2075 2080

Pro Gly Asp Pro Arg Ser Glu Leu Leu Leu His Val Phe His Ser Asp

2085 2090 2095

Ile Gly Leu Pro Leu Ile Leu His Gly Leu Pro Ile Ser Gly Ser Gly

2100 2105 2110

Arg Pro Ser Pro Ser Leu Val Ala Ser Ile Ser Pro Leu Ala Arg Leu

2115 2120 2125

Arg Asn Gly Ser Tyr Thr Ile Pro Thr Phe Ile Ile His Gly Thr Lys

2130 2135 2140

Asp Val Ile Ala Pro Tyr Ala Ala Ala Glu Arg Phe Val Lys Ile Met

2145 2150 2155 2160

Ser Glu Lys Gly Val Lys Ser Gly Phe Leu Ser Leu Ser Gly Thr Gly

2165 2170 2175

His Val Phe Asp Val Thr Met Lys Pro Asp Ser Lys Gly Trp Glu Asp

2180 2185 2190

Lys Val Lys Pro Gly Leu Asp Phe Leu Ile Gln Asn Ala

2195 2200 2205

<210> 1

<211> 6618

<212> DNA/RNA

<213> Artificial sequence ()

<400> 1

atgtctaatt ctacacgtga ctatcccata tccgcggcct tcttttgtcc acagagtagg 60

gcacccccag cggaatacct ccacgccctt tattcttttc tcagccaaaa cacccttgga 120

aaggctttcc ttcgccacat tgcgtccctt gacgaagtct ggcccatctt ctccgaagcg 180

agggacgata ttctcagact gcctgatgcg cgccaaaata tcaatgtgct cgtcgattgg 240

gcaaagggtg gctcttccac tcccattgct gaagcccgat ctggagtgat cgctcttcca 300

tcagtcttca ttgtccagct tggacaatac tttcgttacc tcgaggcaaa tcggctatcc 360

cacggcgact ttatcggcca gctcaaggat attggtggtg ttcatggata ctgtggaggc 420

gctgctgcag cactctccgt tgcgtgtgca gccgatgaga cccagctcat tgaccatgct 480

gcagtgttgc tacgtttatt cgttggtatc ggctgttgca tcgaggcagt ggatgattgg 540

accacaactg agtccactgt cattgcctgc cgtctcaaat acgaaggaca gggtgatgaa 600

ctctgtagcc gatttccagg tacatacgtc tctgccatta cagagcctaa gtcaataagc 660

attactggca atgcccgcac actgtcagag ctttttgatt atgcggtggg ccttggactt 720

cccacccata agatggaaat caccggtaaa gcacacaacc cagaaaacgc tgaactggcc 780

aaagatttta tcaacttata tcgtcggact ccggctctgc aactgccccc taccttcaag 840

ttgcaagcaa cagtgcgctc aaatcgtact gcggagaagt tgaccaacga aggcattatt 900

gaggacatga tcacgatgat tatagcgtcc caatgtgact ggaacacgct tctgacaaga 960

gtcgccgagg acatgaaggt ctctggtcga ccatttcaca agatggtgtc ctttgggatg 1020

aacgattgcg ttcctgtaac acctttcaat cgacagcggc ttaagaccac caaatttgag 1080

gctcatgtcc tcatcgagcc cctgaagcct tcgcgtattt ccgctgcgca gtatcctacc 1140

ttctcagatg atgcaatcgc cataacaggc gcttctttgc gcctaccagg tgcaaataat 1200

cttgacgaat tatgggacct gatctccaaa ggtaccgact gtcacaggga aataccaaaa 1260

gacagattcg atccgcacaa catttatcgg acctctcaga gtggcttcag caaagcccag 1320

aagtattttg gcaactttct tgaggacatc aaagggttcg atagggcgta tttcagcatg 1380

ggtgtacggg aagctgccaa catcgaccca caacagcgat tactcctgga gcttgcagtt 1440

gaagcccttg aggcaagtgg ctatctcgcc aaccatgtac gagaagctgg tgacccggtc 1500

ggctgctttg ttggagccag ctttatagaa tacctggaaa atactggtgc ccaccctcca 1560

acagcttaca ccgctcccgg aaccatcaga gcctttttat gtggcagact cagctattac 1620

tttggatgga cagccccagc ggaagttatc gatactgcct gctcggcttc catggtcgcg 1680

atcaaccgcg cagtcaaatc tatccaagca ggagaatgtg agatggcgct tgctggaggc 1740

gtaaacctga tcactggaat gaacaactat ctcgatttgg ccaaagccgg atttctgagc 1800

ccaacaggcc aatgcaagcc attcgaccaa tctggagatg gctattgtcg ctctgatgga 1860

gcaggatttg ttgttctgaa gaagttgtcg caggctctgg taaatggcga tccgatcatg 1920

ggtgttattc ccagtatcga aaccaatcaa ggtggtctat ccgggtcact cactgttcca 1980

tcatccactg cactacaagc actttacaaa cgcgtccttt cgaaatctgg tctggaacct 2040

gcacagatta cctatgttga agcccatggt acaggaaccc aagcaggtga cccgattgag 2100

gtggagagcg ttcgtgcagt tctcggagac cccacgcgag cccattctct ttccctaggc 2160

tctgtgaaag gaaacattgg ccattgcgaa actggcgctg gcgtcgctgg tctgctgaaa 2220

gtacttgcaa tgatcaaaca tggaggtatc ccgcctctgg caagccacaa agcgctcaat 2280

cctaaaatac cagccttgga gacacatcac atggaaattg caaagcagct taagccttgg 2340

gatgtcccac tgagggcagc atttgtcaac agttatggcg ctgctggctc gaatgctgcc 2400

gtgatctgtg tcgagccacc accagtcgtc accgacggat catctttgat tggcactgag 2460

cctcaaaagg taacgctccc agtcatcgtc agtggcgcta caagaaagag cttggtcctg 2520

aatgcacgag cattggcaag ctacctctcg caagacggat cacacctcag catacatgat 2580

gttgcgttta ccgtcaacca acggagaaaa cggaatagat tttgcgccga ggtctctggc 2640

accgatttgc catctttggt tcagtcactc cgcgctgtag actctcccag tttcgagagc 2700

cctggaaagt ccaagcctgt ggtgctcgtc ttcagcggac aaaacaccaa tgcagtagcc 2760

ttggaccgca cgatatacga tacctaccca gtatttaaag cctatattga cgcctgtgac 2820

tccgaaattg tgaaacttgg cttcccaagt atcatggagg ccattttcca gaaggagccc 2880

ataagcactg ctgttgcttt gcaaggcagt atcttcgcaa tgcagtatgc gtgtgcccgt 2940

tcttggatcg acgcaggcct caaacctcga gcaatcatcg gccacagttt cggcgaactt 3000

actgctttgg ctgtctctgg agctctgtca cttgcagaca gcttgaagct ggtcacatgc 3060

cgcggtcacc tcatcgacac taagtggggt gaggaaaggg gcggtatgct tgtcattcat 3120

gcggatgtgg ccacggtcga acgcttccaa tcccggttca aagcgcagca tgacggagct 3180

gaactggaga ttgcttgcta taactctcca actaccacag tggttgctgg gccagtggca 3240

tacatggatg cagctgagca gatgctggcc acagatccag atttccaggg ccttcgtaag 3300

ctgcgcattg taacgagtaa cgccttccat tcttcgttgt cggatccgat cctagccgat 3360

ttggactcca tggcagatac cttgacctgg aatgagccca gtatcccact ggaagcttgc 3420

acgagtgaag gcctggcgtc gatcaaagag tggagtgcct ccagacatac cagaggctca 3480

gtgtacttca ccaaagcggt ggagcgtatc gaaggacgac tgggtgcatg catctgggtt 3540

gaagccggcc ttgactccgc catcatagca atggctcgaa aagcttcttc aaagccagac 3600

tcgcaggtct ttcaatctgt cagcacgaag gctggagcta cttctttcat tgatggaatc 3660

gtaaacaatc tttggcgtca aggcgtgcct ctttctcact tgaacgcgct atcggcgact 3720

gtcaaaccca accctgtctg gcttccacca taccagttcg aaagagaaca gcattggacc 3780

gagcacatcg atagggcgac cgaagcaagt caagcaagca ccacaagcga cactattcag 3840

tcgactccaa cgcaaaccgt ccaaagtcca ccaaagctga tttccagact cgcatctctg 3900

cagtatcaga tcaacacgca gtgcgagcgc ttccaaaaga tcaccgaagg ccatgcggtg 3960

ttgtatgaac ccttatgccc tgcatccttg tacatggaat gtgttgtcat ggctctccaa 4020

gaactagcag gcgaccttgg ttcccgcact cttgacttcg aaaatctgga cttccatgcg 4080

gggttgggcc tacagactga ccgccgtgtg cttctcgact tggaagaagc gcgccctcac 4140

tcatggactt tcaaggtgca atccacgaaa gctgggtcat ctcggtcatt gttacattgc 4200

tcgggccggg tgatcctaac tgagagttcg gtacctacca cgttccagcg tctggtcgat 4260

ggccccagat cacgtctcga ccaggacaag gatgctgaga agcttatgtc gtctcgcgcc 4320

tacggtctgt tttccaacat catgacctat tcggaattcc tcaagccgat ttcgtcgatc 4380

atcctgcgcg aaaacgaatc cttggctacc atcaaacttc caccgaacca gcctggtctg 4440

catgaaagca cggcttggaa aagatgcgac gcagtgttcc tggatggctt catctcctct 4500

tcgggacttc tgctcaatag cagcagcgtg gttcagtcag gccatgtttt gatcgctgtt 4560

ggagtcgagc gggccatcct cacagctgct ttccaagcat cccttgcttc ctcatggcag 4620

gcgtacgcaa catttaccat ggttggcgaa actcacgctc tctgcgatgt tttcgcttgc 4680

actcctgacg gtgaagtggt agccatgatg acaggcgtga ggttcaacaa gatggagatc 4740

tcaaagttag cgaaatcgct ctcgtcggtc aacgcctcgt caccgacagg tggaagaact 4800

cagccaccag ccgcgccaaa aacccaagct cagccgatgg cttctagacc ctcacctacc 4860

ccactgcagg tttcctttgc aacggcagaa cctgccgcac cagagcctgt tcaacagtca 4920

acagctgccc tagcgcgtaa tgacataggt ccagtcctta agtctctcat ctccaactac 4980

accggcctaa tcgaagagga tgtctcggaa gatagtcctc tcgtcgactt aggtcttgac 5040

tcgctctctt ccgttgagtt cgcgtctgaa atcggaacca agttcggagt cactctggat 5100

gcggatacag tgggagactt gacgttacac tcgctttgcc agaggctcag tggcacctcg 5160

aacgttgtat cccagaagat gtccgagaca cctgcagcgg ctccggtgaa ggagctgatt 5220

gaaactgtac catcacccat cgtgactttc agcagtcctg tgtcaaacag catcacatcg 5280

gtcctaaagt ctcttctcgg gagctacacc ggcttacaag aagaagacat gcccgacgat 5340

gtacctctta ttgatcttgg actggattcc ttgtcatccg tcgagtttgc gtcggaactg 5400

aacgacaaaa tgggagcaga tatcgactcc gctgttgttg cagacatgac cttatctgcg 5460

ctcgagcaac agctgggagc atctgcgaca ccacctagca ccaccggctc atccactcct 5520

ggggatattt caactgctgc cacgacccct tatgctacgg gagcctctac gccagactac 5580

ttggtgcatg gaaataaacc gtctatttca aatggagttg tcgctgcaaa agactcctat 5640

caagtcaaga ccgtcgaata caaacgggta agtggtgttc ccatccacgc agacatctac 5700

gtccctcttg ttcaacgcgt ctctcctatg cccttagcgc tcatgatcca cggcggtggc 5760

cacatgacct tgtctcggaa ggccgtccgt cccacacaag ccaaatatct tctgtcgcac 5820

ggcttcctcc ccattagtat tgactaccgt ctgtgtccgg aggtcaatct catcgatgga 5880

cccatcgccg acgtacgtga tgcttatgtg tgggcttgtc aaaatctggg gacgcatctt 5940

gcggaacaca gtatctctgt cgatggtggc cgcgtggtgg tggtggggtg gtcgactgga 6000

ggtcatttgg caatgtcact gggatggtcg cttgaagagg ctggcgtgcc gccaccaaag 6060

gctgtgttga gcttctatgc cccagtggat tttgagtctg gtgagctcga taatcaaaag 6120

aaccccgctc tcccgaaacc gcgcatgact ttggaccaaa ttacaaaggc acttcctagg 6180

accccagtga cgcagtatgg tgcctcttcc acggacgaaa caaatctcgg atggctgcac 6240

ccaggcgatc cgcgatccga actccttctc cacgtgttcc actccgatat tggcttaccg 6300

ctcatactac acggtcttcc catttcgggt tctggcaggc catcgccttc ccttgttgct 6360

tccataagtc cgctggcgcg cttgcgcaac ggctcataca ctatcccgac cttcatcatt 6420

catggcacga aggacgtcat cgctccatat gcagcagcgg agcgatttgt caagattatg 6480

tccgagaaag gggtaaagag tggatttcta tcgttgtcag ggacgggtca tgttttcgat 6540

gttacgatga agcctgatag taaggggtgg gaggacaagg ttaagccggg gcttgacttt 6600

ttgattcaga acgcttga 6618

<210> 3

<211> 60

<212> DNA

<213> Artificial sequence ()

<400> 3

gactacaaag acgatgacga caagcttcat atgtctaatt ctacacgtga ctatcccata 60

<210> 4

<211> 52

<212> DNA

<213> Artificial sequence ()

<400> 4

catgtttgat cattgcaagt actttcagca gaccagcgac gccagcgcca gt 52

<210> 5

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 5

gctgaaagta cttgcaatga tcaaacatgg aggtatcccg cct 43

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 6

tcggcttgag gaattccgaa taggt 25

<210> 7

<211> 56

<212> DNA

<213> Artificial sequence ()

<400> 7

tcatgaccta ttcggaattc ctcaagccga tttcgtcgat catcctgcgc gaaaac 56

<210> 8

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 8

agtgatggtg atggtgatgt ccgtttaaac tcaagcgttc tgaatcaaaa agtcaagcc 59

Claims (9)

1. Polyketide synthase PreuA, characterized in that the amino acid sequence of the polyketide synthase PreuA is shown in SEQ ID NO. 1.

2. A gene encoding the polyketide synthase PreuA of claim 1.

3. The gene of claim 2, wherein the gene sequence is the nucleotide sequence shown in SEQ ID No. 2.

4. A recombinant vector comprising the gene according to claim 2 or 3.

5. A recombinant genetically engineered bacterium transformed with the recombinant vector of claim 4.

6. Use of the polyketide synthase PreuA according to claim 1 for the preparation of lichen ruber acid.

7. A construction method of a yeast mutant strain capable of efficiently producing pinoceric acid is characterized by comprising the following steps:

transforming saccharomyces cerevisiae by using the recombinant vector of claim 4 by using a LiAc/PEG4000 mediated transformation method, uniformly coating the transformation liquid on an SC-Ura solid plate, and culturing in an incubator at 30 ℃ to obtain the yeast mutant strain.

8. A method for producing bryozoan, which is a yeast mutant strain constructed by the construction method according to claim 7, comprising the steps of:

inoculating a yeast mutant strain into an SC-Ura liquid culture medium, and culturing at 30 ℃ in a shaking table; adding 1% YPD medium to the cultured 2d, and further culturing for 3-4d to obtain fermentation broth;

wherein, 1% of the YPD medium and the SC-Ura liquid medium have the same volume;

step (2), extracting the fermentation liquor obtained by culturing in the step (1) by using ethyl acetate with the same volume, and concentrating the extract liquor to obtain a crude extract;

and (3) purifying the crude extract obtained in the step (2) to obtain the pinoceric acid.

9. The method of preparing Bryoid acid red powder according to claim 8, wherein:

in the step (3), the crude extract obtained in the step (2) is purified, and the purification method comprises the following steps: sequentially washing the crude extract with n-hexane, chloroform, dichloromethane, ethyl acetate and methanol, recovering ethyl acetate and methanol washing solutions, concentrating, and mixing; wherein the volume of n-hexane, chloroform, dichloromethane, ethyl acetate and methanol is one tenth of that of SC-Ura liquid culture medium.

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