CN110117582A

CN110117582A - Fusion protein, its encoding gene and the application in biosynthesis

Info

Publication number: CN110117582A
Application number: CN201910392068.1A
Authority: CN
Inventors: 刘春生; 李妍芃; 尹艳; 高伟; 姜丹
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-05-13
Filing date: 2019-05-13
Publication date: 2019-08-13
Anticipated expiration: 2039-05-13
Also published as: CN110117582B

Abstract

The present invention relates to a kind of fusion proteins, the fusion protein includes chalcone synthase and chalcone reductase, chalcone synthase is connected with chalcone reductase by a connexon GGGS, and the gene of fusion protein of the present invention and the recombination engineering comprising the gene are encoded, fusion protein of the present invention can be used for the biosynthesis of chalcone and isoliquiritigenin.

Description

Fusion protein, its encoding gene and the application in biosynthesis

Technical field

The present invention relates to a kind of fusion protein and the fusion protein encoding gene, include the fusion protein encoding gene The application in isoliquiritigenin synthesis of expression vector and recombinant bacterium and the fusion protein or recombination, belong to medicinal Ingredient synthetic biology field.

Background technique

As the ancient plant medicine for having thousands of years history simply, Radix Glycyrrhizae (Glycyrrhizae Radix et Rhizoma) It is one of most common large Precious, Rare, Endangered medicinal material, a variety of diseases is demonstrated by with good and safe preventive and therapeutic action, it is several It appears in all tcm prescriptions, patent medicine, and more and more health care products even food.Isoliquiritigenin be in Radix Glycyrrhizae content compared with A kind of high chalcone and a kind of common natural pigment, structure is simple, research be found to have anti-inflammatory, anticancer, antihistaminicum, A variety of remarkable activities such as anti-oxidant, anti-platelet aggregation, anticancer, antiallergy, antiviral and estrogen-like, wherein, anticancer activity It is more prominent, it is able to suppress a variety of cancer cell multiplications and induces their apoptosis.Its glycoside compounds isoliquiritin is able to suppress Tumor vascular newborn, antidepressant effect and anti-oxidant.These all imply isoliquiritigenin in terms of have it is more preferable Development and application prospect.But isoliquiritigenin content is limited in plant in Radix Glycyrrhizae, and due to the great demand amount in market, early in Chinese Wild Radix Glycyrrhizae reserves are less than 500,000 t within 2009.It is predatory to wild licorice etc. to excavate, it is big not only to cause Radix Glycyrrhizae The width underproduction, also leads to the destruction and desertification of Kinds of Licorice growing area environment, and Radix Glycyrrhizae population is difficult to be restored after being destroyed.To alleviate Medicine source is insufficient, has largely in such a way that chemical method is come synthesizing isoliquiritigenin, but chemical synthesis process needs are used largely Organic solvent, it is unfavorable to environmental protection, and there are certain risk of explosion.Therefore the methods of histocyte culture has also been developed to obtain Isoliquiritigenin is obtained, but yield is too low, it is time-consuming too long.Heterologous organisms synthesizing isoliquiritigenin becomes having for Licorice sustainable development Effect strategy.Isoliquiritigenin belongs to 5- oxidation chalcone, and biosynthesis pathway originates in phenylpropyl alcohol alkane approach, in phenylalanine solution ammonia Enzyme (phenylalanine ammo-nialyase, PAL), cinnamic acid 4-hydroxylase (cinnamate 4-hydroxylase, C4H) and under the gradually catalysis of coumaric acyl-CoA ligase (4-coumarate:CoA ligase, 4CL), phenylalanine is converted For coumaric acyl CoA, subsequent 3 molecule malonyl coenzyme A and 1 molecule coumaroyl A are in chalcone synthetase (chalcone Synthase, CHS) and chalcone reductase (chalcone reductase, CHR) under the action of generate isoliquiritigenin.Radix Glycyrrhizae The clone of middle isoliquiritigenin biosynthesis pathway genes (PAL, C4H, 4CL, CHS and CHR), to utilize Fermentation Engineering mass production Active constituent isoliquiritigenin provides important foundation.

Summary of the invention

Specifically, first aspect present invention is related to a kind of fusion protein, and the fusion protein includes:

(1) chalcone synthase (CHS)；With

(2) chalcone reductase (CHR)；

Between the chalcone synthase and chalcone reductase by a connexon connect, the connexon be GGGS, Any one in GSG, GSGGGGS, GSGEAAAK, GSGEAAAKEAAAK or GSGMGSSSN.

In one embodiment of the invention, it is related to a kind of fusion protein, which includes SEQ ID The CHR of amino acid sequence shown in CHS the and SEQ ID NO:10 of amino acid sequence shown in NO:8, passes through between the CHS and CHR The connection of one connexon, the connexon are in GGGS, GSG, GSGGGGS, GSGEAAAK, GSGEAAAKEAAAK or GSGMGSSSN Any one, such as can be GGGS.

In the second aspect of the present invention, the invention further relates to the polynucleotide sequence for encoding fusion protein of the present invention, The polynucleotide sequence includes: chalcone synthase encoding gene (CHS) described in SEQ ID NO:7 and SEQ ID NO:9 institute State chalcone reduction enzyme coding gene (CHR), it is preferable that connect between the CHS and CHR by a linker sequence, such as Linker sequence can be GGTGGTGGTTCT, more specifically, such as can be in CHS in 3 ' ends (removal SEQ ID NO:7 institute After showing terminator codon TGA) by linker sequence GGTGGTGGTTCT be connected to CHR 5 ' end form CHS::CHR.

Third aspect present invention further relates to recombinant expression carrier, and it includes promoters, coding fusion protein of the present invention Polynucleotides and transcription terminator, the expression vector be preferably by promoter, coding fusion protein of the present invention it is more Nucleotide, terminator and episomal vector are stitched together using the method for yeast homologous recombination, and wherein episomal vector is yeast Expression vector is such as selected from pESC, pYX212, pYES2.0, pRS425, pRS426 and p424；Preferably pESC expression vector, The pESC expression vector is selected from pESC-Leu, pESC-His or pESC-Trp.

In one preferred embodiment, table of the present invention can be any one in following in carrier:

Recombinant expression carrier pYM3, it includes CHS and CHR, the CHS holds (nucleotides sequence shown in SEQ ID NO:7 3 ' Arrange after removing terminator codon TGA) CHS::CHR is formed by the 5 ' ends that linker sequence GGTGGTGGTTCT is connected to CHR, The CHS::CHR is inserted into the downstream promoter GAL10 of expression vector pESC-Leu；

Recombinant expression carrier pYM2 comprising the coumaric acyl-CoA connection of amino acid sequence shown in coding SEQ ID NO:6 Enzyme coding gene (4CL), CHS and CHR, at 3 ' ends, (nucleotide sequence shown in SEQ ID NO:7 removes termination codon to the CHS After sub- TGA) by the 5 ' ends that linker sequence GGTGGTGGTTCT is connected to CHR form CHS::CHR, the 4CL is inserted into yeast The downstream promoter GAL1 of expression vector pESC-Leu, the CHS::CHR are inserted into the promoter of expression vector pESC-Leu The downstream GAL10；Coumaric acyl-CoA ligase encoding gene of amino acid sequence shown in the coding SEQ ID NO:6 can be The nucleotide sequence as shown in SEQ ID NO:5.

The fourth aspect of the present invention also relates to a kind of recombination yeast engineering bacteria, the recombination yeast engineering bacteria, comprising compiling Recombinant expression carrier described in the polynucleotide sequence or fourth aspect present invention of code fusion protein of the present invention.

In the fifth aspect of the invention, it is related to a kind of preferred recombination yeast engineering bacteria, the preferred recombination ferment Female engineering bacteria, to further include expression vector pYM1, the expression vector in fourth aspect present invention Yeast engineering bacteria PYM1 includes the phenylalanine lyase encoding gene (PAL) and coding SEQ of amino acid sequence shown in coding SEQ ID NO:2 The cinnamic acid 4-hydroxylase encoding gene (C4H) of amino acid sequence shown in ID NO:4, the PAL are inserted into expression vector pESC- The downstream of the promoter GAL1 of His, the C4H are inserted into the downstream of the promoter GAL10 of expression vector pESC-His.

Specifically, the phenylalanine lyase encoding gene (PAL) for encoding amino acid sequence shown in SEQ ID NO:2 can be with It is the nucleotide sequence as shown in SEQ ID NO:1；The cinnamic acid 4-hydroxylase for encoding amino acid sequence shown in SEQ ID NO:4 is compiled Code gene (C4H) can be the nucleotide sequence as shown in SEQ ID NO:3.

The sixth aspect of the present invention is related to a kind of method for constructing recombination yeast engineering bacteria of the present invention, the ferment The construction method of female engineering bacteria are as follows:

Recombinant expression carrier pYM3 is transferred in Yeast engineering bacteria WAT11, bacterial strain WM4 is obtained；Or

Recombinant expression carrier pYM2 is transferred in Yeast engineering bacteria WAT11, bacterial strain WM3 is obtained；Or

Recombinant expression carrier pYM1 and pYM2 are transferred in Yeast engineering bacteria WAT11, bacterial strain WM2-1 is obtained；Or

Recombinant expression carrier pYM1, pYM2 and pYM3 are transferred in Yeast engineering bacteria WAT11, bacterial strain WM2-2 is obtained.

The seventh aspect of the present invention is related to fusion protein of the present invention or encodes the more of fusion protein of the present invention Nucleotide, the recombinant expression carrier comprising polynucleotides of the present invention or recombination yeast engineering bacteria of the present invention are in life It produces the application in chalcone or isoliquiritigenin, after galactolipin induction fermentation obtained strains, is extracted with ethyl acetate fermentation liquid, passes through LC-MS detection, can be detected isoliquiritigenin.

The invention further relates to PAL described above, C4H, 4CL, application of the CHS or CHR gene in fermentation by saccharomyces cerevisiae, tools Body can be applied to the preparation of Fermentation Engineering synthesizing isoliquiritigenin and other flavonoids biosynthesis intermediates.Further, described Other flavonoids biosynthesis intermediates be cinnamic acid, p-Coumaric Acid, to coumaroyl A and naringenin chalcone.

Isoliquiritigenin and flavonoids biosynthesis intermediate meat can be generated by biosynthesis technology using the present invention Cinnamic acid, p-Coumaric Acid have a good application prospect coumaroyl A and naringenin chalcone.

The present invention also provides phenylalanine lyase of the present invention, cinnamic acid 4-hydroxylase, coumaric acyl-CoA connections Enzyme, chalcone synthetase and chalcone reductase or coding phenylalanine lyase of the present invention, cinnamic acid 4-hydroxylase, perfume (or spice) Beans acyl-CoA ligase, chalcone synthetase and chalcone reductase gene, in the plant breeding containing flavonoids In utilization.With phenylalanine lyase of the present invention, cinnamic acid 4-hydroxylase, coumaric acyl-CoA ligase, chalcone Synzyme and chalcone reductase or its encoding gene can improve flavones in plant by being applied in plant cell Content.

Detailed description of the invention

Fig. 1 is that different genes encode proteins carry product analysis figure.EIC (extracted ion chromatogram) is i.e. Ion flow graph is extracted, m/z 164,147,163,271 and 255 is respectively phenylalanine, cinnamic acid, p-Coumaric Acid, shaddock ped in figure Plain chalcone and isoliquiritigenin [M-H]^-Nucleocytoplasmic ratio.

Fig. 2 is recombinant yeast WM1, WM2-1 and WM2-2 tunning analysis chart.M/z 163 and 255 distinguishes in Fig. 2A For p-Coumaric Acid and isoliquiritigenin [M-H]^-Nucleocytoplasmic ratio.

Fig. 3 is recombinant yeast WM3, WM4 and WM5 tunning analysis chart.M/z 163 and 255 is respectively to perfume (or spice) in figure Beans acid and isoliquiritigenin [M-H]^-Nucleocytoplasmic ratio.

Specific embodiment

Various aspects and features of the invention, the skill of this field are illustrated below by way of preferred embodiment and in conjunction with attached drawing Art personnel are not intended to limit the scope of the invention it should be understood that these embodiments are only intended to illustrate.Without departing substantially from claim Under conditions of book range, those skilled in the art can carry out various modifications and improve to various aspects of the present invention, these Modification and improvement also belong to protection scope of the present invention.For example, institute's Practical Expression carrier and host strain in embodiment are replaced with Other expression vectors commonly used in the art and host strain are that those skilled in the art can understand and realize.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The clone of isoliquiritigenin biosynthesis pathway enzyme gene in 1 Glycyrrhiza Uralensis of embodiment

1. design of primers

It is screened to obtain full length gene sequence fragment according to Glycyrrhiza Uralensis transcript profile data notes, designs upstream and downstream gram Grand primer, primer sequence are as follows:

2.PCR amplification

Using QuantScript RT Kit (Tiangeng biochemical technology Co., Ltd, Beijing, China) by Glycyrrhiza Uralensis RNA Reverse transcription is at cDNA.

Using cDNA as template, PCR amplification is carried out.

Amplification system are as follows: 2 × KAPA HiFi Hotstart ReadyMix (Kapa Biosystems, Wilmington, USA each 1.5 μ L of) 25 μ L, primer P1 and P2,2 μ L of template, distilled water supply 50 μ L.Reaction condition: 98 DEG C of initial denaturation 3min, 98 DEG C 20s, 62 DEG C of annealing 15s, 72 DEG C of extension 1.5min, 72 DEG C of extensions 5min after 35 circulations, 4 DEG C save.

Sequencing result show the sequence of pcr amplification product respectively as shown in SEQ ID No.1,3,5,7 and 9, by sequence 1, 3, gene shown in 5,7 and 9 is respectively designated as PAL, C4H, 4CL, CHS and CHR, and the albumen of coding is respectively designated as PAL, The amino acid sequence of C4H, 4CL, CHS and CHR, corresponding albumen are respectively shown in SEQ ID No.2,4,6,8 and 10.

Below in an example, gene PAL, C4H, 4CL, CHS and CHR and albumen PAL, C4H, 4CL, CHS and CHR, It is all identical as upper corresponding nucleic acid or amino acid sequence.

2 PAL of embodiment, the prokaryotic expression of CHS and CHR gene and external enzymatic reaction

1, prokaryotic expression

PAL, CHS and CHR are utilized respectively EasyGeno Assembly Cloning kit (the limited public affairs of Tiangeng biochemical technology Department, Beijing, China) it is inserted between the KpnI and XhoI of pET-32a (+), and be transferred in E.coli BL21 (DE3).Transformant is used The LB plate screening and picking monoclonal sequence verification of the ampicillin containing 100mg/mL.Recombinant expression cell contains in 200mL In the LB culture medium of 100mg/mL ampicillin, 37 DEG C of shake cultures to OD600=0.6-1.0, with the IPTG of 0.2mM 16 DEG C induction 10h.5000rpm, 4 DEG C thalline were collected by centrifugation.Thallus is resuspended in 3mL PBS buffer (pH 8.0), and ice-bath ultrasonic is broken Supernatant is collected by centrifugation in bacterium.Recombinant protein (health is century Biotechnology Co., Ltd, Beijing, China) purifying, is used Bradford method detectable concentration.

1. external enzymatic reaction

The external enzymatic reaction system of 1mL respectively includes:

Recombinant protein PAL about 50ng/ μ L (10mM PBS, pH=8.0), dithiothreitol (DTT) (DTT) 1mM, substrate phenylpropyl alcohol ammonia Sour 1mM；

Recombinant protein c HS about 50ng/ μ L (10mM PBS, pH=8.0), DTT 1mM, substrate 1mM (are for molal weight ratio The coumaroyl A and malonyl coenzyme A of 1:3)；

Recombinant protein c HS and CHR about 50ng/ μ L (10mM PBS, pH=8.0), DTT 1mM, substrate 1mM are (for a mole matter Amount is than the coumaroyl A and malonyl coenzyme A for 1:3)；

Recombinant protein c HS and CHR about 50ng/ μ L (10mM PBS, pH=8.0), DTT 1mM, substrate 1mM are (for a mole matter Amount is than the coumaroyl A and malonyl coenzyme A for 1:3), and, the NADPH of 1mM；

Respective system in 30 DEG C of incubation 12h, is added 200 μ L methanol and terminates reaction.Catalysate 12000g centrifugation, supernatant 0.22 μm of PTFE filter membrane is crossed, is detected with HPLC-MS.As a result such as Figure 1A and Fig. 1 D.Compared with zero load control, it is with phenylalanine Cinnamic acid (Figure 1A) is generated in the PAL1 recombinant protein vitro enzyme rush system of substrate, therefore assert that PAL1 has catalysis phenylalanine Generate the activity of cinnamic acid.

Using molal weight than in the external enzymatic system of coumaroyl A and malonyl coenzyme A as substrate for 1:3, Naringenin chalcone can be generated by being only added when CHS is incubated for；When equivalent CHS, CHR recombinant protein and NADPH is added simultaneously, The generation (Fig. 1 D) of isoliquiritigenin can also be detected other than naringenin chalcone；Determine CHS, CHR chalcone synthetase and Chalcone reductase activity.

However, equivalent CHS, CHR recombinant protein is added simultaneously, but when being added without NADPH in substrate, fail to detect different sweet The generation of careless element.

The Yeast expression and characterization of 3 C4H and 4CL gene of embodiment

Yeast expression carrier building, utilizes EasyGeno Assembly Cloning kit (the limited public affairs of Tiangeng biochemical technology Department, Beijing, China), C4H or CHS is inserted into Yeast expression carrier pESC-His (Anjelen Sci. & Tech. Inc, Sheng Ke respectively The city La La, the U.S.) (or pESC-Leu (Anjelen Sci. & Tech. Inc, Santa Clara, the U.S.) can also be inserted into respectively) The site SpeI and NotI between, 4CL insertion had connected CHS recombinant vector the site NheI and BamHI between.Recombinate matter Grain is transferred to host strain WAT11 using Yeast Transformation Kit (Zymo Research Corporation, Irvine, USA), turns Beggar was at corresponding defect culture medium (SC-His or-Leu, 2% glucose and 2% agar) upper 30 DEG C of screening and culturings 4 days.It is positive It is cloned in corresponding liquid defect culture medium (2% glucose), 30 DEG C of concussions to OD600 about 0.8.It is replaced with 2% galactolipin It changes the induced medium of glucose, 30 DEG C, after 220rpm inducing expression 6h, 20 μM of cinnamic acids is added or p-Coumaric Acid continues to cultivate 12h.Culture solution is extracted three times with isometric ethyl acetate, after extract volatilizes solvent, is redissolved with methanol, and 0.22 μm of PTFE is crossed Filter membrane detects product under HPLC-MS negative ion mode.As a result such as Figure 1B and Fig. 1 C.It include the recombination ferment of C4H with cinnamic acid feeding It is female and with after galactolipin inducing expression, p-Coumaric Acid (Figure 1B) can be detected in culture solution, illustrate that C4H can be catalyzed cinnamic acid and give birth to At p-Coumaric Acid.Because 4CL catalysate is unstable, by 4CL and CHS using binary expression vector pESC-Leu in WAT11 Coexpression, is fed with p-Coumaric Acid, can also detect naringenin chalcone (Fig. 1 C), explanation in culture liquid extract 4CL can generate coumaroyl A using the coacetylase combination p-Coumaric Acid of yeast entogenous, provide substrate for CHS synthesizing chalcone.

Embodiment 4 produces the building of the Yeast engineering bacteria of isoliquiritigenin

It utilizes EasyGeno Assembly Cloning kit (Tiangeng biochemical technology Co., Ltd, Beijing, China) point Other:

(1) by the downstream promoter GAL1 of gene PAL insertion double base Yeast expression carrier pESC-His, gene C 4H insertion The downstream promoter GAL10 of double base Yeast expression carrier pESC-His, obtains plasmid YM1 (pHIS-^GAL1PAL-^GAL10C4H)；

(2) by the downstream promoter GAL1 of gene 4CL insertion double base Yeast expression carrier pESC-Leu, fusion (gene C HS::CHR is to remove CHS in the downstream promoter GAL10 of CHS::CHR insertion double base Yeast expression carrier pESC-Leu Fusion (the SEQ that 3 ' ends are formed by the end linker sequence GGTGGTGGTTCT connection CHR 5 ' after terminator codon TGA ID NO:21)), obtain plasmid pYM2 (pLEU-^GAL14CL-^GAL10CHS::CHR)；

(3) it by the downstream of fusion CHS::CHR insertion double base Yeast expression carrier pESC-TrpGAL10 promoter, obtains To plasmid pYM3 (pTRP-^GAL10CHS::CHR)。

(4) by the downstream promoter GAL1 of gene C HS insertion double base Yeast expression carrier pESC-His, gene C HR insertion The downstream promoter GAL10 of double base Yeast expression carrier pESC-His, obtains plasmid pYM4 (pHIS-^GAL1CHS-^GAL10CHR)

It is incited somebody to action respectively using Yeast Transformation Kit (Zymo Research Corporation, Irvine, USA):

(1) recombinant expression carrier pYM1 is transferred in Yeast engineering bacteria WAT11, obtains transformant 1；

(2) recombinant expression carrier pYM1 and pYM2 is transferred in Yeast engineering bacteria WAT11, obtains transformant 2；

(3) recombinant expression carrier pYM1, pYM2 and pYM3 is transferred in Yeast engineering bacteria WAT11, obtains transformant 3；

(4) recombinant expression carrier pYM2 is transferred in Yeast engineering bacteria WAT11, obtains transformant 4；

(5) recombinant expression carrier pYM3 is transferred in Yeast engineering bacteria WAT11, obtains transformant 5；

(6) recombinant expression carrier pYM4 is transferred in Yeast engineering bacteria WAT11, obtains transformant 6；

Each transformant is according to carrier tag in corresponding defect culture medium (SC-His (transformant 1, transformant 6), SC- His-Lue (transformant 2), SC-His-Lue-Trp (transformant 3), SC-Leu (transformant 4) or SC-Trp (transformant 5), 2% Glucose and 2% agar) it is 30 DEG C of screening and culturings 4 days upper.The positive colony restructuring yeast strains of acquisition are respectively designated as WM1 (packet Containing pYM1), WM2-1 (include pYM1 and pYM2), WM2-2 (including pYM1, pYM2 and pYM3), WM3 (including pYM2), WM4 (packet Containing pYM3) and WM5 (including pYM4).

Recombination yeast is respectively at corresponding liquid defect culture medium (SC-His (WM1), SC-His-Lue (WM2-1) or SC- His-Lue-Trp (WM2-2), 2% glucose) in, 30 DEG C of concussions to OD600 about 0.8.Glucose is replaced with 2% galactolipin Induced medium, 30 DEG C, 220rpm inducing expression 12-48h.Culture solution is extracted three times with isometric ethyl acetate, extract It after volatilizing solvent, is redissolved with methanol, crosses 0.22 μm of PTFE filter membrane, detect product under HPLC-MS negative ion mode.As a result as schemed 2.Recombination yeast WM1 can be generated p-Coumaric Acid (Fig. 2A) under galactolipin induction fermentation, and culture 36h or so detection p-Coumaric Acid produces Amount is at 7.59umol/L (Fig. 2 B).When being transferred to pYM2 into WM1, can be detected in fermentation liquid a small amount of isoliquiritigenin (Fig. 2, WM2-1).And being overexpressed CHS::CHR can make the isoliquiritigenin yield of WM2-2 rise 18.2 times (Fig. 2 B) compared with WM2-1.

Recombinant yeast WM3, WM4 and WM5 be corresponding in turn to liquid defect culture medium (SC-Leu, SC-Trp or SC-His, 2% glucose) in, 30 DEG C of concussions to OD600 about 0.8.With 2% galactolipin replacement glucose induced medium, 30 DEG C, After 220rpm inducing expression 6h, 20 μM of p-Coumaric Acids are added and continue to cultivate WM3,20 μM of coumaroyl A are added and continue to cultivate WM4 And WM5.The isometric ethyl acetate of culture solution is extracted three times after 12h, after extract volatilizes solvent, is redissolved with methanol, mistake 0.22 μm of PTFE filter membrane detects product under HPLC-MS negative ion mode.The result shows that with p-Coumaric Acid (WM3) or coumaric acyl Coacetylase (WM4) is fed, and isoliquiritigenin can be also detected in culture liquid extract, and feeds WM5 with coumaroyl A Isoliquiritigenin (Fig. 3) cannot be obtained.

Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff Range, protection scope of the present invention are subject to claims.

SEQUENCE LISTING

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His Ser Leu Asn Thr Phe Leu Leu Ser Phe Arg Arg Asp Asn Ser Ser

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Asn Met Asp Ala Thr Thr Ala Asn Gly His Val Val Asp Gly Val Asn

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Ser Phe Cys Leu Lys Ser Gly Ser Gly Gly Gly Asp Pro Leu Asn Trp

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Gly Ala Ala Ala Glu Ser Met Lys Gly Ser His Leu Asp Glu Val Lys

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Arg Met Val Ala Glu Tyr Arg Lys Pro Val Val Arg Leu Gly Gly Glu

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

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Val Arg Val Glu Leu Ser Glu Ser Ala Arg Ala Gly Val Lys Ala Ser

145 150 155 160

Ser Asp Trp Val Met Asp Ser Met Asn Asn Gly Thr Asp Ser Tyr Gly

165 170 175

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

180 185 190

Gly Ala Leu Gln Lys Glu Leu Ile Arg Phe Leu Asn Ala Gly Ile Phe

195 200 205

Gly Asn Gly Thr Glu Ser Asn Cys Thr Leu Pro His Thr Ala Thr Arg

210 215 220

Ala Ala Met Leu Val Arg Ile Asn Thr Leu Leu Gln Gly Tyr Ser Gly

225 230 235 240

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

245 250 255

Ile Thr Pro Cys Leu Pro Leu Arg Gly Thr Ile Thr Ala Ser Gly Asp

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Glu Gly Leu Ala Leu Val Asn Gly Thr Ala Val Gly Ser Gly Leu Ala

325 330 335

Ser Ile Val Leu Phe Glu Ala Asn Ile Leu Ala Val Leu Ser Glu Val

340 345 350

Ile Ser Ala Ile Phe Ala Glu Val Met Gln Gly Lys Pro Glu Phe Thr

355 360 365

Asp Tyr Leu Thr His Lys Leu Lys His His Pro Gly Gln Ile Glu Ala

370 375 380

Ala Ala Ile Met Glu His Val Leu Asp Gly Ser Ser Tyr Val Lys Ala

385 390 395 400

Ala Lys Lys Leu His Glu Val Asp Pro Leu Gln Lys Pro Lys Gln Asp

405 410 415

Arg Tyr Ala Leu Arg Thr Ser Pro Gln Trp Leu Gly Pro Leu Ile Glu

420 425 430

Val Ile Arg Phe Ser Thr Lys Ser Ile Glu Arg Glu Ile Asn Ser Val

435 440 445

Asn Asp Asn Pro Leu Ile Asp Val Ser Arg Asn Lys Ala Leu His Gly

450 455 460

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

465 470 475 480

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

485 490 495

Leu Val Asn Asp Phe Tyr Asn Asn Gly Leu Pro Ser Asn Leu Ser Gly

500 505 510

Gly Arg Asn Pro Ser Leu Asp Tyr Gly Phe Lys Gly Ala Glu Ile Ala

515 520 525

Met Ala Ser Tyr Cys Ser Glu Leu Gln Tyr Leu Ala Asn Pro Val Thr

530 535 540

Ser His Val Gln Ser Ala Glu Gln His Asn Gln Asp Val Asn Ser Leu

545 550 555 560

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

565 570 575

Leu Met Ser Ser Thr Phe Leu Ile Ala Leu Cys Gln Ala Ile Asp Leu

580 585 590

Arg His Leu Glu Glu Asn Leu Arg Asn Thr Val Lys Asn Thr Val Ser

595 600 605

Gln Val Ala Lys Arg Thr Leu Thr Thr Gly Val Asn Gly Glu Leu His

610 615 620

Pro Ser Arg Phe Cys Glu Lys Asp Leu Leu Lys Val Val Asp Arg Glu

625 630 635 640

Tyr Val Phe Ala Tyr Ile Asp Asp Pro Cys Ser Ala Thr Tyr Pro Leu

645 650 655

Met Gln Lys Leu Arg Gln Val Leu Val Asp His Ala Leu Val Asn Gly

660 665 670

Glu Ser Glu Lys Ser Leu Asn Thr Ser Ile Phe Gln Lys Ile Ala Thr

675 680 685

Phe Glu Asp Glu Leu Lys Ala Leu Leu Pro Lys Glu Val Glu Gly Ala

690 695 700

Arg Val Ala Tyr Glu Asn Gly Gln Cys Ala Ile Pro Asn Lys Ile Lys

705 710 715 720

Glu Cys Arg Ser Tyr Pro Leu Tyr Lys Phe Val Arg Glu Glu Leu Gly

725 730 735

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

740 745 750

Asp Lys Leu Phe Ile Ala Met Cys Gln Gly Lys Ile Ile Asp Pro Leu

755 760 765

Leu Glu Cys Leu Gly Glu Trp Asn Gly Ala Pro Leu Pro Ile Cys

770 775 780

<210> 3

<211> 1518

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 3

atggatctcc tccttctgga gaagacccta ttgggtctct tcatcgccgc cataaccgcc 60

attgcaatct caaagctccg aggccggcga ttcaagctcc caccgggacc aatcccggta 120

ccaatcttcg gtaactggct ccaagtcggc gacgacctca accaccgcaa cctcaccgac 180

ctagcgaaac gcttcggcga catcttcctc ctccgaatgg gacagcgaaa cctcgtcgtc 240

gtttcatcgc cggagctagc caaggaggtc ctccacacac agggcgtgga attcggatcc 300

cgaacacgaa acgtcgtatt cgacatcttc accggaaagg gacaagacat ggtgttcacc 360

gtctacggcg aacactggcg gaagatgagg aggatcatga cggtgccctt tttcaccaac 420

aaggttgttc agcagtaccg gttcgggtgg gaatctgagg ctgctagtgt cgtcgatgat 480

gttcggcgta accccgatgc agccgccggc gggattgtac tccgccggag acttcagctc 540

atgatgtata acaatatgta tcggattatg tttgatagga ggtttgagag tgaggaggat 600

cctctgttta tgaagctgaa ggctctgaat ggggagagga gtcgtttggc acagagtttt 660

gagtataact atggggattt cattcctatt ttgagaccct tcttgaaagg ttacttgacg 720

atttgtaagg aggttaagga gaggaggttg aagctcttca aggactattt cgttgatgag 780

aggatgaagc ttgaaagcac aaagagcacc agcaacgaag gacttaaatg cgctattgat 840

cacattttgg acgctcagaa gaagggtgag atcaacgaag acaacgtcct ttacattgtt 900

gagaacatca acgttgctgc aattgaaaca actctatggt caattgaatg gggaattgct 960

gagcttgtga accacccaga gatccaaaag aaagtgaggg atgagattga cagagttctt 1020

ggaccaggac accaagtgac tgagccagat atgcagaagc taccttacct tcaggcagtg 1080

atcaaggaga cactccggct ccgaatggcg atcccgctcc tcgtcccaca catgaacctc 1140

cacgacgcaa agctcggtgg gtacgacatt ccggcggaga gcaagatatt ggtgaatgca 1200

tggtggcttg caaacaaccc tgctaattgg aaaaggccag aggagtttag gccagagagg 1260

ttcttagagg aagagtcaca tgttgaggct aatgggaatg actttaggta ccttccattt 1320

ggtgttggta gaaggagttg ccctggaatc attcttgctt tgcctatcct tggtattact 1380

ttgggacgtt tggttcaaaa ttttgagcta ttgcctcctc ctggacagtc caaacttgac 1440

actgctgaga aaggagggca attcagtttg cacatactca aacactcaac cattgttgcc 1500

aagccaagat cattttag 1518

<210> 4

<211> 505

<212> PRT

<213> Artificial Sequence

<220>

<223> aritficial sequence

<400> 4

Met Asp Leu Leu Leu Leu Glu Lys Thr Leu Leu Gly Leu Phe Ile Ala

1 5 10 15

Ala Ile Thr Ala Ile Ala Ile Ser Lys Leu Arg Gly Arg Arg Phe Lys

20 25 30

Leu Pro Pro Gly Pro Ile Pro Val Pro Ile Phe Gly Asn Trp Leu Gln

35 40 45

Val Gly Asp Asp Leu Asn His Arg Asn Leu Thr Asp Leu Ala Lys Arg

50 55 60

Phe Gly Asp Ile Phe Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val

65 70 75 80

Val Ser Ser Pro Glu Leu Ala Lys Glu Val Leu His Thr Gln Gly Val

85 90 95

Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr Gly

100 105 110

Lys Gly Gln Asp Met Val Phe Thr Val Tyr Gly Glu His Trp Arg Lys

115 120 125

Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln

130 135 140

Gln Tyr Arg Phe Gly Trp Glu Ser Glu Ala Ala Ser Val Val Asp Asp

145 150 155 160

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

165 170 175

Arg Leu Gln Leu Met Met Tyr Asn Asn Met Tyr Arg Ile Met Phe Asp

180 185 190

Arg Arg Phe Glu Ser Glu Glu Asp Pro Leu Phe Met Lys Leu Lys Ala

195 200 205

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

210 215 220

Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Lys Gly Tyr Leu Thr

225 230 235 240

Ile Cys Lys Glu Val Lys Glu Arg Arg Leu Lys Leu Phe Lys Asp Tyr

245 250 255

Phe Val Asp Glu Arg Met Lys Leu Glu Ser Thr Lys Ser Thr Ser Asn

260 265 270

Glu Gly Leu Lys Cys Ala Ile Asp His Ile Leu Asp Ala Gln Lys Lys

275 280 285

Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn

290 295 300

Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala

305 310 315 320

Glu Leu Val Asn His Pro Glu Ile Gln Lys Lys Val Arg Asp Glu Ile

325 330 335

Asp Arg Val Leu Gly Pro Gly His Gln Val Thr Glu Pro Asp Met Gln

340 345 350

Lys Leu Pro Tyr Leu Gln Ala Val Ile Lys Glu Thr Leu Arg Leu Arg

355 360 365

Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu His Asp Ala Lys

370 375 380

Leu Gly Gly Tyr Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn Ala

385 390 395 400

Trp Trp Leu Ala Asn Asn Pro Ala Asn Trp Lys Arg Pro Glu Glu Phe

405 410 415

Arg Pro Glu Arg Phe Leu Glu Glu Glu Ser His Val Glu Ala Asn Gly

420 425 430

Asn Asp Phe Arg Tyr Leu Pro Phe Gly Val Gly Arg Arg Ser Cys Pro

435 440 445

Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Leu Gly Arg Leu

450 455 460

Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Lys Leu Asp

465 470 475 480

Thr Ala Glu Lys Gly Gly Gln Phe Ser Leu His Ile Leu Lys His Ser

485 490 495

Thr Ile Val Ala Lys Pro Arg Ser Phe

500 505

<210> 5

<211> 1653

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 5

atggccattc agaacgagat gaagcagcag caacaacaga tcaaagaaga gttcatattc 60

aagtcgaagc ttcccgatat ccacattccc aaacacctcc ctctgcattc ctactgcttc 120

cagaatcttc cagagttcgg ttcacgtccc tgtctcatca acgccccaac gggtgaaata 180

ttcacctact ccgacgtgga actcgccgca cggagagtcg catcggggct aaaaaaacta 240

ggcatccaac acggcgatgt aatcatggtc ctcctcccaa attgccctga attcgttttc 300

tccttcctcg gcgcttcctt ttgcggcgca atcaccaccg ccgcgaaccc gttcttcacc 360

gccgcggaaa ttgccaaaca ggccaaagcc tcccatggga aggtgatcgt aacacaggct 420

tgttactacg agaaggtgaa ggacttgggt gtgaccaatc tcgtgttcgt ggattctccc 480

cctgaggggc acatgcattt cagcgagttg atggctgatg atgatgacgt catcaccggt 540

gatgaaatta agatccaccc tgatgacgtg gtggctttgc cttattcttc cgggacgacg 600

ggtctcccca aaggggtgat gctgacacac aaggggttgg taacgagcat agcacagcag 660

gtggatgggg agaacccaaa cctttactac cacagcgagg atgtgatcct ctgtgtgctt 720

cccctgtttc acatatactc cctcaactct gttctcctct gtgggttaag ggccaaagcc 780

tccatcttgt tgatgcccaa gttcgacatt catgctttct tgggtctggt tcacaggcac 840

agggtcacca ttgcaccact tgtgcccccc attgttctcg ccattgccaa gtcacctgat 900

cttgataaat atgacctctc atccattagg gtcctcaaat ctggaggggc tccccttggt 960

aaagaacttg aagacactgt cagggccaaa ttcccccaag ccaaacttgg acagggatat 1020

gggatgacgg aggcaggtcc agtgttgaca atgtgcttat catttgcaaa agtgccaata 1080

gatgtaaaac caggtgcatg tggaaccgtc gtcaggaatg cggagatgaa gattgtggat 1140

cctgaaaccg atacttcttt gcctcgaaat caacccggtg aaatctgtat tagaggcgac 1200

caaatcatga aaggttatct gaacgacccg gaagctacag agagaacaat agacaaagaa 1260

ggttggttgc atacgggtga cattgggtac attgacaatg atgatgagtt gttcatcgtt 1320

gataggctga aggaattgat taaatacaaa gggtttcaag tggctccagc tgaactcgaa 1380

gcccttattc tctcacaccc taagatctcc gatgttgctg tggtcccaat gaaggatgaa 1440

gcagctggtg aggtcccagt tgcatttgtg gtgagagcaa atggtcatat cgacacaact 1500

gaggatgaaa ttaagcaatt cgtctccaaa caggtggtgt tttacaaaag aataaacaga 1560

gtattcttca ttgatgccat tcccaagtca ccctcaggca aaatcttacg aaaggaccta 1620

agggctaagc ttgcagcggg tcttccaaat tga 1653

<210> 6

<211> 550

<212> PRT

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 6

Met Ala Ile Gln Asn Glu Met Lys Gln Gln Gln Gln Gln Ile Lys Glu

1 5 10 15

Glu Phe Ile Phe Lys Ser Lys Leu Pro Asp Ile His Ile Pro Lys His

20 25 30

Leu Pro Leu His Ser Tyr Cys Phe Gln Asn Leu Pro Glu Phe Gly Ser

35 40 45

Arg Pro Cys Leu Ile Asn Ala Pro Thr Gly Glu Ile Phe Thr Tyr Ser

50 55 60

Asp Val Glu Leu Ala Ala Arg Arg Val Ala Ser Gly Leu Lys Lys Leu

65 70 75 80

Gly Ile Gln His Gly Asp Val Ile Met Val Leu Leu Pro Asn Cys Pro

85 90 95

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

100 105 110

Thr Ala Ala Asn Pro Phe Phe Thr Ala Ala Glu Ile Ala Lys Gln Ala

115 120 125

Lys Ala Ser His Gly Lys Val Ile Val Thr Gln Ala Cys Tyr Tyr Glu

130 135 140

Lys Val Lys Asp Leu Gly Val Thr Asn Leu Val Phe Val Asp Ser Pro

145 150 155 160

Pro Glu Gly His Met His Phe Ser Glu Leu Met Ala Asp Asp Asp Asp

165 170 175

Val Ile Thr Gly Asp Glu Ile Lys Ile His Pro Asp Asp Val Val Ala

180 185 190

Leu Pro Tyr Ser Ser Gly Thr Thr Gly Leu Pro Lys Gly Val Met Leu

195 200 205

Thr His Lys Gly Leu Val Thr Ser Ile Ala Gln Gln Val Asp Gly Glu

210 215 220

Asn Pro Asn Leu Tyr Tyr His Ser Glu Asp Val Ile Leu Cys Val Leu

225 230 235 240

Pro Leu Phe His Ile Tyr Ser Leu Asn Ser Val Leu Leu Cys Gly Leu

245 250 255

Arg Ala Lys Ala Ser Ile Leu Leu Met Pro Lys Phe Asp Ile His Ala

260 265 270

Phe Leu Gly Leu Val His Arg His Arg Val Thr Ile Ala Pro Leu Val

275 280 285

Pro Pro Ile Val Leu Ala Ile Ala Lys Ser Pro Asp Leu Asp Lys Tyr

290 295 300

Asp Leu Ser Ser Ile Arg Val Leu Lys Ser Gly Gly Ala Pro Leu Gly

305 310 315 320

Lys Glu Leu Glu Asp Thr Val Arg Ala Lys Phe Pro Gln Ala Lys Leu

325 330 335

Gly Gln Gly Tyr Gly Met Thr Glu Ala Gly Pro Val Leu Thr Met Cys

340 345 350

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

355 360 365

Thr Val Val Arg Asn Ala Glu Met Lys Ile Val Asp Pro Glu Thr Asp

370 375 380

Thr Ser Leu Pro Arg Asn Gln Pro Gly Glu Ile Cys Ile Arg Gly Asp

385 390 395 400

Gln Ile Met Lys Gly Tyr Leu Asn Asp Pro Glu Ala Thr Glu Arg Thr

405 410 415

Ile Asp Lys Glu Gly Trp Leu His Thr Gly Asp Ile Gly Tyr Ile Asp

420 425 430

Asn Asp Asp Glu Leu Phe Ile Val Asp Arg Leu Lys Glu Leu Ile Lys

435 440 445

Tyr Lys Gly Phe Gln Val Ala Pro Ala Glu Leu Glu Ala Leu Ile Leu

450 455 460

Ser His Pro Lys Ile Ser Asp Val Ala Val Val Pro Met Lys Asp Glu

465 470 475 480

Ala Ala Gly Glu Val Pro Val Ala Phe Val Val Arg Ala Asn Gly His

485 490 495

Ile Asp Thr Thr Glu Asp Glu Ile Lys Gln Phe Val Ser Lys Gln Val

500 505 510

Val Phe Tyr Lys Arg Ile Asn Arg Val Phe Phe Ile Asp Ala Ile Pro

515 520 525

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

530 535 540

Ala Ala Gly Leu Pro Asn

545 550

<210> 7

<211> 1179

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 7

atggtgagtg tagctgaaat tcgcaaagct caaagggcag aaggccctgc aaacatcttg 60

gccattggca ctgcaaaccc accaaactgt gttgatcaga gtacttatcc tgatttttac 120

tttaagatca caaatagtga gcacaagacc gagcttaagg aaaaatttca gcgcatgtgt 180

gataaatcta tgatcaagaa gcgatatatg tacctaacgg aagagatttt gaaagagaat 240

cctaacattt gcgcttatat ggcaccttct ttggacgcta ggcaagacat ggtggtcgta 300

gaggtgccta gactagggaa ggaagctgcg gtcaaagcta taaaagaatg gggccaacca 360

aagtcgaaga ttacccactt aatcttttgc actactagtg gtgtggacat gcctggcgct 420

gattaccagc ttactaaact cttgggtctt cgcccatatg tgaaaaggta tatgatgtac 480

caacaagggt gctttgcagg tggcacggtg cttcgcttgg ccaaagactt ggcggagaac 540

aacaaaggtg ctcgtgtgct agttgtttgt tctgaagtta ctgcagtcac attccgtggc 600

cctactgata ctcacctaga tagccttgtg ggacaagcat tatttggaga tggagcagct 660

gcagtcattg ttggttctga cccaataccc gaaattgaga agcctatatt tgagttggtt 720

tggactgcac aaacaatagc tccagatagt gaaggagcca ttgatggtca ccttcgtgaa 780

gttgggctca catttcatct tcttaaagat gttcccggga ttgtctcaaa gaacattgat 840

aaagcactga ctgaggcatt ccaaccatta ggcatctctg attacaactc aatcttttgg 900

attgcacacc caggtggacc ggcaattctt gaccaagttg agcaaaagtt agctttgaaa 960

cctgaaaaga tgaaggccac tagggatgtg cttagtgatt atggtaacat gtcaagtgca 1020

tgtgtcctat tcatcttgga tgagatgaga aagaaatccg ctcaaaatgg acttaagacc 1080

actggcgaag ggctcgaatg gggtgtgtta ttcggctttg gacctggact taccatcgaa 1140

actgttgttt tgcacagtgt ggctacatga 1170

<210> 8

<211> 389

<212> PRT

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 8

Met Val Ser Val Ala Glu Ile Arg Lys Ala Gln Arg Ala Glu Gly Pro

1 5 10 15

Ala Asn Ile Leu Ala Ile Gly Thr Ala Asn Pro Pro Asn Cys Val Asp

20 25 30

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

35 40 45

Lys Thr Glu Leu Lys Glu Lys Phe Gln Arg Met Cys Asp Lys Ser Met

50 55 60

Ile Lys Lys Arg Tyr Met Tyr Leu Thr Glu Glu Ile Leu Lys Glu Asn

65 70 75 80

Pro Asn Ile Cys Ala Tyr Met Ala Pro Ser Leu Asp Ala Arg Gln Asp

85 90 95

Met Val Val Val Glu Val Pro Arg Leu Gly Lys Glu Ala Ala Val Lys

100 105 110

Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr His Leu Ile

115 120 125

Phe Cys Thr Thr Ser Gly Val Asp Met Pro Gly Ala Asp Tyr Gln Leu

130 135 140

Thr Lys Leu Leu Gly Leu Arg Pro Tyr Val Lys Arg Tyr Met Met Tyr

145 150 155 160

Gln Gln Gly Cys Phe Ala Gly Gly Thr Val Leu Arg Leu Ala Lys Asp

165 170 175

Leu Ala Glu Asn Asn Lys Gly Ala Arg Val Leu Val Val Cys Ser Glu

180 185 190

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

195 200 205

Leu Val Gly Gln Ala Leu Phe Gly Asp Gly Ala Ala Ala Val Ile Val

210 215 220

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

225 230 235 240

Trp Thr Ala Gln Thr Ile Ala Pro Asp Ser Glu Gly Ala Ile Asp Gly

245 250 255

His Leu Arg Glu Val Gly Leu Thr Phe His Leu Leu Lys Asp Val Pro

260 265 270

Gly Ile Val Ser Lys Asn Ile Asp Lys Ala Leu Thr Glu Ala Phe Gln

275 280 285

Pro Leu Gly Ile Ser Asp Tyr Asn Ser Ile Phe Trp Ile Ala His Pro

290 295 300

Gly Gly Pro Ala Ile Leu Asp Gln Val Glu Gln Lys Leu Ala Leu Lys

305 310 315 320

Pro Glu Lys Met Lys Ala Thr Arg Asp Val Leu Ser Asp Tyr Gly Asn

325 330 335

Met Ser Ser Ala Cys Val Leu Phe Ile Leu Asp Glu Met Arg Lys Lys

340 345 350

Ser Ala Gln Asn Gly Leu Lys Thr Thr Gly Glu Gly Leu Glu Trp Gly

355 360 365

Val Leu Phe Gly Phe Gly Pro Gly Leu Thr Ile Glu Thr Val Val Leu

370 375 380

His Ser Val Ala Thr

385

<210> 9

<211> 948

<212> DNA

<213> Artificial Sequence

<220>

<223> aritificial sequence

<400> 9

atggctgctg cccctacagt ccctgtaata gttctccctt cctcctctgg acagcggaag 60

atgccggtga tgggactcgg cacggcgccg gaagcaacca gtaaggttac cacaaaggat 120

gctgtccttg aggccatcaa gcagggttac aggcactttg atgctgctgc tgcatatggg 180

gttgagaaat cagtaggaga agccatagca gaagcactta aacttggact acttgcatcc 240

agagatgagg tcttcattac ttccaaactt tgggtcactg acaaccaccc tgaaaccatt 300

gttcctgctc tgaagaaatc tctcaggact cttcaactag aatacttaga cctcattttg 360

atccactggc ccattgctac aaaaccagga gaagttaaat accctattga tgtatcagat 420

attgtggagt ttgacatgaa gggtgtgtgg ggatcattgg aggaatgtca aagacttggt 480

ctcaccaaag ccattggagt cagcaacttc tctatcaaga agcttgaaaa attgctctcc 540

tttgccacca tccctcctgc agtaaatcaa gtggaagtca accttggttg gcaacaagag 600

aaacttagag ctttctgcaa ggaaaagggt attgtcataa ctgctttctc acccctgagg 660

aaaggtgcca gtaggggttc taatttggtg atggacaatg atgtgctgaa agaaattgca 720

gatgctcatg gcaagactat agctcagatt tgtcttcgat ggttatatga acaaggcttg 780

acatttgtgg tgaagagcta tgacaaggag aggatgaatc aaaacttgca gatctttgat 840

tggtcattga ctgaggatga ctacaagaaa ataagtgaaa tctatcaaga gaggctcatc 900

aaaggtccaa ccaagcctct tcttgatgac ctgtgggatg aagaatga 948

<210> 10

<211> 315

<212> PRT

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 10

Met Ala Ala Ala Pro Thr Val Pro Val Ile Val Leu Pro Ser Ser Ser

1 5 10 15

Gly Gln Arg Lys Met Pro Val Met Gly Leu Gly Thr Ala Pro Glu Ala

20 25 30

Thr Ser Lys Val Thr Thr Lys Asp Ala Val Leu Glu Ala Ile Lys Gln

35 40 45

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

50 55 60

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

65 70 75 80

Arg Asp Glu Val Phe Ile Thr Ser Lys Leu Trp Val Thr Asp Asn His

85 90 95

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

100 105 110

Leu Glu Tyr Leu Asp Leu Ile Leu Ile His Trp Pro Ile Ala Thr Lys

115 120 125

Pro Gly Glu Val Lys Tyr Pro Ile Asp Val Ser Asp Ile Val Glu Phe

130 135 140

Asp Met Lys Gly Val Trp Gly Ser Leu Glu Glu Cys Gln Arg Leu Gly

145 150 155 160

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

165 170 175

Lys Leu Leu Ser Phe Ala Thr Ile Pro Pro Ala Val Asn Gln Val Glu

180 185 190

Val Asn Leu Gly Trp Gln Gln Glu Lys Leu Arg Ala Phe Cys Lys Glu

195 200 205

Lys Gly Ile Val Ile Thr Ala Phe Ser Pro Leu Arg Lys Gly Ala Ser

210 215 220

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

225 230 235 240

Asp Ala His Gly Lys Thr Ile Ala Gln Ile Cys Leu Arg Trp Leu Tyr

245 250 255

Glu Gln Gly Leu Thr Phe Val Val Lys Ser Tyr Asp Lys Glu Arg Met

260 265 270

Asn Gln Asn Leu Gln Ile Phe Asp Trp Ser Leu Thr Glu Asp Asp Tyr

275 280 285

Lys Lys Ile Ser Glu Ile Tyr Gln Glu Arg Leu Ile Lys Gly Pro Thr

290 295 300

Lys Pro Leu Leu Asp Asp Leu Trp Asp Glu Glu

305 310 315

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> aitificial sequence

<400> 11

atgccccatt ctctctccct cttcc 25

<210> 12

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 12

ttaacaaatt ggaagaggtg caccattc 28

<210> 13

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 13

taacatttaa ctccctaccc atttgc 26

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 14

ccacccctcc attttcccac tac 23

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 15

cttccaccac ccttacccac tc 22

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 16

gaagcataag cgggcatcat aaaat 25

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 17

atggtgacag ttgaagagat ccgc 24

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 18

tcaattagcc tgcaagggaa cactg 25

<210> 19

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 19

ttacaatacc aagagcagct accact 26

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 20

gttcattctt catcccacag gtcat 25

<210> 21

<211> 2127

<212> DNA

<213> Artificial Sequence

<220>

<223> artificial sequence

<400> 21

atggtgagtg tagctgaaat tcgcaaagct caaagggcag aaggccctgc aaacatcttg 60

gccattggca ctgcaaaccc accaaactgt gttgatcaga gtacttatcc tgatttttac 120

tttaagatca caaatagtga gcacaagacc gagcttaagg aaaaatttca gcgcatgtgt 180

gataaatcta tgatcaagaa gcgatatatg tacctaacgg aagagatttt gaaagagaat 240

cctaacattt gcgcttatat ggcaccttct ttggacgcta ggcaagacat ggtggtcgta 300

gaggtgccta gactagggaa ggaagctgcg gtcaaagcta taaaagaatg gggccaacca 360

aagtcgaaga ttacccactt aatcttttgc actactagtg gtgtggacat gcctggcgct 420

gattaccagc ttactaaact cttgggtctt cgcccatatg tgaaaaggta tatgatgtac 480

caacaagggt gctttgcagg tggcacggtg cttcgcttgg ccaaagactt ggcggagaac 540

aacaaaggtg ctcgtgtgct agttgtttgt tctgaagtta ctgcagtcac attccgtggc 600

cctactgata ctcacctaga tagccttgtg ggacaagcat tatttggaga tggagcagct 660

gcagtcattg ttggttctga cccaataccc gaaattgaga agcctatatt tgagttggtt 720

tggactgcac aaacaatagc tccagatagt gaaggagcca ttgatggtca ccttcgtgaa 780

gttgggctca catttcatct tcttaaagat gttcccggga ttgtctcaaa gaacattgat 840

aaagcactga ctgaggcatt ccaaccatta ggcatctctg attacaactc aatcttttgg 900

attgcacacc caggtggacc ggcaattctt gaccaagttg agcaaaagtt agctttgaaa 960

cctgaaaaga tgaaggccac tagggatgtg cttagtgatt atggtaacat gtcaagtgca 1020

tgtgtcctat tcatcttgga tgagatgaga aagaaatccg ctcaaaatgg acttaagacc 1080

actggcgaag ggctcgaatg gggtgtgtta ttcggctttg gacctggact taccatcgaa 1140

actgttgttt tgcacagtgt ggctacaggt ggtggttcta tggctgctgc ccctacagtc 1200

cctgtaatag ttctcccttc ctcctctgga cagcggaaga tgccggtgat gggactcggc 1260

acggcgccgg aagcaaccag taaggttacc acaaaggatg ctgtccttga ggccatcaag 1320

cagggttaca ggcactttga tgctgctgct gcatatgggg ttgagaaatc agtaggagaa 1380

gccatagcag aagcacttaa acttggacta cttgcatcca gagatgaggt cttcattact 1440

tccaaacttt gggtcactga caaccaccct gaaaccattg ttcctgctct gaagaaatct 1500

ctcaggactc ttcaactaga atacttagac ctcattttga tccactggcc cattgctaca 1560

aaaccaggag aagttaaata ccctattgat gtatcagata ttgtggagtt tgacatgaag 1620

ggtgtgtggg gatcattgga ggaatgtcaa agacttggtc tcaccaaagc cattggagtc 1680

agcaacttct ctatcaagaa gcttgaaaaa ttgctctcct ttgccaccat ccctcctgca 1740

gtaaatcaag tggaagtcaa ccttggttgg caacaagaga aacttagagc tttctgcaag 1800

gaaaagggta ttgtcataac tgctttctca cccctgagga aaggtgccag taggggttct 1860

aatttggtga tggacaatga tgtgctgaaa gaaattgcag atgctcatgg caagactata 1920

gctcagattt gtcttcgatg gttatatgaa caaggcttga catttgtggt gaagagctat 1980

gacaaggaga ggatgaatca aaacttgcag atctttgatt ggtcattgac tgaggatgac 2040

tacaagaaaa taagtgaaat ctatcaagag aggctcatca aaggtccaac caagcctctt 2100

cttgatgacc tgtgggatga agaatga 2127

Claims

1. a kind of fusion protein, the fusion protein includes:

(1) chalcone synthase (CHS)；With

(2) chalcone reductase (CHR)；

Between the chalcone synthase and chalcone reductase by a connexon connect, the connexon be GGGS, GSG, Any one in GSGGGGS, GSGEAAAK, GSGEAAAKEAAAK or GSGMGSSSN.

2. fusion protein according to claim 1, the fusion protein includes:

(1) CHS of amino acid sequence shown in SEQ ID NO:8；With

(2) CHR of amino acid sequence shown in SEQ ID NO:10；

Between the CHS and CHR by a connexon connect, the connexon be GGGS, GSG, GSGGGGS, GSGEAAAK, Any one in GSGEAAAKEAAAK or GSGMGSSSN, preferably GGGS.

3. encoding the polynucleotides of fusion protein as claimed in claim 1 or 2.

4. polynucleotides described in claim 3, wherein the polynucleotides include: chalcone synthase described in SEQ ID NO:7 The reduction of chalcone described in encoding gene (CHS) and SEQ ID NO:9 enzyme coding gene (CHR).

5. recombinant expression carrier, it includes promoter, the polynucleotides of claim 3 or 4 and transcription terminator, the expression Promoter, the polynucleotides of claim 3 or 4, terminator and episomal vector are preferably used yeast homologous weight by carrier Group method be stitched together, the episomal vector be Yeast expression carrier, as pESC, pYX212, pYES2.0, pRS425, PRS426 or p424；Preferably, the pESC expression vector is selected from pESC-Leu, pESC-His or pESC-Trp.

6. recombinant expression carrier according to claim 5, wherein the expression vector is any one in following:

(1) recombinant expression carrier pYM3, it includes CHS and CHR, the CHS passes through linker sequence at 3 ' ends 5 ' the ends that GGTGGTGGTTCT is connected to CHR form CHS::CHR, and the CHS::CHR is inserted into expression vector pESC-Trp's The downstream promoter GAL10；

(2) coumaric acyl of amino acid sequence shown in recombinant expression carrier pYM2, packet encoder SEQ ID NO:6-CoA ligase is compiled Code gene (4CL), CHS and CHR, the CHS are connected to 5 ' the end shapes of CHR at 3 ' ends by linker sequence GGTGGTGGTTCT At CHS::CHR, the downstream promoter GAL1 of the 4CL insertion Yeast expression carrier pESC-Leu, the CHS::CHR is inserted into The downstream promoter GAL10 of expression vector pESC-Leu.

7. the yeast engineering comprising the polynucleotides of claim 3 or 4 or the recombinant expression carrier of claim 5 or 6 Bacterium.

8. Yeast engineering bacteria according to claim 7, wherein the Yeast engineering bacteria further includes expression vector pYM1, The expression vector pYM1 includes the phenylalanine lyase encoding gene of amino acid sequence shown in coding SEQ ID NO:2 (PAL) and coding SEQ ID NO:4 shown in amino acid sequence cinnamic acid 4-hydroxylase encoding gene (C4H), the PAL and C4H is inserted into the downstream of the promoter GAL1 and GAL10 of expression vector pESC-His according to this.

9. Yeast engineering bacteria according to claim 7 or 8, the construction method of the Yeast engineering bacteria are as follows:

(1) recombinant expression carrier pYM3 is transferred in Yeast engineering bacteria WAT11, obtains bacterial strain WM4；

(2) recombinant expression carrier pYM2 is transferred in Yeast engineering bacteria WAT11, obtains bacterial strain WM3；

(3) recombinant expression carrier pYM1 and pYM2 are transferred in Yeast engineering bacteria WAT11, obtain bacterial strain WM2-1；

(4) recombinant expression carrier pYM1, pYM2 and pYM3 are transferred in Yeast engineering bacteria WAT11, obtain bacterial strain WM2-2.

10. institute in fusion protein as claimed in claim 1 or 2 or the polynucleotides of claim 3 or 4 or claim 5 or 6 Yeast engineering bacteria described in any one claim is stated in recombinant expression carrier or claim 7-9 in production chalcone or different sweet Application in careless element.