CN106701800B - A kind of Aureobasidium pullulans polyketide synthases gene and its application - Google Patents

Abstract

The present invention relates to a kind of polyketide synthases gene, more particularly to a kind of related polyketide synthases gene of fungi B16 cell and its application, belong to gene engineering technology field.A kind of polyketide synthases gene, nucleotide sequence is as shown in SEQ ID No.1.The polyketide synthases of the gene code, amino acid sequence is as shown in SEQ ID No.2.Polyketide synthases gene of the present invention can be used for external synthesis of melanin.After testing, melanin content is 35.6g/L in external enzyme reaction solution.

Description

A kind of Aureobasidium pullulans polyketide synthases gene and its application

Technical field

The invention belongs to genetic engineering field, and in particular to a kind of Aureobasidium pullulans (Aureobacidium Pullulans) polyketide synthases gene and its application.

Background technology

Melanin is the protectiveness pigment being widely present in organism, with bio-semiconductor, removes free radical, UV resistance The functions such as beta radiation, chelating heavy metal ion, in various fields such as daily cosmetics industry, food industry, medical industry, agriculturals There is important purposes.According to source, melanin can be divided into Animal melanin, plant melanin and microorganism melanin；According to change Eumelanin (eumelanin), pheomelanins (phaeomelanin) and allpmelanin can be divided into by learning composition (allomelanin).Eumelanin is carbon containing, hydrogen, oxygen, nitrogen, not sulfur atom-containing, and color is dark-brown or black, is primarily present in It is that tyrosine is poly- via the conversion of DOPA (Dopa) approach in the black plumage of animal, poultry or blue plumage, eye, skin and related tissue Symphysis into.Pheomelanins are carbon containing, hydrogen, oxygen, nitrogen, sulphur atom, and color is reddish brown and yellow, is primarily present in poultry In the tissue such as rufous, light yellow feather, hair and fine hair, route of synthesis and eumelanin are approached, difference be cystine or Glutathione has participated in building-up process.Allpmelanin is carbon containing, hydrogen, oxygen, not nitrogenous, sulphur atom, color be brown or black, mainly It is present in plant and microorganism, is that aldehydes matter aggregated and oxidation under polymerase and oxidation enzyme effect is formed, studies table Melanin majority is using dihydroxy naphthlene (DHN) type melanin as main structural framework in bright plant and fungi.

Prepared by current melanin mainly plants skin and animal hair, epidermis separation and Extraction, yield from black soya bean, Semen sesami nigrum etc. Low, purity is low, cost is high.With the continuous intensification that people are understood melanin, its functional study is also increasingly wide with application It is wealthy so that people increasingly increase the demand of melanin, only the market demand can not be met by limited extraction, in the urgent need to opening The preparation method for sending out new.Black yeast fermentation is extracted has the cycle short with the external synthesis of melanin of enzyme process, can overcome weather and the place of production Limitation, substantially reduces production cost, thus receives significant attention.

Polyketide synthases (polyketide synthase) are the complicated multi-enzyme system of a class, the polyketone having now been found that Synzyme can be roughly divided into 3 types, the i.e. modular type of I types, II type iterative types, its alloytype of type III.Type III polyketide synthases include 1, 3,6,8- tetrahydroxy naphthalenes synzyme, chalcone synthase etc..The first step of DHN type B16 cells is 1 in polyketide synthases, 3,6,8- tetrahydroxy naphthalenes synthesis enzymatic malonyl coenzyme A synthesizes 1,3,6,8- tetrahydroxy naphthalenes, and 1,3,6,8- tetrahydroxy naphthalene is successively In small spore post ketone dehydratase (scytalone dehydratase), hydroxyl naphthalene reductase (HN reductase) and polyphenol oxidase The lower synthesis DHN type melanin of (polyphenol oxidase) effect.

It is relevant poly- in fungi but the polyketide synthases gene report related to DHN B16 cells focuses mostly in bacterium The report of ketone synthase gene and DHN B16 cells is seldom, and Aureobasidium pullulans belongs to mould in taxonomy, is fungi One kind, the report of the polyketide synthases related on DHN B16 cells in Aureobasidium pullulans few at present.And it is international The upper polyketide synthases for carrying out melanin PRODUCTION TRAITS are less, limited source, and new melanin is efficiently synthesized accordingly, it would be desirable to develop Enzyme source.

The content of the invention

For above-mentioned prior art, it is an object of the present invention to provide a kind of Aureobasidium pullulans polyketide synthases gene.

The Aureobasidium pullulans polyketide synthases gene that the present invention is provided, is the DNA molecular of following (1) or (2)：

(1) DNA molecular in sequence table shown in SEQ ID NO.1；

(2) divide under strict conditions with the DNA sequence dna hybridization shown in SEQ ID NO.1 and the DNA of coding polyketide synthases Son.

Above-mentioned polyketide synthases gene source is in (the purchase of Aureobasidium pullulans (Aureobasidium pullulans) 3.3984 In China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium numbering is that 3.3984), full length gene is 6492bp。

It is a further object of the present invention to provide the polyketide synthases by above-mentioned Aureobasidium pullulans polyketide synthases gene code.

The polyketide synthases that the present invention is provided, are the protein of following (a) or (b)：

(a) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；

(b) by the amino acid sequence shown in SEQ ID NO.2 is by the substitution of one or several amino acid residues and/or lacks Lose and/or addition and the protein as derived from sequence 1 with polyketide synthases activity.

Albumen shown in SEQ ID NO.2 is made up of 2163 amino acid residues.

Recombinant expression carrier containing above-mentioned Aureobasidium pullulans polyketide synthases gene falls within protection scope of the present invention.

The recombinant expression carrier can contain the recombinant expression carrier of the gene with existing expression vector establishment.

Above-mentioned Aureobasidium pullulans polyketide synthases gene is concretely inserted into pGAPZ α-A by the recombinant expression carrier The multiple cloning sites of expression vector obtain recombinant plasmid.

Expression cassette, transgenic cell line and recombinant bacterium containing above-mentioned Aureobasidium pullulans polyketide synthases gene belong to this The protection domain of invention.

The recombinant bacterium can be that the recombinant expression carrier is converted into the recombinant bacterium that Pichia pastoris SMD1168H is obtained.

The present invention also provides above-mentioned Aureobasidium pullulans polyketide synthases gene and/or polyketide synthases are closed outside melanosome Application in.

Third object of the present invention is to provide a kind of method synthesized outside melanosome.

The method that the present invention is provided, comprises the following steps：Recombinant bacterium described in fermented and cultured, collects fermented supernatant fluid, precipitation Protein in supernatant, then desalination and concentration are carried out to albumen precipitation, produce polyketide synthases crude enzyme liquid；By polyketide synthases Crude enzyme liquid is mixed with Aureobasidium pullulans crude enzyme liquid, malonyl coenzyme A, adjusts reaction under the conditions of pH to 8.0,38~40 DEG C, reaction Period carries out abundant oxygen supply, reacts 40~60 minutes；After reaction terminates, pH to 2.0~3.0 is adjusted, centrifugation is collected precipitation, obtained To melanin crude product.

The Aureobasidium pullulans crude enzyme liquid is the new fresh thalli of Aureobasidium pullulans through liquid nitrogen grinding broken wall, 50mM sodium phosphate buffers Liquid (pH8.0) weight bacteriolyze powder, 4000rpm centrifuges 5 minutes supernatants obtained.Because DHN B16 cells are related to much instead Step is answered, polyketide synthases crude enzyme liquid catalyzes and synthesizes the first step that 1,3,6,8- tetrahydroxy naphthalenes are reactions, and Aureobasidium pullulans is thick Enzyme liquid includes a variety of enzymes required for DHN B16 cells, by adding Aureobasidium pullulans crude enzyme liquid, it is therefore an objective to provide DHN black The enzyme of pigment subsequently required for synthesis, makes 1,3,6,8- tetrahydroxy naphthalenes be converted into melanin.

The method that the present invention is provided, in addition to：The step of melanin crude product is purified, it is specific as follows：By black Plain crude product is dissolved in 1mol/L NaOH, adds 2 times of volume acidic methanols (pH=2), stands 4h, 6000r/min centrifugations After 10min, precipitation is collected, precipitation is washed with distilled water to neutrality, 60 DEG C of drying produce melanin.

Beneficial effects of the present invention：

(1) the isolated polyketide synthases gene from Aureobasidium pullulans first of the invention, polyketide synthases gene warp Be cloned on pGAPZ α-A expression vectors, convert Pichia pastoris SMD1168H, obtain polyketide synthases, available for melanosome outside Synthesis, new enzyme source and synthetic method are provided for synthesis outside melanosome.

(2) present invention also offers a kind of method of external synthesis of melanin, synthesized by fermentation containing polyketone of the present invention The recombinant bacterial strain of enzyme gene, obtains polyketide synthases crude enzyme liquid；Further mixed with Aureobasidium pullulans crude enzyme liquid, malonyl coenzyme A Reaction is closed, and by the optimization to reaction condition, substantially increases the yield of the melanin of external enzyme reaction synthesis.

(3) polyketide synthases of the invention are with a wide range of applications, available for fields such as pharmacy, cosmetics, food.

Brief description of the drawings

Fig. 1：The restructuring external B16 cell reaction product figure of polyketide synthases of the present invention；

Wherein, before reaction：0.5mol/L is added after restructuring polyketide synthases crude enzyme liquid and the mixing of Aureobasidium pullulans crude enzyme liquid Malonyl coenzyme A, reacts 0 minute.

Control group：The α of pGAPZ containing empty carrier-A Pichia pastoris SMD1168H zymotic fluids crude enzyme liquid and the thick enzyme of Aureobasidium pullulans 0.5mol/L malonyl coenzyme As are added after liquid mixing, 38 DEG C are reacted 60 minutes；

Experimental group：0.5mol/L malonyl is added after recombinating polyketide synthases crude enzyme liquid and the mixing of Aureobasidium pullulans crude enzyme liquid Coacetylase, 38 DEG C are reacted 60 minutes.

Embodiment

Involved instrument, reagent, material etc. in following embodiments, are existing in the prior art unless otherwise noted Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Involved experimental method in following embodiments, inspection Survey method etc., is existing normal experiment method, detection method in the prior art unless otherwise noted.

Embodiment 1：The PCR amplifications of Aureobasidium pullulans polyketide synthases gene

By China Committee for Culture Collection of Microorganisms's common micro-organisms center (CMGCC), bacterium numbering is As3.3984 Aureobasidium pullulans nutrient solution 5mL, Total RNAs extraction is carried out with RNA extracts kits, extraction step is with reference to Takara The RNA extracts kit specifications of company.Reverse transcription is carried out with reverse transcription reagent box by template of the total serum IgE of extraction, is obtained CDNA, reverse transcription reagent box specification of the reverse transcription step with reference to Takara companies.

Retrieval belongs to the nucleotide sequence of different strains polyketide synthases gene, design pair of primers F-I together from GenBank And R-I.Primer sequence is as follows：

F-I：5’-ATGTCTAACGTTCTTCTTTT-3’ SEQ ID NO.3；

R-I：5’-GATCTGAAGACCTTCTTGGA-3’ SEQ ID NO.4；

Expanded, expanded using above-mentioned primer by template of cDNA.Cumulative volume is 50 μ L, ultra-pure water 18 μ L, 10 × LAtaq buffer (contain MgCl₂) 5 μ L, dNTP (each 2.5mmol/L) 4 μ L, primer (20 μm of ol/L) each 1 μ L, cDNA 25ng, TaKaRa LA Taq enzymes 2.5U.PCR amplification conditions：95 DEG C of pre-degenerations 5 minutes, react 30 circulations (95 DEG C of denaturation 30 Second, 55 DEG C are annealed 30 seconds, and 72 DEG C extend 6.5 minutes), 72 DEG C extend 10 minutes after 30 circulations terminate.PCR primer is through agarose Gel electrophoresis, ethidium bromide staining, uv analyzer detection carries out PCR fragment with Bioflux gel reclaims kits and cuts glue time Receive, sequencing size is 6492bp.Products therefrom is Aureobasidium pullulans polyketide synthases genetic fragment, measures sequence such as SEQ ID No.1。

This section of nucleotide sequence total length 6492bp, is a complete ORF, encodes 2163 amino acid.The 6492bp's ORF is Aureobasidium pullulans polyketide synthases gene, and the amino acid sequence of its nucleotide sequence and coding is respectively SEQ ID No.1 and SEQ ID No.2.

Embodiment 2：Expression of the Aureobasidium pullulans polyketide synthases gene in Pichia pastoris SMD1168H

Primers F-I-A and R-I-A according to SEQ ID No.1, pGAPZ α-A matter can be inserted respectively by introducing SacII, XbaI enzyme cutting site (shown in the underscore) of grain (being purchased from Invitrogen companies of the U.S.), sequence is as follows

F-I-A：5’-GCGCCGCGGCATGTCTAACGTTCTTCTTTT-3’ SEQ ID NO.5；

R-I-A：5’-GCGTCTAGAATGATCTGAAGACCTTCTTGGA-3’ SEQ ID NO.6；

Aureobasidium pullulans cDNA using reverse transcription enters performing PCR amplification as template using above-mentioned primer.PCR amplification conditions and PCR primer recovery method be the same as Example 1.After PCR primer is reclaimed plus NdeI, SalI (NEB, USA) were in 37 DEG C of digestions 1 hour, enter Row DNA is purified, and pGAPZ α-A plasmid vectors are handled using same enzymatic cleavage methods.By the enzyme gene fragment prepared and pGAPZ α-A Carrier is mixed by a certain percentage, is connected 18 hours in 16 DEG C using T4 ligases (NEB, USA), then using CaCl₂Conversion method Convert Host Strains Escherichia coli Top10, transformed bacteria solution coating zeocin (25 μ g/mL) resistance LB flat boards, 37 DEG C of incubated overnights.Choose Take well-grown single bacterium to drop down onto in LB fluid nutrient mediums 37 DEG C to cultivate 10 hours, extract plasmid, tested by enzyme cutting method and PCR methods Positive transformant is demonstrate,proved, recombinant plasmid pGAPZ α-A-alb are obtained.

Above-mentioned recombinant plasmid pGAPZ α-A-alb were carried out with AvrII restriction endonucleases (NEB, USA) in 37 DEG C of digestions 1 hour DNA is purified.Then the pGAPZ α-A-alb of linearisation are converted into host Pichia pastoris SMD1168H, conversion using electric method for transformation Bacterium solution is coated with zeocin (100 μ g/mL) resistance YPD flat boards, and 30 DEG C are cultivated 3 days.The well-grown single bacterium of picking drops down onto YPD liquid Cultivated 3 days for 30 DEG C in culture medium, extract genome, positive transformant is verified by PCR methods, recombinant yeast pichia pastoris is obtained SMD1168H-alb。

Above-mentioned LB nutrient solutions composition：Peptone 10g/L, dusty yeast 5g/L, NaCl 5g/L, pH7.0~7.5,121 DEG C goes out Bacterium 20 minutes.

Above-mentioned YPD nutrient solutions composition：Peptone 20g/L, dusty yeast 10g/L, glucose 20g/L, natural pH, 115 DEG C go out Bacterium 30 minutes.Wherein glucose individually prepares sterilizing, and 115 DEG C sterilize 30 minutes, are mixed after taking-up with other compositions.

Embodiment 3：Aureobasidium pullulans polyketide synthases catalysed in vitro synthesis of melanin

The recombinant yeast pichia pastoris SMD1168H-alb obtained in embodiment 2 is inoculated into YPD fluid nutrient mediums, 30 DEG C are shaken Bed culture 3 days, rotating speed 200rpm.Cultured zymotic fluid 8000rpm centrifuges to collect for 5 minutes slowly to be added in supernatant, supernatant Plus ammonium sulfate, to 80% concentration, 8000rpm is centrifuged 5 minutes and is collected albumen precipitation.Albumen precipitation 50mM sodium phosphate buffers (pH8.0) desalination and concentration are carried out with 100KD super filter tubes after weight is molten, finally makes protein solution body compared with above-mentioned fermented supernatant fluid 1000 times of product concentration.The protein solution obtained by desalination and concentration is restructuring polyketide synthases crude enzyme liquid.

Aureobasidium pullulans As3.3984 inoculations are into YPD fluid nutrient mediums, 30 DEG C of shaking table cultures 2 days, rotating speed 200rpm.Cultured zymotic fluid 8000rpm is centrifuged 5 minutes, abandons supernatant, is collected bacterial sediment and is placed in mortar.In mortar plus Enter appropriate liquid nitrogen, quickly grind thalline, liquid nitrogen is rejoined after liquid nitrogen volatilization is finished, continue to be ground to thalline in powdered. With 50mM sodium phosphate buffers (pH8.0) the weight bacteriolyze powder of 1/1000 fermentating liquid volume, 4000rpm is centrifuged 5 minutes, and supernatant is For Aureobasidium pullulans crude enzyme liquid.

Take above-mentioned restructuring polyketide synthases crude enzyme liquid 1mL, Aureobasidium pullulans crude enzyme liquid 1mL, 2.5mol/L malonyl coenzyme A 0.5mL is mixed, and NaOH adjusts pH to 8.0, and 38 DEG C of insulations are reacted for 60 minutes, during which carries out abundant oxygen supply.Reaction mixture face Color is gradually deepened until black.After question response terminates, adjusted after pH to 2.5,6000r/min centrifugations 10min, received with 6mol/L HCl Collection precipitation, obtains melanin crude product.Melanin crude product is dissolved in 1mol/L NaOH, 2 times of volume acidic methanols are added (pH=2), stand after 4h, 6000r/min centrifugations 10min, collect precipitation, precipitation is washed with distilled water to neutrality, 60 DEG C of bakings It is dry, melanin is produced, the yield of melanin is 35.6g/L.

Experimental example：The external enzyme reaction of control group

It is related to polyketide synthases by the synthesis of the external melanin of control group experimental verification to set the experimental example purpose.

Experimental group vitro reactions enzyme liquid and reaction condition be the same as Example 3.

Control group difference compared with experimental group is：With the Pichia pastoris SMD1168H fermentations containing empty carrier pGAPZ α-A Liquid crude enzyme liquid is mixed with Aureobasidium pullulans crude enzyme liquid described in embodiment 3 instead of restructuring polyketide synthases crude enzyme liquid and the short stalk that sprouts Mould crude enzyme liquid.

The above-mentioned Pichia pastoris SMD1168H zymotic fluid crude enzyme liquid preparation methods containing empty carrier pGAPZ α-A are：By zero load Electricity conversion Pichia pastoris SMD1168H after body pGAPZ α-A are linearized with AvrII restriction endonucleases, obtains recombinant bacterium, recombinant bacterium inoculation The culture of YPD fluid nutrient mediums collects supernatant after 3 days, is produced after ammonium sulfate precipitation, super filter tube desalination and concentration.Concrete operations are walked Rapid reference embodiment 2 and embodiment 3.

After reaction terminates, the reaction solution color regime of observation contrast control group and experimental group, control group color is light gray, Experimental group color is black (Fig. 1).Melanin in reaction solution is carried out to weigh after purifying is dried, control group obtains melanin 2mg, Experimental group obtains melanin 89mg.Vitro reactions system is 2.5mL, and melanin content is respectively pair in vitro reactions liquid after conversion According to a group 0.8g/L, experimental group 35.6g/L.

Interpretation of result：Control group vitro reactions also create a small amount of melanin, during this is due to Aureobasidium pullulans crude enzyme liquid Containing a small amount of polyketide synthases, melanin can be catalyzed and synthesized under the conditions of enzyme reaction in vitro.By adding weight in experimental group Group polyketide synthases, the melanin production of external enzyme reaction synthesis is 35.6g/L, is 44.5 times of control group.

Comparative example 1：

With the Aureobasidium melanogenum strain XJ5-1polyketide described in NCBI Synthase gene as a comparison, the Sequence ID of the gene:KT429644.1, sequence length is 6498bp.By implementation The method of example 2 expresses the gene in Pichia pastoris SMD1168H；Catalyze and synthesize black in vitro as described in Example 3 Element.After testing, the yield of melanin is 14.6g/L.

Comparative example 2：

With the Aureobasidium namibiae CBS 147.97polyketide synthase described in NCBI Partial mRNA as a comparison, the Sequence ID of the gene:XM_013576562.1, sequence length is 6495bp.By reality The method for applying example 2 expresses the gene in Pichia pastoris SMD1168H；Catalyze and synthesize black in vitro as described in Example 3 Element.After testing, the yield of melanin is 0.9g/L.

The Aureobasidium pullulans polyketide synthases coded by said gene of the present invention is poly- it can be seen from comparative example 1 and comparative example 2 Ketone synzyme has very high catalytic activity, by by the Aureobasidium pullulans polyketide synthases gene of the present invention through recombinating table Reach, and for external synthesis of melanin, the yield of melanin can be significantly improved.

The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto, It is any to be familiar with those skilled in the art in the technical scope that the present invention is disclosed, technique according to the invention scheme and its invention Design is subject to equivalent substitution or change, should all be included within the scope of the present invention.

SEQUENCE LISTING

<110>Shandong University；Shandong Fu Ruida Pharmaceutical Groups company

<120>A kind of Aureobasidium pullulans polyketide synthases gene and its application

<130> 2016

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 6492

<212> DNA

<213>Aureobasidium pullulans（Aureobasidium pullulans）

<400> 1

atgtctaacg ttcttctttt cggcgaccag accgccgagc agtaccctct gctccgcaag 60

gtggtcctga gaactaagaa tgctctcgtt ctcaacttcc tggagcgcac cgtcgtcgct 120

ctgcgaggag aaattgccca actctctcgc cctcagcgcg atgccattcc ggatttcatg 180

accctcaaca acttggtcga tctctactac gagaagggat tgaagcttcc tcaccttgag 240

agtgccctgg tcactatcgc ccagcttggc cactacatcg gatacttctc cgagaagact 300

aacgagcttc ctcctgctgc caacactcgc attcttgctc tctgcactgg atcactcgcc 360

gctgccgctg tcgcttccgc cagaactctt gatgagctcg tcactcttgg tgtcgagacc 420

gtccgcatcg ccttccgcac tggtgtccgc gtcaatgaag ccagaactgc tttgggccag 480

gacctcatgt ccagggagag ctggtctact attgtcactg gcattaacga gcagtccgcc 540

aaggaggctc tcaacgcttt ccacgagtct gctggcattc ccactaccag ccacgcttac 600

atcagcgctg ttagcaccat ggccatcact atcagtggtc ctcccaccat cactaagcgc 660

ttcttcgagg agtctgaggc tgttcgcaag aacaaccgtg ttaagatccc cgtttacgct 720

ccttaccacg ccgagcacct ctacagcagt gccgatatcg agaccgtcat tggcgaggag 780

tctgctaagc tcctccaggc ttaccagcct cgttcgttgg tccactccgg ctcatctggc 840

atgtgccaca ttgccgagaa caccttcgag ctgttcaagt tgtccctgaa cgacatgctc 900

agatcccctg ttggctggaa caacctcctc gaggagagtg tttcgcagat caccgccaac 960

accaacgctc ctgctaaggt cttctgcatt ggtgtcagca acgtatccaa cagcctcgtc 1020

tcggccatca aagctggtgg ccaggcctcg gtctccctcg ttgaccactc tgcctgggag 1080

tctgccgtca ctgatgccag ccacggtcgc acccagaacg acaagattgc catcgttggt 1140

atgtctggtc gtttccccag cgctgccagc accgaggctc tttgggaact tctcgagaag 1200

ggtctcgatg tccaccgcaa gattccctct gaccgtttcg atgccgacgc tcactgcgac 1260

ccctctggca agggcaagaa caagtcgcac actccttacg gttgctttat cgacgagcct 1320

ggtcttttcg accctcgctt cttcaacatg tctcctcgtg aggctgcaca gactgatcct 1380

atgggtcgtc tggctctcgt caccgcttac gaggctcttg agcaatctgg ttacgtcccc 1440

aaccgtactc cctccaccaa gcttcaccgc atcggtacct tctacggtca gacctctgac 1500

gattggcgtg agatcaacgc ttccgagaat gttgacacct acttcatcac tggtggtgtg 1560

cgtgctttcg cacctggtcg catcaactac tacttcaagt tcagtggtcc ttcgttctcg 1620

atcgatactg cttgttcgtc atctttggct gctattcagc ttgcctgcac ttcgctgtgg 1680

gccggtgact gcgacactgc ctgtgctggt ggtctgaacg tccttaccaa ccctgacatt 1740

ttctctggtc tttccaaggg tcaattcttg tccaagacag gttcttgcaa gacttacgac 1800

aacgctgccg atggttactg ccgtggtgat ggttgtggta ccgttgtcct caagagatac 1860

gaggatgcgc ttgccgacaa ggacaacatc ctcggatgca ttcttggcgc tgctaccaac 1920

cactctgctg aggctgtttc tattactcac cctcacgctg gcgctcagga gttcctttac 1980

aagaaggtcc tcgccaacgc tggtgtcgat gcccatgaga ttagctacgt cgagatgcac 2040

ggtactggta cccaggctgg tgatggtatt gagatgacct cagtcaccaa cgtcttcgct 2100

ccccgtcacc gccagcgcaa gcctgaggag aagctccacc ttggtgccat caaggccaac 2160

atcggccacg gtgaagccgc ttctggtatc aactccctct gcaaggtctt gatgatgatg 2220

aagaagaacg ccattcctgc caacgttggt atcaagggcg agatcaacaa gactttcccc 2280

aaggacttga aggaccgcaa tgtccacatt cctcagcagc agatcgactt cccccgcaac 2340

ggtgctgaga agcgcaagat cttcctcaac aacttctccg ctgctggtgg taacaccgcc 2400

atcctcctcg aggatggtcc tcttcgtgag gctcctaagg ctgttgaccc tagaggcact 2460

cttcccgtca ctgtaactgc ccgttcgatc gctgccttga agaacaacat tcagcgtctg 2520

cagggatacc tccgcgagaa ccccgagacc actcttccca gcttgtccta cacttcgact 2580

gcccgccgca tccagcacaa ctaccgcatc gccttcccca ttgctgatat tgccaaggtc 2640

agcgatgctc tgcaggccca aactaaggac acctacagcc cagtccccat ggtttctacc 2700

aagaccgcct tctgcttcac cggacagggc tcacagtaca ctgctcttgg tcagaagctc 2760

taccaagacc tcaagtcttt ccgtgacgac atcaaccagc tcgacaacat ggccaccatt 2820

cagggccttc cttcgttcat tgagctgctt gacggcactg atgtcgctac tctttcccct 2880

gtcaaggttc agctgggtat ggcttgtatt caggttgctc tcgctcgcat gtgggcttca 2940

tggggtatca ctcctaccgc tgtcattggc cacaacttgg gcgagtacgc tgctctccac 3000

gttgctggtg tcatctctgc cagcgacatg atcaccctcg ttggtcgtcg tgctgagctc 3060

cttgtcaagg actgcactcc tcacacccac ggtatgctcg ctgtcaaggg tggcgctgaa 3120

gccatccgtg acgtcctcgg ctccaagatg accgagattg cctgcatcaa cggtcctgag 3180

gagactgtac tttgcggtag tggcgaggtt gttagtgctg ccaacgagac tctttccgct 3240

cgcggcttca aggccaccaa gctcaatgtt cccttcgcct tccactcggc ccaggtcgac 3300

cctatcctcg agtccttcaa gaaggttgct tcctgcgtca ccttcaacaa gcccgctgtc 3360

cctgtcctct ctcccctttc cggagatgtc attcgcgagg ctggtgttat tggcccagac 3420

tacctcgcta gacacgccag agagactgtc aacttctgga ccgctctgac cactggtcag 3480

aaggagaaga tcttcgatga gaagactgct tggctcgagg ttggcgctca ccccgtctgc 3540

tctggcatgg tcaaggcttc tgtcggtgcc accgtcaccg ctccttcgct ccgtcgcaac 3600

gaggacccct ggaagactat cgccaccagt gtttgcgccc tcttcaacgc tggtgtccag 3660

atcaacttcg atgagtacca ccgtgaattc aatgctgctc aggaacttct tcctctgccc 3720

acctactctt tcgacaacaa gaagtactgg ctcgactacc agaacaactg gactcttacc 3780

aaaggtgagg cccgcgaggt ccagaagact attgaggccg cccctcaagc tgcccctgtc 3840

gtcgagaagc ccgctaagcg cctctccact tcctgtcaga aggttatcca cgaggagttc 3900

gaggccaact ctggtaccgt tgtcattcag tccaacgttg ccgagcccaa gctcttccag 3960

gctgtcattg gtcaccaagt caacgacact gccctctgcc cctcgtccct gtacgctgac 4020

atggcattga ctgtctgcga ctacctttac aagcagctca gaccctctgc ccccaagatt 4080

ggcatgaacg tctgcgagat ggaggtccct aagcccttca tcgccaagat ggagcagccc 4140

gctgaaggtc agcacatcca actcgaggcc aaggctgacc tcgacgaggg tgtcgctcat 4200

ttgaagttca gaagtgtcac tcccgatggc aagctcatcc aagaccatgc tcactgcctc 4260

gtcaagttcg aggacatcaa ctcctggaac gaggactggg agcgtatcaa cttcatggtc 4320

aagtctcaag tcgatgttct caaggctaag actaagactc aggaggctca tgtcatgcag 4380

cgcggtatgg cttacaagct cttcaagtgc ttcgtcaact acaacgagaa gtaccgtggt 4440

atggaggagg tcattgtcaa cagccagacc actgaggcta ctgcctccat caagttccag 4500

aacggcccca acgacggtga ctggtaccaa gccccttacc agattgacag catgtgccac 4560

atctcgggtt tcattgtcaa cgctactgac ctgattgact ctgacaacaa cgtctgcatc 4620

tcgcacggct ggggttcgat gagaattgcc aagcagatga ctcctgaggg caactatcgc 4680

tcctgcgttc gcatgcttcc caagcctggt aacatttacc aaggtgatgt ttacatcatg 4740

gagggagatg agatcgttgc cgtctgcggt ggtcttaagt tccagcagat ctttcgccgt 4800

gtcctgaacg tcttcctgcc tcctcagaag gctggagcac ctgccaaggc tgctgcccct 4860

gctgcccccg ctgctgcacc caaggcagtc gctgccccca aggctgccgc tcctaaggcc 4920

caggccccta agcctgttgt caaggctgct aaggctccca agaaggtcgc tgccaagaag 4980

cctcaaggcg ctaccatcac ttctcgtgtt atgcaaatca ttgctaccga gaccgatgtt 5040

gagcagtctg agcttgttga tgaggctgca ttcgagaatc ttggtgtcga ctccctcatg 5100

tccctgaccg tctctgcgaa gttcagagag gatcttgaca tggagatcag ctctactctt 5160

ttcactgact accctaccgt tggtgagatg aagaagtact tctctcagtt cgatggaaac 5220

gtcggcccag ttgaggatga ctctgaggac gagtctgagc cttcggacat cgccactcct 5280

tacgaggacg acctcagcac tcccgccagc tctgctccta gcactgctcc cagtgatgca 5340

ggaaagcctg acattgactc tcccactcgt gagatccctg actccgaggt tggtgaagtc 5400

agcctggctc gccacatcat cgttcaggag atgggaattg acagctccga acttactgac 5460

gaggctgacc tttctgagct cggtatggac tctttgatga gcttgaccat ccttagtgag 5520

cttcgcgaga agacgggtat cgatcttccc tcgaccttcc tctccaccaa ctctaccatc 5580

ttggacattg agaatgctct cggtatgcgc ccaaagccca aggccaaggc tgctgctgcc 5640

cccaaggccg caaagcagac ttcgcctcaa cttgacaaag tcaacgccaa gcttaccgat 5700

gtctccaagt tccccgccgc aacctcagtt cttctccagg gcaatgctaa gattgctacc 5760

aagaagatct tcttcctgcc cgacggttct ggctctgcta catcctatgt cagcattcct 5820

aacattggtc ctgatgttgc tgcctttgga ctcaactgcc ctttcatgaa gagccccact 5880

gactggactt gtggtatcga gactgtctct ctcctctacc ttgctgagat caagagacgc 5940

cagcctcaag gcccctacat catcggtggt tggtctgctg gtggtgtcat tgcctacgcc 6000

gttgctcagg ctctgcttgc caacggtgaa gaagtcgagc gcctgcttct tcttgactct 6060

ccttgccctg tcaaccttgc acctcttcct caacgtctcc acgtcttctt caacgagatt 6120

ggtctccttg gtactggtga cccgtccaag actcccaagt ggttgcttcc ccacttcagc 6180

gccgccattc gttcgctctc tgattacgag cctcaacctt cgttgaagcc tatcaagacc 6240

tacgctatct ggtgccgtga tggtgtcgct ggtaaccccg gtgaccctcg ccctccacca 6300

gctgaggagg aggaccctgc acccatgaag tggctgttga accaccgcac tgacttcact 6360

gacaacggtt gggctcagct ctgcggcaat gagatgaagt tcggtgttat gggcggcaac 6420

cacttcacca tgatgaagga gcctcatgca caagaactcg gcaagctcat ccaagaaggt 6480

cttcagatct aa 6492

<210> 2

<211> 2163

<212> PRT

<213>Aureobasidium pullulans（Aureobasidium pullulans）

<400> 2

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

1 5 10 15

Leu Leu Arg Lys Val Val Leu Arg Thr Lys Asn Ala Leu Val Leu Asn

20 25 30

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

35 40 45

Ser Arg Pro Gln Arg Asp Ala Ile Pro Asp Phe Met Thr Leu Asn Asn

50 55 60

Leu Val Asp Leu Tyr Tyr Glu Lys Gly Leu Lys Leu Pro His Leu Glu

65 70 75 80

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

85 90 95

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

100 105 110

Ala Leu Cys Thr Gly Ser Leu Ala Ala Ala Ala Val Ala Ser Ala Arg

115 120 125

Thr Leu Asp Glu Leu Val Thr Leu Gly Val Glu Thr Val Arg Ile Ala

130 135 140

Phe Arg Thr Gly Val Arg Val Asn Glu Ala Arg Thr Ala Leu Gly Gln

145 150 155 160

Asp Leu Met Ser Arg Glu Ser Trp Ser Thr Ile Val Thr Gly Ile Asn

165 170 175

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

180 185 190

Ile Pro Thr Thr Ser His Ala Tyr Ile Ser Ala Val Ser Thr Met Ala

195 200 205

Ile Thr Ile Ser Gly Pro Pro Thr Ile Thr Lys Arg Phe Phe Glu Glu

210 215 220

Ser Glu Ala Val Arg Lys Asn Asn Arg Val Lys Ile Pro Val Tyr Ala

225 230 235 240

Pro Tyr His Ala Glu His Leu Tyr Ser Ser Ala Asp Ile Glu Thr Val

245 250 255

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

260 265 270

Leu Val His Ser Gly Ser Ser Gly Met Cys His Ile Ala Glu Asn Thr

275 280 285

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

290 295 300

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

305 310 315 320

Thr Asn Ala Pro Ala Lys Val Phe Cys Ile Gly Val Ser Asn Val Ser

325 330 335

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

340 345 350

Leu Val Asp His Ser Ala Trp Glu Ser Ala Val Thr Asp Ala Ser His

355 360 365

Gly Arg Thr Gln Asn Asp Lys Ile Ala Ile Val Gly Met Ser Gly Arg

370 375 380

Phe Pro Ser Ala Ala Ser Thr Glu Ala Leu Trp Glu Leu Leu Glu Lys

385 390 395 400

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

405 410 415

Ala His Cys Asp Pro Ser Gly Lys Gly Lys Asn Lys Ser His Thr Pro

420 425 430

Tyr Gly Cys Phe Ile Asp Glu Pro Gly Leu Phe Asp Pro Arg Phe Phe

435 440 445

Asn Met Ser Pro Arg Glu Ala Ala Gln Thr Asp Pro Met Gly Arg Leu

450 455 460

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

465 470 475 480

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

485 490 495

Gln Thr Ser Asp Asp Trp Arg Glu Ile Asn Ala Ser Glu Asn Val Asp

500 505 510

Thr Tyr Phe Ile Thr Gly Gly Val Arg Ala Phe Ala Pro Gly Arg Ile

515 520 525

Asn Tyr Tyr Phe Lys Phe Ser Gly Pro Ser Phe Ser Ile Asp Thr Ala

530 535 540

Cys Ser Ser Ser Leu Ala Ala Ile Gln Leu Ala Cys Thr Ser Leu Trp

545 550 555 560

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

565 570 575

Asn Pro Asp Ile Phe Ser Gly Leu Ser Lys Gly Gln Phe Leu Ser Lys

580 585 590

Thr Gly Ser Cys Lys Thr Tyr Asp Asn Ala Ala Asp Gly Tyr Cys Arg

595 600 605

Gly Asp Gly Cys Gly Thr Val Val Leu Lys Arg Tyr Glu Asp Ala Leu

610 615 620

Ala Asp Lys Asp Asn Ile Leu Gly Cys Ile Leu Gly Ala Ala Thr Asn

625 630 635 640

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

645 650 655

Glu Phe Leu Tyr Lys Lys Val Leu Ala Asn Ala Gly Val Asp Ala His

660 665 670

Glu Ile Ser Tyr Val Glu Met His Gly Thr Gly Thr Gln Ala Gly Asp

675 680 685

Gly Ile Glu Met Thr Ser Val Thr Asn Val Phe Ala Pro Arg His Arg

690 695 700

Gln Arg Lys Pro Glu Glu Lys Leu His Leu Gly Ala Ile Lys Ala Asn

705 710 715 720

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

725 730 735

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

740 745 750

Gly Glu Ile Asn Lys Thr Phe Pro Lys Asp Leu Lys Asp Arg Asn Val

755 760 765

His Ile Pro Gln Gln Gln Ile Asp Phe Pro Arg Asn Gly Ala Glu Lys

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

Leu Lys Asn Asn Ile Gln Arg Leu Gln Gly Tyr Leu Arg Glu Asn Pro

835 840 845

Glu Thr Thr Leu Pro Ser Leu Ser Tyr Thr Ser Thr Ala Arg Arg Ile

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

Tyr Thr Ala Leu Gly Gln Lys Leu Tyr Gln Asp Leu Lys Ser Phe Arg

915 920 925

Asp Asp Ile Asn Gln Leu Asp Asn Met Ala Thr Ile Gln Gly Leu Pro

930 935 940

Ser Phe Ile Glu Leu Leu Asp Gly Thr Asp Val Ala Thr Leu Ser Pro

945 950 955 960

Val Lys Val Gln Leu Gly Met Ala Cys Ile Gln Val Ala Leu Ala Arg

965 970 975

Met Trp Ala Ser Trp Gly Ile Thr Pro Thr Ala Val Ile Gly His Asn

980 985 990

Leu Gly Glu Tyr Ala Ala Leu His Val Ala Gly Val Ile Ser Ala Ser

995 1000 1005

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

1010 1015 1020

Asp Cys Thr Pro His Thr His Gly Met Leu Ala Val Lys Gly Gly

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

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

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

Arg His Ala Arg Glu Thr Val Asn Phe Trp Thr Ala Leu Thr Thr

1145 1150 1155

Gly Gln Lys Glu Lys Ile Phe Asp Glu Lys Thr Ala Trp Leu Glu

1160 1165 1170

Val Gly Ala His Pro Val Cys Ser Gly Met Val Lys Ala Ser Val

1175 1180 1185

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

1190 1195 1200

Trp Lys Thr Ile Ala Thr Ser Val Cys Ala Leu Phe Asn Ala Gly

1205 1210 1215

Val Gln Ile Asn Phe Asp Glu Tyr His Arg Glu Phe Asn Ala Ala

1220 1225 1230

Gln Glu Leu Leu Pro Leu Pro Thr Tyr Ser Phe Asp Asn Lys Lys

1235 1240 1245

Tyr Trp Leu Asp Tyr Gln Asn Asn Trp Thr Leu Thr Lys Gly Glu

1250 1255 1260

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

1265 1270 1275

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

1280 1285 1290

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

1295 1300 1305

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

1310 1315 1320

Gly His Gln Val Asn Asp Thr Ala Leu Cys Pro Ser Ser Leu Tyr

1325 1330 1335

Ala Asp Met Ala Leu Thr Val Cys Asp Tyr Leu Tyr Lys Gln Leu

1340 1345 1350

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

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

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

1400 1405 1410

Gln Asp His Ala His Cys Leu Val Lys Phe Glu Asp Ile Asn Ser

1415 1420 1425

Trp Asn Glu Asp Trp Glu Arg Ile Asn Phe Met Val Lys Ser Gln

1430 1435 1440

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

1445 1450 1455

Met Gln Arg Gly Met Ala Tyr Lys Leu Phe Lys Cys Phe Val Asn

1460 1465 1470

Tyr Asn Glu Lys Tyr Arg Gly Met Glu Glu Val Ile Val Asn Ser

1475 1480 1485

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

1490 1495 1500

Asn Asp Gly Asp Trp Tyr Gln Ala Pro Tyr Gln Ile Asp Ser Met

1505 1510 1515

Cys His Ile Ser Gly Phe Ile Val Asn Ala Thr Asp Leu Ile Asp

1520 1525 1530

Ser Asp Asn Asn Val Cys Ile Ser His Gly Trp Gly Ser Met Arg

1535 1540 1545

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

1550 1555 1560

Arg Met Leu Pro Lys Pro Gly Asn Ile Tyr Gln Gly Asp Val Tyr

1565 1570 1575

Ile Met Glu Gly Asp Glu Ile Val Ala Val Cys Gly Gly Leu Lys

1580 1585 1590

Phe Gln Gln Ile Phe Arg Arg Val Leu Asn Val Phe Leu Pro Pro

1595 1600 1605

Gln Lys Ala Gly Ala Pro Ala Lys Ala Ala Ala Pro Ala Ala Pro

1610 1615 1620

Ala Ala Ala Pro Lys Ala Val Ala Ala Pro Lys Ala Ala Ala Pro

1625 1630 1635

Lys Ala Gln Ala Pro Lys Pro Val Val Lys Ala Ala Lys Ala Pro

1640 1645 1650

Lys Lys Val Ala Ala Lys Lys Pro Gln Gly Ala Thr Ile Thr Ser

1655 1660 1665

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

1670 1675 1680

Glu Leu Val Asp Glu Ala Ala Phe Glu Asn Leu Gly Val Asp Ser

1685 1690 1695

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

1700 1705 1710

Met Glu Ile Ser Ser Thr Leu Phe Thr Asp Tyr Pro Thr Val Gly

1715 1720 1725

Glu Met Lys Lys Tyr Phe Ser Gln Phe Asp Gly Asn Val Gly Pro

1730 1735 1740

Val Glu Asp Asp Ser Glu Asp Glu Ser Glu Pro Ser Asp Ile Ala

1745 1750 1755

Thr Pro Tyr Glu Asp Asp Leu Ser Thr Pro Ala Ser Ser Ala Pro

1760 1765 1770

Ser Thr Ala Pro Ser Asp Ala Gly Lys Pro Asp Ile Asp Ser Pro

1775 1780 1785

Thr Arg Glu Ile Pro Asp Ser Glu Val Gly Glu Val Ser Leu Ala

1790 1795 1800

Arg His Ile Ile Val Gln Glu Met Gly Ile Asp Ser Ser Glu Leu

1805 1810 1815

Thr Asp Glu Ala Asp Leu Ser Glu Leu Gly Met Asp Ser Leu Met

1820 1825 1830

Ser Leu Thr Ile Leu Ser Glu Leu Arg Glu Lys Thr Gly Ile Asp

1835 1840 1845

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

1850 1855 1860

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

1865 1870 1875

Ala Ala Pro Lys Ala Ala Lys Gln Thr Ser Pro Gln Leu Asp Lys

1880 1885 1890

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

1895 1900 1905

Ser Val Leu Leu Gln Gly Asn Ala Lys Ile Ala Thr Lys Lys Ile

1910 1915 1920

Phe Phe Leu Pro Asp Gly Ser Gly Ser Ala Thr Ser Tyr Val Ser

1925 1930 1935

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

1940 1945 1950

Pro Phe Met Lys Ser Pro Thr Asp Trp Thr Cys Gly Ile Glu Thr

1955 1960 1965

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

1970 1975 1980

Gly Pro Tyr Ile Ile Gly Gly Trp Ser Ala Gly Gly Val Ile Ala

1985 1990 1995

Tyr Ala Val Ala Gln Ala Leu Leu Ala Asn Gly Glu Glu Val Glu

2000 2005 2010

Arg Leu Leu Leu Leu Asp Ser Pro Cys Pro Val Asn Leu Ala Pro

2015 2020 2025

Leu Pro Gln Arg Leu His Val Phe Phe Asn Glu Ile Gly Leu Leu

2030 2035 2040

Gly Thr Gly Asp Pro Ser Lys Thr Pro Lys Trp Leu Leu Pro His

2045 2050 2055

Phe Ser Ala Ala Ile Arg Ser Leu Ser Asp Tyr Glu Pro Gln Pro

2060 2065 2070

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

2075 2080 2085

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

2090 2095 2100

Glu Asp Pro Ala Pro Met Lys Trp Leu Leu Asn His Arg Thr Asp

2105 2110 2115

Phe Thr Asp Asn Gly Trp Ala Gln Leu Cys Gly Asn Glu Met Lys

2120 2125 2130

Phe Gly Val Met Gly Gly Asn His Phe Thr Met Met Lys Glu Pro

2135 2140 2145

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

2150 2155 2160

<210> 3

<211> 20

<212> DNA

<213>It is artificial synthesized

<400> 3

atgtctaacg ttcttctttt 20

<210> 4

<211> 20

<212> DNA

<213>It is artificial synthesized

<400> 4

gatctgaaga ccttcttgga 20

<210> 5

<211> 30

<212> DNA

<213>It is artificial synthesized

<400> 5

gcgccgcggc atgtctaacg ttcttctttt 30

<210> 6

<211> 31

<212> DNA

<213>It is artificial synthesized

<400> 6

gcgtctagaa tgatctgaag accttcttgg a 31

Claims (11)

1. a kind of Aureobasidium pullulans polyketide synthases gene, it is characterised in that nucleotide sequence is as shown in SEQ ID NO.1.

2. as the polyketide synthases of the Aureobasidium pullulans polyketide synthases gene code described in claim 1, it is characterised in that Amino acid sequence is as shown in SEQ ID NO.2.

3. the recombinant expression carrier containing the Aureobasidium pullulans polyketide synthases gene described in claim 1.

4. recombinant expression carrier as claimed in claim 3, it is characterised in that the recombinant expression carrier is by claim 1 Aureobasidium pullulans polyketide synthases gene be inserted into the multiple cloning sites of pGAPZ α-A expression vectors and obtain recombinant plasmid.

5. the expression cassette containing the Aureobasidium pullulans polyketide synthases gene described in claim 1.

6. transgenic cell line or recombinant bacterium containing the recombinant expression carrier described in claim 3 or 4.

7. recombinant bacterium as claimed in claim 6, it is characterised in that the recombinant bacterium is by the restructuring described in claim 3 or 4 The recombinant bacterium that expression vector conversion Pichia pastoris SMD1168H is obtained.

8. the polyketide synthases described in Aureobasidium pullulans polyketide synthases gene and/or claim 2 described in claim 1 exist Application in being synthesized outside melanosome.

9. a kind of method synthesized outside melanosome, comprises the following steps：Recombinant bacterium described in fermented and cultured claim 6, is collected Protein in fermented supernatant fluid, precipitation supernatant, then desalination and concentration are carried out to albumen precipitation, produce the thick enzyme of polyketide synthases Liquid；Polyketide synthases crude enzyme liquid is mixed with Aureobasidium pullulans crude enzyme liquid, malonyl coenzyme A, pH to 8.0,38 ~ 40 DEG C of bars is adjusted Reacted under part, abundant oxygen supply is carried out during reaction, reacted 40 ~ 60 minutes；After reaction terminates, pH to 2.0 ~ 3.0 is adjusted, is centrifuged, Precipitation is collected, melanin crude product is obtained.

10. method as claimed in claim 9, it is characterised in that the Aureobasidium pullulans crude enzyme liquid is that Aureobasidium pullulans is fresh Thalline is through liquid nitrogen grinding broken wall, with sodium phosphate buffer weight bacteriolyze powder, centrifugation, the supernatant of acquisition.

11. method as claimed in claim 9, it is characterised in that also include：The step of melanin crude product is purified, It is specific as follows：Melanin crude product is dissolved in NaOH, acidic methanol is added, stood, precipitation is collected in centrifugation, and precipitation is steamed Distilled water is washed to neutrality, drying, produces melanin.