The specific implementation mode is as follows:
example 1 construction of yarrowia lipolytica engineering bacteria producing Glycyrrhetinic acid
(1) Synthesis of coding sequence of glycyrrhetinic acid synthesis related protein
Artificially synthesizing beta-amyrin synthase bAS, wherein the nucleotide sequence is shown in SEQ ID NO. 001, and the amino acid sequence is shown in SEQ ID NO. 002, and constructing a pUC19 plasmid vector skeleton to obtain a pUC19-bAS plasmid; artificially synthesizing an NADPH-cytochrome P450 reductase gene CPR, wherein the nucleotide sequence is shown in SEQ ID NO. 003, the amino acid sequence is shown in SEQ ID NO. 004, and constructing the CPR to a pUC19 vector skeleton to obtain a pUC19-CRP plasmid; artificially synthesizing cytochrome P450 oxidase CYP88D6 gene, wherein the nucleotide sequence is shown in SEQ ID NO:005, and the amino acid sequence is shown in SEQ ID NO:006, constructing the cytochrome P450 oxidase CYP88D6 gene into a pUC19 vector skeleton, and obtaining a pUC19-CYP88D6 plasmid; artificially synthesizing 11/30-oxidation-beta-balsamic alcohol synthase gene CYP72A154, the nucleotide sequence of which is shown in SEQ ID NO. 007, and the amino acid sequence of which is shown in SEQ ID NO. 008, and obtaining pUC19-CYP72A 154.
(2) Construction of expression module plasmid integrating glycyrrhetinic acid biosynthesis
Constructing a beta-Amyrin synthse expression module, using PmeI-bAS-up and BamHI-bAS-down as primers and pUC19-bAS plasmid as a template, and amplifying to obtain a PCR fragment containing a PmeI enzyme cutting site, a beta-Amyrin synthase gene and a BamHI enzyme cutting site; the pINA1269 plasmid is subjected to double enzyme digestion by PmeI and BamHI, and the two ends of the obtained vector skeleton have PmeI and BamHI cleavage sites. The PCR fragment carrying the PmeI and BamHI enzyme cutting sites is enzymatically connected with a carrier skeleton, and is transferred into an escherichia coli transformant to obtain a correct positive transformant plasmid, and the obtained integrated plasmid is named pINA1269-Php4d-bAS-xpr2 plasmid.
Construction of cytochrome P450 oxidase 88D6(CYP88D6) expression Module Using PEXP1-up/PEXP1Down and lip2t-up/lip2t-down as primers and yarrowia lipolytica genome as a template respectively amplify the fragments of a PEXP1 promoter and a lip2t terminator; CYP88D6-up/down is used as a primer, pUC19-CYP88D6 plasmid is used as a template, a fragment III containing cytochrome P450 oxidase gene CYP88D6 is obtained through amplification, PmeI-PEXE1-up and ClaI-lip2t-down are used as primers, and the three fragments are used as templates to obtain a fragment IV containing a PmeI enzyme cutting site, a PEXE1 promoter, a CYP88D6 gene, a lip2t terminator and a ClaI enzyme cutting site through amplification. PmeI and ClaI enzymes are used for carrying out double enzyme digestion on pINA1269 plasmid to obtain a linear vector fragment V, the vector fragment and the IV fragment are transferred into escherichia coli after enzyme digestion, a correct positive transformant is obtained, plasmid is extracted, and the obtained integrative plasmid is named pINA1269-PEXP1-CYP88D6-lip2t。
Constructing an 11/30-oxidation-beta-balsamic alcohol synthase gene CYP72A154 expression module, and respectively amplifying a PGPD2 promoter and a migt terminator by using pGPD2-up/PGPD2-down and migt-up/migt-down as primers and yarrowia lipolytica genome as a templateTwo fragments of (a); CYP72A154-up/down is used as a primer, and pUC19-CYP72A154 plasmid is used as a template, so that the CYP72A154 fragment is obtained through amplification. PmeI-PGPD2-up/ClaI-migt is used as a primer, the three fragments are used as templates, a fragment containing a PmeI enzyme cutting site, a PGPD2 promoter, a CYP72A154 gene, a migt terminator and a claI enzyme cutting site is obtained by amplification, and the integrated plasmid named pINA1269-P is obtained by connecting the fragment and a linear carrier fragment in the same enzyme cutting and enzyme connecting modeGPD2-CYP72A154-migt。
Constructing a cell P450 reductase CPR expression module, using pTEF-up/down and natr-up/down as primers and yarrowia lipolytica genome as a template, and amplifying to obtain a pTEF promoter fragment and a natr terminator fragment; and amplifying to obtain Cpr gene segment with Cpr-up/down as primer and pUC19-Cpr plasmid as template. PmeI-PTEF-up/ClaI-natr0down is used as a primer, the three fragments are used as a template, a fragment containing a PmeI enzyme cutting site, a PTEF promoter, a Cpr gene, a natr terminator and a claI enzyme cutting site is obtained through amplification, and the fragment and a linear carrier fragment are connected IN the same enzyme cutting enzyme connection mode to form IN1269-PTEF-Cpr-natr integrative plasmid.
To construct the resulting Php4d-bAS-xpr2、PEXP1-CYP88D6-lip2t、PGPD2CYP72A154-migt and-PTEFthe-Cpr-natr is integrated into the pINA1269 plasmid, and the resulting plasmid is named pINA1269-bAS-88D6-Cpr-72A 154.
(3) Construction of yarrowia lipolytica engineering bacterium for producing glycyrrhetinic acid
The pINA1269-bAS-88D6-Cpr-72A154 integrated plasmid obtained in the last step is cut by NotI enzyme to obtain the plasmid carrying pINA1269-Php4d-bAS-xpr2、PEXP1-CYP88D6-lip2t、PGPD2CYP72A154-migt and PTEF-a linear plasmid vector of the Cpr-natr expression cassette, the linear vector carrying these 4 expression cassettes being integrated into the pBR site in yarrowia lipolytica YL-003 strain by the lithium acetate method. The YL-003 strain is obtained by genetic engineering on the basis of the po1f strain, the po1f strain can be obtained by purchasing, and the specific genetic engineering genotype of the YL-003 strain is that the ku70 and ku80 sites are knocked out on the basis of the po1f strain. Specific method for lithium acetate conversionThe method comprises the following steps:
the lithium acetate conversion method comprises the following steps: starting yarrowia lipolytica was cultured in 2ml YPD medium for 18 hours, and 200ul of the cultured yarrowia lipolytica was added to fresh YPD medium and cultured for 4-5 hours. Centrifuging at 6000rpm at room temperature for 5min, collecting thallus, and sterilizing with ddH2And O, re-suspending the bacteria, then centrifuging again to collect the bacteria, and discarding the supernatant. Then 1mL of 100mM lithium acetate precooled at 4 ℃ is added into the centrifuged thallus, after standing treatment for 5min at room temperature, the thallus is centrifuged and collected at 6000rpm at normal temperature, and the preparation of the yarrowia lipolytica competent cell is completed. The transformation mix system included (1)240ul PEG (50% W/v); (2)36ul of 1.0M lithium acetate solution; (3)10ul salmon sperm ss-DNA; (4) the linearized vector-integrated fragments were added in equal amounts of about 400ng each; (5) sterile water is filled to 360 ul. Adding the above into competent cells sequentially, vortex and shake for 1min, water bath at 30 deg.C for 30min, water bath at 42 deg.C for 30min, and centrifuging at 6000rpm for 5 min. After removing the supernatant, 1ml of YPD medium was added and cultured at 30 ℃ and 200rpm for 2 hours to recover the cells. And then centrifuging at 6000rpm for 5min, removing supernatant, washing the thalli for 2 times by using sterile water, finally resuspending by using 100ul of sterile water, coating and screening in an SD solid culture medium lacking leucine, wherein the screening condition is 30 ℃, and performing genome extraction verification on a single colony on a plate after culturing for 48h, wherein the yarrowia lipolytica strain correctly integrating the glycyrrhetinic acid synthesis way is named as GA 001.
Example 2 fermentation and detection of Glycyrrhetinic acid
(1) And (3) shaking flask fermentation: the YPD medium is used for the culture and fermentation of yarrowia lipolytica, for single fermentation in a shake flask, a single clone of GA001 strain is picked from a plate, inoculated into 10mL of YPD medium for activating seed liquid, then the activated seed liquid is transferred to a 250mL shake flask to be loaded with 30mL of YPD medium, and the shake culture is carried out for 4 days under the conditions of 30 ℃ shaking table and 220 rpm. All shake flask fermentation experiments were set up in 3 parallel experimental groups. After the fermentation is finished, detecting the product according to the mode shown in example 2 (2);
(2) centrifuging the cells at 6000rpm for 10min, separating supernatant and thallus, adding 4ml 1-butanol and quartz sand into the thallus, vortex oscillating for 20min, and extracting intracellular product. The butanol was evaporated and the residue was dissolved in methanol and detected by LC-MS after passage through a membrane.
Detection conditions are as follows: the extracted metabolites were detected using an ACQUITY UPLC/MS system (Waters, Milford, MA, USA) with an ACQUITY UPLC HSS C18 column (2.1X 150mm column and 2.1X 5mm VanGuard pre-column; particle size, 1.8 μm, Waters, Milford, MA, USA) and an ACQUITY TQ detector (Waters) [ using electrospray ionisation anion mode with Selective Ion Monitoring (SIM) ]. The mobile phase consisted of 0.025% (v/v) aqueous acetic acid (solvent 1) and 0.025% (v/v) acetonitrile acetate (solvent 2). The sample was separated by gradient elution (elution with 30% solvent 2 for 6 minutes followed by increasing solvent concentration with time, after 22 minutes the concentration reached 100%: 40% at 6 minutes, 50% at 18 minutes and 100% at 28 minutes) at a flow rate of 0.20 mL/min. The final conditions were maintained for 3.5 minutes and then returned to the initial conditions, resulting in a total chromatographic run time of 38.5 minutes. The sample management conditions and the column were maintained at 15 ℃ and 30 ℃ respectively. For MS detection, the capillary voltage was set to 2.5kV, the cone voltage was set to 80V, the extraction cone voltage was set to 3V, the source temperature was set to 150 ℃, the desolvation temperature was set to 350 ℃, the cone gas flow rate was set to 50L/h and the desolvation gas flow rate was set to 600L/h, respectively. The amount of the extracted target compound in the peak area was determined using MassLynx software (Waters).
(3) The GA001 strain was cultured in the fermentation manner described above, and a glycyrrhetinic acid production of 53.2mg/L was finally detected.
Example 3 attenuation of lanosterol synthase and enhancement of squalene synthase and squalene epoxidase expression
Acid yield
(1) Construction of expression modules for squalene synthase (ERG9) and squalene epoxidase (ERG1)
Construction of the Php4d-ERG9-xpr2 expression Module: respectively amplifying to obtain a Php4d promoter and a xpr2t terminator by using hp4d-up/down and xpr2t-up/down as primers and a pINA1269 plasmid as a template; the gene of squalene synthase ERG9 is obtained by amplification by taking ERG9-F/R as a primer and yarrowia lipolytica genome as a template, and the expression module Php4d-ERG9-xpr2 is obtained by constructing by using an Overlap method.
Construction of the pTEFin-ERG1-lip2t expression Module: amplifying to obtain a PTEFin promoter and a lip2t terminator by taking pTEFin-up/down and lip2t-up/down as primers and pMT015 plasmid as a template; the gene of squalene epoxidase ERG1 is amplified by taking ERG1-F/R as a primer and yarrowia lipolytica genome as a template, and the pTEFin-ERG1-lip2t expression module is constructed by utilizing an Overlap method. Wherein the sequence of the pTEFin promoter is shown in SEQ ID No. 009.
The two expression modules were assembled by the Gibson assembly method to obtain a plasmid of pUC19-ERG9-ERG1 expression cluster.
(2) Expression of ERG9 and ERG1 genes at lanosterol synthase (ERG7) site
Using del-ERG7-1-up/del-ERG7-1-down as primer and yarrowia lipolytica genome as template to amplify to obtain segment of 500bp upstream of ERG7 gene; using HisG-F/R as a primer and pUC19-HisG-Ura-HisG plasmid as a template, and amplifying to obtain a HisG-Ura-HisG uracil screening marker loss module; using hp4d-up-hisG/lip2t as a primer and pUC19-ERG9-ERG1 as a plasmid, and amplifying to obtain an ERG9-ERG1 expression module gene cluster; and (3) amplifying by taking del-ERG7-2-up/del-ERG7-2-down as a primer and yarrowia lipolytica genome as a template to obtain a 500bp fragment at the downstream of the ERG7 gene. The transformation of the gene expression module is carried out by adopting a yeast self-assembly mode, the length of homologous fragments of two adjacent fragments is about 80bp, the transformation method refers to a lithium acetate transformation method described in example 1, the transformed strain is YL-003, the addition amount of each fragment is 400ng, an SD-delta Ura plate is coated with the incubated bacterial liquid, and the engineering bacteria YL-103-Ura which excessively express squalene synthase (ERG9) and squalene epoxidase (ERG1) at the ERG7 site are obtained by screening.
(3) Loss of URA selection marker
The YL-103-Ura strain simultaneously carries a uracil loss module HisG, Ura and His, and can screen a marker gene through 5-fluoroorotic acid loss Ura, and the specific operation steps are as follows: screening the positive monoclone obtained in the last step to 10ml of YPD culture medium, culturing overnight for 16h, properly diluting, coating 100ul of 5-fluoroorotic acid YPD plate with the final concentration of 1mg/ml, culturing at 30 ℃ for 2-3 days, selecting 30 larger monoclonals, simultaneously streaking on an SD plate and an SD-delta Ura plate respectively, continuously placing in an incubator at 30 ℃ for standing and culturing for 2-3 days, selecting bacterial colonies growing on the SD plate and not growing on the SD-delta Ura plate according to the growth conditions of the two plates, and obtaining the engineering bacteria successfully losing the Ura screening marker, wherein the bacterial strain is named as YL-103.
Example 4 regulated expression of mevalonate pathway Key enzymes (HMGS and trHMGR)
FBA1in-up/down is used as a primer, pRFBA1in plasmid is used as a template, and the FBA1in promoter is obtained through amplification; using HMGS-F/R as a primer and yarrowia lipolytica genome as a template to amplify to obtain a hydroxymethyl glutarate-CoA synthase (HMGS) gene segment; and (3) amplifying by using the natR-up/down as a primer and using the yarrowia lipolytica genome as a template to obtain the natR terminator. And assembling the obtained 3 fragments in an Overlap mode to construct and obtain a Pfbain-HMGS-natR expression module.
Using hp4d-up/down as a primer and pINA1269 plasmid as a template to amplify to obtain an hp4d promoter; using trHMGR-F/R as a primer and yarrowia lipolytica genome as a template, and amplifying to obtain a gene fragment with 500 amino acids truncated by hydroxymethyl glutarate reductase; and (3) amplifying by using xpr2t-up/down as a primer and pINA1269 plasmid as a template to obtain a xpr2t terminator. The 3 fragments obtained were assembled by means of Overlap to construct an expression module of Php4d-trHMGR-xpr2 t.
And constructing the PFBA1in-HMGS-natR expression module and the expression module of Php4d-trHMGR-xpr2t on a pUC19 plasmid in a Gibson assembly mode to obtain the pUC19-HMGS-trHMGR plasmid. Wherein the promoter sequence of FBA1in is shown in SEQ ID NO: 010.
And constructing a Pfbain-HMGS-natR expression module and an expression module of Php4d-trHMGR-xpr2t on a pUC19 plasmid in a Gibson assembly mode to obtain the pUC19-HMGS-trHMGR plasmid.
(1) Regulated expression of HMGS and trHMGR
Using del-poxB1-up/down as primer and yarrowia lipolytica genome as template to amplify to obtain poxB gene upstream 500bp segment; using HisG-up/down as a primer and pUC19-HisG-URA-HisG plasmid as a template, and amplifying to obtain a screening marker Ura loss module; amplifying to obtain regulated HMGS and trHMGR gene expression clusters by using fba1-1/xpr2t-2 as a primer and pUC19-HMGS-trHMGR plasmid as a template; using del-poxB2-up/down as primer and yarrowia lipolytica genome as template to amplify and obtain the downstream 500bp segment of poxB gene.
The transformation of the gene expression module adopts a yeast self-assembly mode, the homologous arm between adjacent modules is about 80bp, the transformation mode refers to example 3(2), the transformed strain is the YL103 strain constructed in example 3, the SD-delta Ura plate is coated with the transformation liquid, and the grown positive clone is verified to be correctly named as YL-108-Ura.
(2) Recovery of YL-108-Ura screening tags
The YL108-108-Ura was screened for marker loss according to the method of example 3(3), and the strain lacking the Ura screening marker was designated YL-108.
(4) Integration of Glycyrrhetinic acid biosynthesis Gene Module
According to the integration type plasmid pINA1269-bAS-88D6-Cpr-72A154 of the glycyrrhetinic acid biosynthesis gene cluster constructed in example 1(3), the glycyrrhetinic acid biosynthesis integration gene cluster is integrated into the pBR site of the YL-108 strain according to the lithium acetate transformation method of yarrowia lipolytica, and thus the engineered strain GA-106 capable of biosynthesizing glycyrrhetinic acid yeast is obtained.
(5) Evaluation of fermentation of GA-106 Strain
The fermentation evaluation of the yarrowia lipolytica engineered strain producing glycyrrhetinic acid was carried out in the same manner as in example 2, and it was confirmed that the production amount of glycyrrhetinic acid was 175.5 mg/L.
Example 5 multicopy integration of Glycyrrhetinic acid Synthesis Gene Cluster
(1) Construction of multicopy integration plasmid pINA1292-bAS-88D6-Cpr-72A154
According to P in example 1hp4d-bAS-xpr2、PEXP1-CYP88D6-lip2t、PGPD2CYP72A154-migt and-PTEFIntegrating each gene expression module into pINA1292 plasmid by using Gibson assembly mode to construct glycyrrhetinic acid biosynthesis multi-copy completeThe synthetic plasmid pINA1292-bAS-88D6-Cpr-72A 154.
(2) Multicopy integration of glycyrrhetinic acid heterologous pathway enzymes
And (3) carrying out enzyme digestion on the multicopy integration plasmid pINA1292-bAS-88D6-Cpr-72A154 obtained in the last step by using NotI enzyme to obtain a linearized pINA1292-bAS-88D6-Cpr-72A154 fragment, integrating the fragment into an YL-108 strain by a lithium acetate conversion method, coating an SD-delta Leu plate on the incubated bacterial liquid, and screening the positive clone obtained by growth on the plate to obtain a GA 205.
(3) Evaluation of fermentation of GA205 Strain
The GA205 strain was subjected to fermentation evaluation in the same manner as in example 2, and the production of glycyrrhetinic acid at 240.3mg/L was examined.
Example 6 integration of cytochrome b5
(1) Construction of cytochrome b5 expression cassette
GPDin-up/down is taken as a primer, a synthesized GPDin sequence is taken as a template, and a GPDin promoter is obtained by amplification, wherein the GPDin promoter sequence is shown in SEQ ID NO: 011; using CYB5-up/down as a primer and synthesized cytochrome b5 as a template to amplify to obtain a cytochrome b5 gene, wherein the nucleotide sequence of the cytochrome b5 is shown in SEQ ID NO:012, and the amino acid sequence is shown in SEQ ID NO: 013; cyc1-up/down is used as a primer, pCFB153 plasmid is used as a template, and Cyc terminator is obtained by amplification. The three fragments are assembled by using an Overlap method to construct an expression module of the PGPDin-CYB5-Cyc gene.
(2) Integration of cellular b5 Synthesis Module
D17-up1/down1 is used as a primer to amplify to obtain a homologous fragment of 500bp at the upstream of the D17 gene; using HisG-up/down as a primer and pUC19HisG-Ura-HisG as a template to obtain a HisG-Ura-HisG screening marker recovery module through amplification; amplifying to obtain a PGPDin-CYB5-Cyc gene expression module by taking PGPDin-up/Cyc-down as a primer and the expression module as a template; d17-up2/down2 is used as a primer to amplify to obtain a homologous fragment of 500bp at the downstream of the D17 gene. The homologous fragments were integrated into GA205 strain by lithium acetate integration using yeast self-assembly, and the resulting incubation fluid was applied to SD-. DELTA.Ura plates and positive clones grown on the plates were selected. The correct strain was glycyrrhetinic acid-producing strain GA208, which incorporated cytochrome b 5.
(3) Fermentation plate of GA208 strain
The GA208 strain was subjected to fermentation evaluation in the same manner as in example 2, and the production of glycyrrhetinic acid at 290.4mg/L was detected.
The above description: integrating glycyrrhetinic acid biosynthesis modules b-AS, CYP88D6, CYA72A154 and CPR into yarrowia lipolytica Chassis bacteria through tool plasmids to construct and obtain a glycyrrhetinic acid biosynthesis primary strain GA001, wherein the strain integrated with the glycyrrhetinic acid biosynthesis modules has the capacity of catalyzing glucose to generate glycyrrhetinic acid through conventional shake flask fermentation verification; starting from a strain which does not integrate a glycyrrhetinic acid biosynthesis module, the synthesis of 2, 3-oxidosqualene is increased by knocking out lanosterol synthase and overexpressing squalene oxidase and squalene epoxidase at the site; enhancing the MVA pathway by integrating a strong promoter-expressing trHMGR and HMGS enzyme at the poxB site; integrating a plurality of glycyrrhetinic acid biosynthesis modules by integrating the glycyrrhetinic acid biosynthesis pathway into the engineered yarrowia lipolytica using a multicopy integrative tool plasmid; the over-expression of cytochrome b5 is also realized, and the yield of the modified integrated strains is remarkably improved compared with that of a glycyrrhetinic acid biosynthesis primary strain GA 001; compared with the original strain before modification, the yield of glycyrrhetinic acid is obviously improved, and the effects of the modification means are fully demonstrated.
In the examples, each plasmid was publicly reported and commercially available; the primers involved therein are designed based on the amplification template and the purpose of amplification, in combination with conventional primer design methods.
Example 7 fed-batch fermentation of GA208 Strain to produce Glycyrrhetinic acid
A single GA208 clone was picked from the plate, inoculated into a test tube containing 2ml of YPD medium, cultured with shaking at 30 ℃ and 220rpm for 24 hours, transferred into a 500ml Erlenmeyer flask containing 100ml of YPD medium, and cultured with shaking for 24 hours to obtain a seed solution required for batch fermentation. Transferring 100ml of the seed solution into 3L of fermentation mediumThe components are as follows: 60g/L glucose, 15g/L (NH)4)2SO4,8g/L KH2PO4,6.15g/L MgSO412ml/L vitamin, 10ml/L trace metal salt. Wherein the trace metal salt solution comprises the following components: 5.75g/L ZnSO4*7H2O,0.32g/LMnCI2,0.32g/L CuSO4,0.47g/L CoCl2,0.48g/L Na2MoO4,2.9g/L CaCl2·2H2O,2.8g/LFeSO4*7H2O, 0.5M EDTA. Wherein the vitamin solution comprises the following components: 0.05g/L of biotin, 1g/L of calcium pantothenate, 1g/L of nicotinic acid, 25g/L of inositol, 1g/L of thiamine hydrochloride, 1g/L of pyridoxal phosphate, and 0.2g/L of p-aminobenzoic acid.
The batch fermentation temperature was 30 ℃ and pH was controlled to 6.0 using NaOH, fed-batch glucose was controlled to a concentration of 30g/L and the fermentation process examined the production of product.
The final GA208 strain was fermented to produce 7g/L glycyrrhetinic acid, which is the highest level reported for de novo synthesis of glycyrrhetinic acid at present, as shown in FIG. 2.
Nucleotide and amino acid sequence listing of the specification
< 110 > Hebei Weidakang Biotech Ltd
Less than 120 yarrowia lipolytica engineering strain for biosynthesis of glycyrrhetinic acid by taking glucose as substrate, construction and application thereof
<160> 3
<210> 1
<211>2289bp
<212> DNA
< 213 > is artificially synthesized
<400> 1
atgtggaggc tgaagatagc ggaaggaggg aaggacccat acatatacag cacaaacaac 60
ttcgtgggga ggcagacatg ggagtatgat cccgatggtg ggagtgccga ggagagagct 120
caggttgatg ccgctcgtct ccatttctat aacaaccgct tccaggtcaa gccctgtggc 180
gacctccttt ggcgttttca gattctgaga gaaaataact tcaaacaaac aatagctagc 240
gtgaagatag gagatggaga ggaaataaca tacgaaaagg cgacaacggc ggtgagaagg 300
gctgcacatc acctatccgc attgcagacc agcgatggcc attggcctgc tcaaattgca 360
ggtcctctgt ttttccttcc ccccttggtt ttttgtatgt acattacagg acatcttgat 420
tcggtattcc cagaagagta tcgcaaagag attcttcgtt acatatacta tcaccagaat 480
gaagatggag ggtgggggct acacatagag ggtcacagca ccatgttttg tactgcactc 540
aactatatat gcatgcgaat tcttggagaa gggcccgatg ggggtcaaga caatgcttgt 600
gctagagcaa gaaagtggat tcatgatcat ggtggtgtaa cacatatacc ttcatgggga 660
aaaacttggc tttcgatact tggtgtattt gataggtgcg gaagcaaccc aatgccccct 720
gagttttgga tcctgccttc atttcttcct atgcatccag ctaaaatgtg gtgttattgc 780
cgattggtat acatgcctat gtcttacttg tatgggaaaa ggtttgtggg tccaatcaca 840
ccactcattc tacagttgag agaagaactc tttactgaac cttatgaaaa agttaattgg 900
aagaaagcac gtcaccaatg tgcaaaggaa gatctttact atccccatcc tttgttacaa 960
gatctgatat gggatagttt atgcttattc actgagccac ttctgactcg ttggcctttc 1020
aacaagctgg ttagagaaaa ggcccttcaa gtaacaatga agcatatcca ctatgaagat 1080
gagactagtc gatacataac cattgggtgt gtggaaaagg ttttatgtat gcttgcttgt 1140
tgggtggaag atccaaatgg agatgctttc aagaagcatc ttgcaagggt cccggattac 1200
ttatgggttt cggaagatgg aatgaccatg cagagttttg gtagtcaaga atgggatgct 1260
ggttttgccg ttcaagcttt gcttgccact aacctagtcg aggaaattgc tcccacactt 1320
gccaaaggac atgatttcat caagaaatct caggttaggg acaacccttc aggagatttt 1380
aagagcatgt atcgtcatgt ttctaaaggc tcatggacct tctccgatca agaccatgga 1440
tggcaagttt ctgattgcac agcagaaggt ttgaagtgtt gtctactttt atcaatgttg 1500
cctccagaga ttgtggggga aaagatggaa cctgaaaggt tatatgattc agtcaatgtc 1560
ctattgtcgc ttcagagtaa aaagggtggt ttatcagcgt gggagccagc aggagctcaa 1620
gaatggttag aactactcaa tcccacggaa ttttttgcgg acattgtagt tgagcatgaa 1680
tacgttgagt gcactggatc agcaattcag gcactagttc tgttcaagaa gctatatcca 1740
gggcatagga agaaagagat agagaatttc attgccaatg cagttcgatt ccttgaagat 1800
acacaaacag ctgatggttc atggtatgga aattggggag tttgcttcac ttacggttct 1860
tggttcgcac ttggtggtct tgcagctgct ggcaagactt ttgccaattg tgctgctatt 1920
cgcaaagctg ttaaatttct tctcacaaca cagagagagg atggtggatg gggggagagc 1980
tatctttcaa gcccaaaaaa gatatatgta cctcttgaag gaagtcgatc aaatgttgta 2040
catactgcat gggcccttat gggtttaatt catgctggcc aggcagagag agaccctgct 2100
cctcttcacc gagctgcaaa attgatcatc aattcccaat tggaagaggg tgattggccc 2160
caacaggaaa tcacgggagt attcatgaaa aattgtatgc tgcattaccc aatgtataga 2220
gatatttatc caatgtgggc tctagctgag tatcgtagac gggttccatt gccttccact 2280
ccagcttaa 2289
<210> 2
<211>762
<212> PRT
< 213 > is artificially synthesized
<400> 1
MWRLKIAEGG KDPYIYSTNN FVGRQTWEYD PDGGSAEERA QVDAARLHFY NNRFQVKPCG 60
DLLWRFQILR ENNFKQTIAS VKIGDGEEIT YEKATTAVRR AAHHLSALQT SDGHWPAQIA 120
GPLFFLPPLV FCMYITGHLD SVFPEEYRKE ILRYIYYHQN EDGGWGLHIE GHSTMFCTAL 180
NYICMRILGE GPDGGQDNAC ARARKWIHDH GGVTHIPSWG KTWLSILGVF DRCGSNPMPP 240
EFWILPSFLP MHPAKMWCYC RLVYMPMSYL YGKRFVGPIT PLILQLREEL FTEPYEKVNW 300
KKARHQCAKE DLYYPHPLLQ DLIWDSLCLF TEPLLTRWPF NKLVREKALQ VTMKHIHYED 360
ETSRYITIGC VEKVLCMLAC WVEDPNGDAF KKHLARVPDY LWVSEDGMTM QSFGSQEWDA 420
GFAVQALLAT NLVEEIAPTL AKGHDFIKKS QVRDNPSGDF KSMYRHVSKG SWTFSDQDHG 480
WQVSDCTAEG LKCCLLLSML PPEIVGEKME PERLYDSVNV LLSLQSKKGG LSAWEPAGAQ 540
EWLELLNPTE FFADIVVEHE YVECTGSAIQ ALVLFKKLYP GHRKKEIENF IANAVRFLED 600
TQTADGSWYG NWGVCFTYGS WFALGGLAAA GKTFANCAAI RKAVKFLLTT QREDGGWGES 660
YLSSPKKIYV PLEGSRSNVV HTAWALMGLI HAGQAERDPA PLHRAAKLII NSQLEEGDWP 720
QQEITGVFMK NCMLHYPMYR DIYPMWALAE YRRRVPLPST PA 762
<210> 3
<211>1989bp
<212> DNA
< 213 > is artificially synthesized
<400> 1
atgtgctgca gacatcctag gtcagttggt ggaactagtg ctgcgcctga aactccccaa 60
agttccgctc ccgcaacacc attgcctgct ccagctccaa cagttcctac gcccgcccct 120
cctactccag catcaacaac agaagctcct gcaccctcta ccccagcttc aactaccggc 180
gcaccaagca ctcctgcttc cacaactggt gccccgagta cccccgcatc aacaacggga 240
agttcagatc aaacccaatt cctagctaac ttattggttc aattgcaagc agccggggca 300
ggtccagaac aacttcaacc aattgtggat gccattgcta agcagttcgt tcaacaacag 360
caacaatctc aagctgtgcc cgatccgcaa caagccgttg atgaaatttg tattgaacta 420
ggagcatctc cggattacac tacccaggtg gctcaaatat tagaacaagg acacgaccct 480
aacagcgctt ctggatctag atccttacaa cctccaccac caccatctgg ttctaatcct 540
ggcggtggga atagtggcca agaagtccct gaacccaagc aaaagaaggc cagagcgccc 600
aaaaaacaag gtagcgaaat ggaaactgat attactacta tggcagctgc tgcggacttt 660
aaactggacg atttcgtgac cacttgggtt ggagcctgta ctacagctga aggtaaggct 720
atggttacca ctatagaatt gttaggtcat tccataatca tgagtgtagg tgttctgttc 780
atgttcgtca aaccgcaaca atactcaaac gcacatgatc ctagattgga gccgttgcag 840
agtcagtccg aattaattaa gatgataaat gcatcctcaa tgggaatggc tgaaatcagg 900
aaggaattgc aagctaaaca aaaaacatta acaattgaag ccttgcaaac aattctgagg 960
aaagccatgc attacaaagg tgtttgggtt tgcattggta tttcatttgg atttactagt 1020
cttggcaaag gtatggaatt cagagggaga tggggcatca atggtcaatc tccaaccgct 1080
tccgcagccc cattcgttga agcttacaag caacactcaa aagtagctgc aaatttgaat 1140
gaggacagta tggatttggt tgagttctgg aataaagcta ggttcgaaga ttggggtgcg 1200
agaacaactc aatgtaaagc aggtgctatg aggaagagga atatgttgat tataagttgg 1260
agtccattaa cctcttcaaa agaatcaatg tatgggaaat acccattttg tgttataaga 1320
tcagactatt tccactatct tggtgatgag aacgttacat tgtctcattt gcaggaagac 1380
ttggttaaaa gtctaaatct gtgcgctgac agaacaaacg ggttaaatgc cacattacat 1440
gtgtgtgccg gtaagggcga ctggaaatgg agacatgaat ggttacaaca gtccaggttc 1500
tatggtagag tgacccacgg ttcaaatggg atctgtcaga gatgcttcgc tgcaaaaaat 1560
gactggttgg acgttcacga aagattcaac aacgctgaag acgtactgcg tgcacaagct 1620
accgcagtag gggaaggcat agccatgaaa caactgtccg gctggacacc taatatggaa 1680
cttccagact tattacattg tatctggcta ggtacgggaa gagatttggt cggtagtttg 1740
tgcatggaaa tggctgaaac aagacaagat ttggtgggtt ctacctatga tgagagactg 1800
gactgtttga gaagggatat tcaagcatgg tgcgcaagtc atgggattag accgagcact 1860
attgatgaat tatctctagc caagctgggt gttgaaaccc tatctcttga ttttccacaa 1920
ggaccatcaa aaggatacgc caataaagtt ttgcaattga gacttattta ctttaacggc 1980
gcttgttaa 1989
<210> 4
<211>662
<212> PRT
< 213 > is artificially synthesized
<400> 1
MCCRHPRSVG GTSAAPETPQ SSAPATPLPA PAPTVPTPAP PTPASTTEAP APSTPASTTG 60
APSTPASTTG APSTPASTTG SSDQTQFLAN LLVQLQAAGA GPEQLQPIVD AIAKQFVQQQ 120
QQSQAVPDPQ QAVDEICIEL GASPDYTTQV AQILEQGHDP NSASGSRSLQ PPPPPSGSNP 180
GGGNSGQEVP EPKQKKARAP KKQGSEMETD ITTMAAAADF KLDDFVTTWV GACTTAEGKA 240
MVTTIELLGH SIIMSVGVLF MFVKPQQYSN AHDPRLEPLQ SQSELIKMIN ASSMGMAEIR 300
KELQAKQKTL TIEALQTILR KAMHYKGVWV CIGISFGFTS LGKGMEFRGR WGINGQSPTA 360
SAAPFVEAYK QHSKVAANLN EDSMDLVEFW NKARFEDWGA RTTQCKAGAM RKRNMLIISW 420
SPLTSSKESM YGKYPFCVIR SDYFHYLGDE NVTLSHLQED LVKSLNLCAD RTNGLNATLH 480
VCAGKGDWKW RHEWLQQSRF YGRVTHGSNG ICQRCFAAKN DWLDVHERFN NAEDVLRAQA 540
TAVGEGIAMK QLSGWTPNME LPDLLHCIWL GTGRDLVGSL CMEMAETRQD LVGSTYDERL 600
DCLRRDIQAW CASHGIRPST IDELSLAKLG VETLSLDFPQ GPSKGYANKV LQLRLIYFNG 660
AC 662
<210> 5
<211>1482 bp
<212> DNA
< 213 > is artificially synthesized
<400> 1
atggaagttc attgggtttg tatgtcagct gcaactttgt tggtttgtta catcttcggt 60
tctaagttcg ttagaaattt gaacggttgg tactacgatg ttaagttgag aagaaaggaa 120
catccattac caccaggtga catgggttgg ccattaattg gtgacttgtt gtcttttatt 180
aaggatttct cttcaggtca tccagattct tttattaaca atttggtttt gaagtacggt 240
agatcaggta tctataagac tcatttgttc ggtaacccat ctatcatcgt ttgtgaacca 300
caaatgtgta gaagagtttt gacagatgat gttaacttca agttgggtta cccaaagtct 360
attaaagaat tggctagatg tagaccaatg attgatgttt caaacgcaga acatagattg 420
tttagaagat taatcacatc tccaatcgtt ggtcataaag ctttagcaat gtacttggaa 480
agattggaag aaatcgttat taattcattg gaagaattgt cttcaatgaa gcatccagtt 540
gaattgttga aggaaatgaa gaaagtttct tttaaagcta tcgttcatgt ttttatgggt 600
tcttcaaacc aagatattat taagaaaatt ggttcttctt ttactgattt gtacaacggc 660
atgttctcta tcccaattaa tgttccaggt tttacattcc ataaagcttt ggaagcaaga 720
aagaaattgg ctaagatcgt tcaaccagtt gttgatgaaa gaagattaat gatcgaaaat 780
ggtccacaag aaggttcaca aagaaaggat ttgatcgata tcttgttaga agttaaagat 840
gaaaatggta gaaaattaga agatgaagat atttctgatt tgttaattgg tttgttattt 900
gctggtcatg aatctactgc aacatcattg atgtggtcta tcacttactt aacacaacat 960
ccacatatct tgaagaaagc taaggaagaa caagaagaaa tcactagaac aagattttct 1020
tcacaaaaac aattgtcatt gaaggaaatt aaacaaatgg tttatttgtc tcaagttatt 1080
gatgaaactt tgagatgtgc aaacattgct tttgcaactt ttagagaagc tacagcagat 1140
gttaacatca acggttacat catcccaaaa ggttggagag ttttgatttg ggctagagca 1200
atccatatgg attcagaata ctacccaaat ccagaagagt ttaatccatc tagatgggat 1260
gattacaatg ctaaagcagg tacattttta ccatttggtg ctggttcaag attgtgtcca 1320
ggtgctgatt tggcaaagtt ggaaatctct attttcttgc attacttttt gttaaattac 1380
agattggaaa gaattaatcc agaatgtcat gttacttcat taccagtttc taaaccaaca 1440
gataactgtt tggctaaagt tattaaagtt tcttgtgcat aa 1482
<210> 6
<211> 493
<212> PRT
< 213 > is artificially synthesized
<400> 1
MEVHWVCMSA ATLLVCYIFG SKFVRNLNGW YYDVKLRRKE HPLPPGDMGW PLIGDLLSFI 60
KDFSSGHPDS FINNLVLKYG RSGIYKTHLF GNPSIIVCEP QMCRRVLTDD VNFKLGYPKS 120
IKELARCRPM IDVSNAEHRL FRRLITSPIV GHKALAMYLE RLEEIVINSL EELSSMKHPV 180
ELLKEMKKVS FKAIVHVFMG SSNQDIIKKI GSSFTDLYNG MFSIPINVPG FTFHKALEAR 240
KKLAKIVQPV VDERRLMIEN GPQEGSQRKD LIDILLEVKD ENGRKLEDED ISDLLIGLLF 300
AGHESTATSL MWSITYLTQH PHILKKAKEE QEEITRTRFS SQKQLSLKEI KQMVYLSQVI 360
DETLRCANIA FATFREATAD VNINGYIIPK GWRVLIWARA IHMDSEYYPN PEEFNPSRWD 420
DYNAKAGTFL PFGAGSRLCP GADLAKLEIS IFLHYFLRNY RLERINPECH VTSLPVSKPT 480
DNCLAKVIKV SCA 493
<210> 7
<211>1437 bp
<212> DNA
< 213 > is artificially synthesized
<400> 1
atggctgcaa cagctgctct tagaccattt gatagtgttc catcattggg taaagctttt 60
gctttcttcg ctaggaataa attatgtaag gaactaatgc aaaaaggtag tgaacacccc 120
agaatgttca gaatgcatgt cggaccacaa accgcgtgcg tcgtaacaca tccagaatta 180
gctagaagcg ttctgacacg tccaaaggat tttataaagt ttcaagtacc tcgtaaccca 240
gcgtcagcat tagactcttt gttaaacaga tctaaagacg gaaccttaag ccttttgatg 300
tctaatggtc atgattgggc ttcccaaagg aagccagtgg atcccgcttt tgagaaacaa 360
gcattacgta atttactacc aaaattcaat gattgtagcg cgagactact ggagacctgg 420
cgtggagctg atgaggtgga cgcgaaacag gatatgtcac gtttcgcttt agatgtttta 480
ggttccgcaa tgctgggtca aagctttggt gcaatcgatg gtaccttttc cgatagttat 540
gaatactaca aatatatcat ggtagagcaa aacaatccac tatatatgag cttccccctt 600
atggagagac taccaacatc ccgtaataga agaatgagag aagctgtaca ccatatgcat 660
ggtgtattgc agggtgctat tgacgctaga gtgcagttaa gaaaagacca acaagcggct 720
ggtacattga gtgatgaacc tcaagacatg ctggacatgc ttcttggtcc aggggccgac 780
gttccaactg atggcgtgct gcctaacgcc ggcgctttgc tacccaattt gtggattttt 840
ttcatcgctg gacacgatac tacagcaatt agtttggcat ggttaatgta ttttttggcc 900
aaatatccag aggttcaaca aaaggcacgt gaggaaactc aaaaagtact tgcgggtcgt 960
aaggatccca cctctgaaga tttagataac gttccatatt tatcagccgt tattaacgaa 1020
ggtttaagag ttagacctcc aatattcaat ttgtataccc gtcaagcagc acatgacaca 1080
gaattagatg gagtgttttt gccaaagggt actggcattg cgttgcacat agctgctatt 1140
aataagcatc ctgaagtgtg gggtgaagct gaagatttta atccagaaag gtttctgcaa 1200
gagaaacaac ctcgtgtctt tcataatctg ccattttctg ctggacctag gagatgtcta 1260
ggggacaagt tctctttgat ggaacaaaag tctttattga tgaagttatt gactgagtac 1320
aaggtcttac cacatgagag tactttggat ggtgataaaa attttacctc ttctatgcta 1380
ggtttgatct ttttacaacc tgaagatata aaggtcagat tgcaggcagt acaataa 1437
<210> 8
<211>478
<212>PRT
< 213 > is artificially synthesized
<400> 1
MAATAALRPF DSVPSLGKAF AFFARNKLCK ELMQKGSEHP RMFRMHVGPQ TACVVTHPEL 60
ARSVLTRPKD FIKFQVPRNP ASALDSLLNR SKDGTLSLLM SNGHDWASQR KPVDPAFEKQ 120
ALRNLLPKFN DCSARLLETW RGADEVDAKQ DMSRFALDVL GSAMLGQSFG AIDGTFSDSY 180
EYYKYIMVEQ NNPLYMSFPL MERLPTSRNR RMREAVHHMH GVLQGAIDAR VQLRKDQQAA 240
GTLSDEPQDM LDMLLGPGAD VPTDGVLPNA GALLPNLWIF FIAGHDTTAI SLAWLMYFLA 300
KYPEVQQKAR EETQKVLAGR KDPTSEDLDN VPYLSAVINE GLRVRPPIFN LYTRQAAHDT 360
ELDGVFLPKG TGIALHIAAI NKHPEVWGEA EDFNPERFLQ EKQPRVFHNL PFSAGPRRCL 420
GDKFSLMEQK SLLMKLLTEY KVLPHESTLD GDKNFTSSML GLIFLQPEDI KVRLQAVQ 478
<210> 9
<211>531 bp
<212> DNA
< 213 > is artificially synthesized
<400> 1
agagaccggg ttggcggcgc atttgtgtcc caaaaaacag ccccaattgc cccaattgac 60
cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttctcc ccacatatca 120
aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 180
cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 240
gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 300
aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 360
cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaaaatgg tgagtttcag 420
aggcagcagc aattgccacg ggctttgagc acacggccgg gtgtggtccc attcccatcg 480
acacaagacg ccacgtcatc cgaccagcac tttttgcagt actaaccgca g 531
<210> 10
<211> 1000bp
<212> DNA
< 213 > is artificially synthesized
<400> 1
aacagtgtac gcagtactat agaggaacaa ttgccccgga gaagacggcc aggccgccta 60
gatgacaaat tcaacaactc acagctgact ttctgccatt gccactaggg gggggccttt 120
ttatatggcc aagccaagct ctccacgtcg gttgggctgc acccaacaat aaatgggtag 180
ggttgcacca acaaagggat gggatggggg gtagaagata cgaggataac ggggctcaat 240
ggcacaaata agaacgaata ctgccattaa gactcgtgat ccagcgactg acaccattgc 300
atcatctaag ggcctcaaaa ctacctcgga actgctgcgc tgatctggac accacagagg 360
ttccgagcac tttaggttgc accaaatgtc ccaccaggtg caggcagaaa acgctggaac 420
agcgtgtaca gtttgtctta gcaaaaagtg aaggcgctga ggtcgagcag ggtggtgtga 480
cttgttatag cctttagagc tgcgaaagcg cgtatggatt tggctcatca ggccagattg 540
agggtctgtg gacacatgtc atgttagtgt acttcaatcg ccccctggat atagccccga 600
caataggccg tggcctcatt tttttgcctt ccgcacattt ccattgctcg gtacccacac 660
cttgcttctc ctgcacttgc caaccttaat actggtttac attgaccaac atcttacaag 720
cggggggctt gtctagggta tatataaaca gtggctctcc caatcggttg ccagtctctt 780
ttttcctttc tttccccaca gattcgaaat ctaaactaca catcacacaa tgcctgttac 840
tgacgtcctt aagcgaaagt ccggtgtcat cgtcggcgac gatgtccgag ccgtgagtat 900
ccacgacaag atcagtgtcg agacgacgcg ttttgtgtaa tgacacaatc cgaaagtcgc 960
tagcaacaca cactctctac acaaactaac ccagctctcc 1000
<210> 11
<211> 1130bp
<212> DNA
< 213 > is artificially synthesized
<400> 1
gacgcagtag gatgtcctgc acgggtcttt ttgtggggtg tggagaaagg ggtgcttgga 60
gatggaagcc ggtagaaccg ggctgcttgg ggggatttgg ggccgctggg ctccaaagag 120
gggtaggcat ttcgttgggg ttacgtaatt gcggcatttg ggtcctgcgc gcatgtccca 180
ttggtcagaa ttagtccgga taggagactt atcagccaat cacagcgccg gatccacctg 240
taggttgggt tgggtgggag cacccctcca cagagtagag tcaaacagca gcagcaacat 300
gatagttggg ggtgtgcgtg ttaaaggaaa aaaaaagaag cttgggttat attcccgctc 360
tatttagagg ttgcgggata gacgccgacg gagggcaatg gcgccatgga accttgcgga 420
tatcgatacg ccgcggcgga ctgcgtccga accagctcca gcagcgtttt ttccgggcca 480
ttgagccgac tgcgaccccg ccaacgtgtc ttggcccacg cactcatgtc atgttggtgt 540
tgggaggcca ctttttaagt agcacaaggc acctagctcg cagcaaggtg tccgaaccaa 600
agaagcggct gcagtggtgc aaacggggcg gaaacggcgg gaaaaagcca cgggggcacg 660
aattgaggca cgccctcgaa tttgagacga gtcacggccc cattcgcccg cgcaatggct 720
cgccaacgcc cggtcttttg caccacatca ggttacccca agccaaacct ttgtgttaaa 780
aagcttaaca tattataccg aacgtaggtt tgggcgggct tgctccgtct gtccaaggca 840
acatttatat aagggtctgc atcgccggct caattgaatc ttttttcttc ttctcttctc 900
tatattcatt cttgaattaa acacacatca acaatggcca tcaaagtcgg tattaacgga 960
ttcggacgaa tcggacgaat tgtgagtacc atagaaggtg atggaaacat gacccaacag 1020
aaacagatga caagtgtcgt cgacccacca gagcccaatt gagctcatac taacagtcga 1080
caacctgtcg aaccaattga tgactccccg acaatgtact aacacaggtc 1130
<210> 12
<211>402bp
<212> DNA
< 213 > is artificially synthesized
<400> 1
atgccgagtt cgaaatttcc caagtacacg tgggaagaaa ttcggaaaca caacaaagac 60
acagattgct gggtggtgct gtacgatcgt gttttggacg ttacaaagtt tttaaacgag 120
catcctggtg gacttgatcc aataaatgat cttggtggct acgacgtcac caattcgttt 180
gagactatcg gacactcctc agccgcgctg gcccgctcaa aagaattcat tgtgggtgac 240
ttggacaagg actcaatccc accacaagtt gttcggaagg aggtgaagaa taaagttcca 300
ctgacacagt accaggcagg tggagaaatg atttcactac aatacatact gggaggtgtc 360
attgcgcttt tgcttcttct ttactacctg atggcggctt aa 402
<210> 13
<211>133
<212> PRT
< 213 > is artificially synthesized
<400> 1
MPSSKFPKYT WEEIRKHNKD TDCWVVLYDR VLDVTKFLNE HPGGLDPIND LGGYDVTNSF 60
ETIGHSSAAL ARSKEFIVGD LDKDSIPPQV VRKEVKNKVP LTQYQAGGEM ISLQYILGGV 120
IALLLLLYYL MAA 133